Download study of the human humoral immune response against rotavirus

STUDY OF THE HUMAN HUMORAL IMMUNE RESPONSE
AGAINST ROTAVIRUS
DANIEL FELIPE HERRERA RODRÍGUEZ
THESIS
Presented as a partial requirement to opt for the Doctoral degree in
Biological Sciences
Tutors
Juana Ángel
Manuel Antonio Franco
Pontificia Universidad Javeriana
School of Sciences
Doctoral Program in Biological Sciences
Bogotá, D.C.
Colombia
January 2014
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Study of the human humoral immune response against rotavirus
Daniel Felipe Herrera Rodríguez
APPROVED
Manuel Antonio Franco Cortés, MD, PhD
Tutor
Juanita Ángel Uribe, MD, PhD
Tutor
John Mario González Escobar, MD, PhD
Jury
Carlos Narváez Rojas, MD, PhD
Jury
Gagandeep Kang, MBBS, MD, PhD
Jury
Gloria Vásquez Duque, MD, PhD
Jury
Deborah Dunn-Walters, PhD
Jury
Study of the human humoral immune response against rotavirus
Daniel Felipe Herrera Rodríguez
Ingrid Schuler, Biol, PhD
Academic Dean
School of Sciences
Manuel Antonio Franco, MD, PhD
Graduate Programs Director
School of Sciences
TABLE OF CONTENTS
1. INTRODUCTION ............................................................................................... 10
2. BACKGROUND ................................................................................................. 14
2.1 ROTAVIRUS ................................................................................................ 14
2.1.1 Structure ................................................................................................ 14
2.1.2 Classification .......................................................................................... 15
2.1.3 Epidemiology ......................................................................................... 16
2.1.4 Life cycle ................................................................................................ 17
2.1.5 Clinical manifestations and pathophysiology in humans ........................ 19
2.2 IMMUNE RESPONSE AGAINST ROTAVIRUS ........................................... 20
2.2.1 Innate immunity ..................................................................................... 20
2.2.2 T cell response ...................................................................................... 21
2.2.3 B cell response ...................................................................................... 23
2.2.4 Correlates of protection after natural infection in children ...................... 23
2.2.5 Rotavirus vaccines ................................................................................. 24
2.2.6 Correlates of protection after RV vaccination in children ....................... 27
2.3 THE MUCOSAL IMMUNE SYSTEM ............................................................ 28
2.3.1 Induction of the mucosal immune response mediated by B cells in the
intestine .......................................................................................................... 28
2.3.2 The polymeric immunoglobulin receptor ................................................ 29
2.3.3 Secretory IgA ......................................................................................... 31
2.3.4 RV secretory IgA (RV-SIgA) in serum .................................................... 32
2.4 B CELLS AND THE MAINTENANCE OF SEROLOGICAL MEMORY........ 33
2.4.1 Phenotype of circulating B cells and current controversies .................... 33
2.4.2 RV-specific B cells and how they are studied ........................................ 37
2.4.3 The serological memory and the theories of how it is maintained .......... 39
2.4.4 Rituximab and its effect on memory B cells and serological memory .... 40
3. HYPOTHESES .................................................................................................. 43
4. OBJECTIVES .................................................................................................... 43
5. MATERIALS AND METHODS .......................................................................... 44
6. JOURNAL ARTICLES ....................................................................................... 44
7. GENERAL DISCUSSION AND CONCLUSIONS .............................................. 48
8. PERSPECTIVES ............................................................................................... 56
9. APPENDIX......................................................................................................... 57
9.1 DETAILED MATERIALS AND METHODS .................................................. 57
9.1.1 Subjects and sample processing: children with gastroenteritis, children
vaccinated with RIX4414, and placebo recipient children ............................... 57
9.1.2 ELISA for measuring plasma RV secretory immunoglobulin .................. 58
9.1.3 ELISA for measuring total plasma secretory IgA ................................... 59
9.1.4 ELISA for measuring plasma RV-IgM .................................................... 59
9.1.5 Recombinant human SC and competitive binding assays ..................... 59
9.1.6 Subjects, sample collection and processing: patients with autoimmune
diseases and healthy volunteers ..................................................................... 60
9.1.7 Production of fluorescent virus like particles (VLPs) .............................. 61
9.1.8 Biotinylated - TT antigen ........................................................................ 61
9.1.9 Flow cytometry assays ........................................................................... 61
9.1.10 Measurement of total immunoglobulins (IgA, IgG and IgM) and IgM
Rheumatoid Factor ......................................................................................... 63
9.1.11 ELISAs for detection of RV-specific IgA, IgG, and IgG1 and TT-specific
IgG in plasma.................................................................................................. 63
9.1.12 Measurement of anti-CCP and anti-dsDNA autoantibodies (IgG isotype)
........................................................................................................................ 63
9.1.13 Statistical analyses .............................................................................. 64
9.2 Figure A1: Schematic representation of the ELISA for measuring
plasma RV secretory immunoglobulin ........................................................... 65
9.3 Figure A2: Schematic representation of the ELISA for measuring total
plasma secretory IgA ....................................................................................... 66
9.4 Relevant data not shown from the first article: “Rotavirus Specific
Plasma Secretory Immunoglobulin in Children with Acute Gastroenteritis
and Children Vaccinated with an Attenuated Human Rotavirus Vaccine” .. 67
9.5 Relevant data not shown from the second article: “Simultaneous
Assessment of Rotavirus-Specific Memory B Cells and Serological Memory
after B Cell Depletion Therapy with Rituximab” ............................................ 68
9.6 Journal paper: “Circulating human rotavirus specific CD4 T cells
identified with a class II tetramer express the intestinal homing receptors
α4β7 and CCR9” ............................................................................................... 68
10. REFERENCES ................................................................................................ 69
ABSTRACT
Rotavirus (RV) vaccines are less efficacious in countries with high mortality rates
by this pathogen; the lack of a widely accepted immunological correlate of
protection hinders the development of new RV vaccines. Secretory immunoglobulin
(SIg) in serum has been proposed to indirectly measure intestinal Ig. Therefore,
plasma RV-SIg was evaluated by ELISA in 50 children vaccinated with RIX4414
and 62 placebo recipients and correlated with protection when both groups were
analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV
vaccines.
A thorough understanding of the relationship between memory B cells (mBc) and
serological memory contributes to identify useful correlates of protection. RV-mBc
are enriched in mBc subpopulations associated with certain autoimmune diseases
pathogenesis. In patients treated with Rituximab (RTX), some autoantibodies
(auto-Abs) decrease, but pathogen-specific IgG remain unchanged; thus,
serological memory may depend on the type of antigen and/or Ab isotype
evaluated. Circulating total, RV-, and tetanus toxoid (TT)-specific mBc and Abs,
and some auto-Abs were evaluated in patients with autoimmunity before and after
RTX therapy. Following treatment, total, RV-, and TT-mBc decreased significantly.
Total IgM levels were significantly lower than total IgA and IgG levels, and the
auto-Abs measured were significantly diminished. In contrast, RV- and TT-Abs
remained unchanged. In conclusion, serological memory against RV and TT seem
to be maintained by long-lived plasma cells (PC), not affected by RTX, and an
important proportion of total IgM and serological memory against some autoantigens seem to be maintained by short-lived PC, dependent on mBc precursors
depleted by RTX.
RESUMEN
Las vacunas contra RV son menos eficaces en países con una alta mortalidad por
este patógeno; la falta de un correlato de protección inmunológico adecuado
dificulta desarrollar nuevas vacunas. La inmunoglobulina secretora (SIg) circulante
evalúa indirectamente la Ig intestinal. La RV-SIg sérica fue medida por ELISA en
50 niños vacunados con RIX4414 y 62 niños que recibieron placebo, y
correlacionó con protección cuando los grupos se analizaron conjuntamente. La
RV-SIg podría ser un correlato de protección valioso.
Entender la relación entre las células B de memoria (cBm) y la memoria serológica
contribuye a identificar correlatos de protección. Las RV-cBm están enriquecidas
en subpoblaciones asociadas con algunas enfermedades autoinmunes. En
pacientes tratados con Rituximab (RTX), ciertos autoanticuerpos (autoAcs)
disminuyen, pero no la IgG específica de patógenos; entonces, la memoria
serológica podría depender del antígeno y/o isotipo del Ac estudiado. Se
evaluaron las cBm y los Acs totales y específicos de RV y toxoide tetánico (TT)
circulantes y algunos autoAcs en pacientes con autoinmunidad antes y después
de recibir RTX. Luego del tratamiento, las cBm totales y específicas de RV y TT
disminuyeron significativamente. La IgM total se redujo significativamente más que
la IgA e IgG total, así como los autoAcs evaluados, mientras que los Acs
específicos de RV y TT permanecieron sin cambios. En conclusión, la memoria
serológica contra RV y TT depende probablemente de células plasmáticas de
larga vida; además, buena parte de la IgM total y la memoria serológica contra
algunos autoantígenos es probablemente mantenida por células plasmáticas de
corta vida dependientes de las cBm.
Abbreviations
RV
SIg
Ig
RVmBc
Bc
RTX
Abs
TT
PC
GE
CI
WHO
Dose 1
Dose 2
pIgR
SC
dsRNA
VP
NSP
DLP
TLP
Ca++
ER
ENS
PAMPs
PRRs
IEC
TLRs
GALT
CSR
GC
pIgs
dsDNA
RF
rhSC
PBMC
Rotavirus
Secretory immunoglobulin
Immunoglobulin
Rotavirus-specific
Memory B cells
B cells
Rituximab
Antibodies
Tetanus toxoid
Plasma cells
Gastroenteritis
Confidence interval
World Health Organization
D1
D2
Polymeric immunoglobulin receptor
Secretory component
Double-stranded RNA
Viral protein
Non-structural protein
Double layered particle
Triple layered particle
Calcium
Endoplasmic reticulum
Enteric nervous system
Pathogen-associated molecular patterns
Pattern recognition receptors
Intestinal epithelial cells
Toll-like receptors
Gut-associated lymphoid tissue
Class-switch recombination
Germinal center
Polymeric immunoglobulins
Double-stranded DNA
Rheumatoid factor
Recombinant human secretory component
Peripheral blood mononuclear cells
1. INTRODUCTION
RV is the main cause of severe gastroenteritis (GE) in young children. In 2008,
before the introduction of routine immunization, it was estimated that RV GE
caused 453,000 deaths (CI 95% 420,000 – 494,000) in children under the age of
five, corresponding to 5% of children’s deaths of all causes and 37% of those
attributable to GE yearly worldwide in this age group [1]. In Colombia it was
estimated that without vaccination the annual number of medical visits and deaths
by RV GE in children under 2 years would be 105,378 and 470 (CI 95% 295 - 560),
respectively [2].
The burden of RV disease is not limited to the underdeveloped world; before
implementation of the RV vaccine program in the United States, 50% of
hospitalized children and 50% of those in the emergency department with acute
GE were infected with RV [3]. This implied that the establishment of public health
measures, such as improvements in water supply, hygiene, and sanitation, was
insufficient and effective vaccines were necessary [4].
Two RV vaccines are commercially available and recommended for infants
worldwide by the WHO [5]: RotarixTM (GlaxoSmithKline Biologicals) and RotaTeqTM
(Merk and Co. Inc.). Nonetheless, both vaccines are less efficacious (39% to 77%)
in some low-income countries in Africa and Asia [6], where 85% of worldwide
mortality occurs [7]. This drawback and the difficulty, given by the cost, to include
RV vaccines in the national immunization programs of the poorest countries make
it necessary to improve them or to develop new RV vaccines [8, 9].
The achievement of this objective implies several difficulties, of which the lack of a
widely accepted immunological correlate of protection is very important. In fact, all
RV vaccine efficacy trials have had a clinical endpoint (protection against RVinduced moderate to severe diarrhea and/or mortality); these studies can be
carried out in a limited array of settings and are very expensive. The use of a
validated correlate of protection as a surrogate endpoint would contribute to the
faster development of a new generation of RV vaccines and the assessment of its
efficacy in a wider number of settings. It would also facilitate the evaluation of
immune interference with other vaccines and support for guiding vaccination
programs and regulatory decisions.
With the present work we aimed to contribute to the correlates of protection for RV
vaccines field in two ways: 1. We propose a new potential correlate of protection
for RV vaccines. 2. In an effort to thoroughly understand the relationship between
mBc and serological memory, we describe the association between RV-mBc and
serological memory in humans, capitalizing the unique opportunity offered by
10
patients with autoimmune diseases treated with Rituximab (RTX). These findings
are presented in two papers in the journal’s articles section.
Currently, serum RV-specific IgA (RV-IgA) measured shortly after natural infection
or vaccination represents the best practically measured correlate of protection
against RV GE in humans [10, 11]. This is probably because serum RV-IgA
transiently reflects the intestinal RV-IgA. Nevertheless, some vaccinees with serum
RV-IgA develop mild RV GE, and protection provided by the vaccines can be
higher or lower than the levels predicted by serum RV-IgA detected in vaccinees
[12, 13].
RV induces an intestinal and systemic immune response, the latter most probably
related to the antigenemia and viremia detected in acutely infected animals and
children [14, 15]. Even so, RV preferentially replicates in the intestine, and local
mucosal immunity is thought to be essential in human RV immunity [13]. In fact,
during an acute RV infection in children, circulating IgD- RV-specific B cells (Bc)
express intestinal-homing receptors (47+, CCR9+) and, as a result, probably
reflect mucosal immunity [16].
Consistent with this finding, in a previous double blind trial of the attenuated
RIX4414 human RV vaccine (which contains the same vaccine strain virus found in
the Rotarix formulation), correlations between protection from disease and
frequencies of RV-memory IgD-CD27+47+CCR9+ circulating Bc measured after
dose 1 (D1) and levels of plasma RV-IgA after dose 2 (D2) were found. However,
the correlation coefficients for both tests were low, suggesting that other factors are
important in explaining protection from disease [17]. Moreover, it is generally
accepted that neutralizing Abs against the RV infecting strain present in the
intestine provide protection [18]; however, assessment of intestinal fluid after RV
vaccination is impractical and measurement of RV-IgA in feces is subject to several
problems [12].
SIg in serum has been proposed as an alternative method for indirectly measuring
intestinal Ig [19]. Polymeric IgA and IgM are recognized by the polymeric
immunoglobulin receptor (pIgR) on the basolateral membrane of the mucosal
epithelial cells [20]. This complex is endocytosed and transcytosed to the apical
membrane, where it is cleaved and part of it (the secretory component [SC])
remains attached to the Ig, forming SIg, which may retro-transcytose across
epithelial cells and eventually enter the circulation [20].
RV-SIg has been detected in serum of children with acute RV infection [19, 21],
and it correlated with the amounts detected in duodenal fluid one week after the
infection [22]. Based on these precedents, we sought to confirm the presence of
plasma RV-SIg in children with natural RV infection and to determine if circulating
RV-SIg could reflect more precisely the intestinal protective immune response
11
induced by the RIX4414 RV vaccine, and be a better correlate of protection than
circulating RV-IgA after vaccination.
Recently, it was confirmed in an IgA deficient mouse model that this Ig is very
important for the intestinal primary immune response against RV, and that it has a
fundamental role in the protection against RV reinfection of the intestine. Even so,
IgA has a minor role in the resolution of systemic primary RV infection and seems
to be dispensable for clearance of antigenemia after reexposure to the virus. These
results suggest that circulating RV- IgG and/or IgM are important in the clearance
of the virus from the systemic compartment [23].
The observed pivotal role of Abs as mediators of protection against RV reinfection
in mice [23, 24], and their correlation with protection in humans [11, 25-27],
indicate that Bc are crucial for the immune response against RV infection. Bc are
also important for the clearance of a primary RV infection in mice [24] and for the
removal of the virus from the systemic circulation [28]. Furthermore, children with B
and/or T immunodeficiencies become chronically infected with RV [29].
A better understanding of the frequencies of antigen-specific mBc subsets and
their association with serological memory –defined as the persistence of Abs levels
in the absence of the antigen [30]– is also important to identify valuable correlates
of protection for vaccines [31, 32].
The mechanisms that lead to the maintenance of serological memory in healthy
individuals are still unclear and, in general, have been studied only with regard to
the IgG isotype and for a limited number of antigens. Pathogen-specific protective
IgG levels following natural infection or vaccination can persist for decades, or in
some cases for a lifetime, without any apparent role of the antigen [30]. In healthy
adults, serological memory seems to be maintained by long-lived PC [30, 33], and
it has been proposed that PC and mBc are independently regulated populations
[34]. Nonetheless, under certain circumstances, such as autoimmunity, short-lived
PC, which need to be constantly replenished by mBc, may also be a factor in the
maintenance of serological memory [35]. Thus, in conditions where short-lived PC
contribute to serological memory, a correlation between numbers of circulating
antigen-specific mBc and levels of antigen-specific serological memory is expected
[36].
Serological memory has been assessed in patients with autoimmunity and treated
with RTX [37-40], an anti-CD20 monoclonal Ab that depletes Bc but does not affect
PC. In such patients, some auto-Abs decrease after treatment [39], but other autoAbs [37] and pathogen specific IgG Abs [37, 38, 40] do not decrease. These
findings suggest that the mechanism of maintenance of serological memory could
depend on the type of antigen and/or Ab isotype evaluated, particularly in patients
with autoimmune diseases. However, antigen-specific mBc and antigen-specific
12
Abs of different isotypes have not been simultaneously assessed in patients that
received Bc depletion therapy with RTX.
Circulating RV-specific Bc, compared to TT-specific Bc, seem to be peculiar
because a subgroup of naïve Bc bind RV-virus like particles (VLPs) [41, 42], and
because RV-mBc are enriched in the CD27+IgM+ and in the CD27-IgG+ mBc
subsets [43, 44]. Additionally, CD27+IgA+ RV-mBc correlated positively with RV-IgA
plasma levels, but a correlation between CD27+IgG+ RV-mBc and RV-IgG in
plasma was absent. In contrast, CD27+IgG+ TT-mBc correlated with TT-IgG
plasma levels [43]. Therefore, the relationship of RV-mBc with serological memory
seems to be somewhat different from that of TT-mBc, making it a relevant model to
study the association between mBc and serological memory.
CD27+IgD+IgM+ mBc (IgM+ mBc) and CD27- mBc, the mBc subsets in which RVmBc are enriched, are relevant in autoimmune diseases pathogenesis. For
example, IgM+ mBc are decreased in patients with systemic lupus erythematosus
(SLE) [45], rheumatoid arthritis (RA) [46], and Sjögren’s Syndrome [47]. In fact,
there is a negative correlation between the circulating number of IgM+ mBc and
auto-Abs levels and disease activity in SLE patients [45]. In contrast, circulating
CD27- mBc are increased in SLE patients and positively correlate with disease
activity [48]. Given that RV-mBc are enriched in these subpopulations, we
hypothesized that RV-mBc are distributed in a distinct manner in patients with
autoimmune diseases and are therefore related to serological memory in a peculiar
way, dissimilar from the proposed manner for pathogens studied thus far.
13
2. BACKGROUND
2.1 ROTAVIRUS
2.1.1 Structure
RVs are non-enveloped double-stranded RNA (dsRNA) viruses that belong to the
Reoviridae family, with a characteristic morphology of wheel-like particles (as seen
by electron microscopy) from which their name is derived. The mature virus particle
has icosahedral symmetry with a diameter of about 100 nm, including the spikes
[49]. The viral genome is constituted by 11 segments of dsRNA encoding six
structural proteins (VPs), which provide structural support and mediate cell entry,
and six non-structural proteins (NSPs) (Figure 1), which are only produced by RVs
in infected cells and are implicated in viral replication, morphogenesis, and evasion
of the host immune response. Each segment encodes a single protein, except
segment 11 that encodes two proteins (NSP5 and NSP6, in some viral strains)
(Figure 1) [49].
The infective virion consists of three concentric protein layers that surround and
cover its genome. The inner layer or core is constituted by 120 copies of VP2
(scaffolding protein), and anchored to each segment of dsRNA are VP1 (RNAdependent RNA-polymerase) and VP3 (guanylyltransferase and methylase), both
proteins implicated in genome transcription. The middle layer surrounds the core
and it is composed of VP6 organized as pentamers and hexamers, giving rise to
132 channels of three classes that play an important role in the entrance of
compounds to the capsid and the export of newly formed mRNAs [50]. VP6 is the
most abundant structural protein and it is assembled to form double layered
particles (DLPs), which are non-infectious but transcriptionally active. The
outermost layer, part of the entire infectious triple layered particle (TLP), is made
up of the calcium-binding glycoprotein VP7, that forms the smooth surface of the
virion, and the protease-sensitive VP4 spikes, involved in cellular attachment
during infection. [51].
14
Figure 1. Rotavirus genome and structure. a. SDS-PAGE showing rotavirus RNA
segments and gene-protein designations. b. Surface of the mature rotavirus
particle (TLP). Arrows denote the three types of aqueous channels labeled I, II and
III. Three protein layers constitute the viral particle. The external layer is made up
of VP7 (yellow) and VP4 (red), which are neutralization antigens and determine the
G and P serotypes, respectively. c. Cut-away of the TLP structure showing the
internal structural characteristics. The middle layer is made of VP6, which is the
major structural protein (blue). The core surrounds the viral genome and contains
the scaffolding protein VP2 (green), the RNA-dependent RNA polymerase VP1,
and VP3 (red). Figure obtained from [49].
2.1.2 Classification
RV strains have a high genomic and antigenic diversity that can be classified into
three different specificities: group, subgroup and serotype [12]. The VP6 protein,
the major capsid viral protein, confers the group specificity; 7 groups (A-G) have
been established according to antigenic properties and seroepidemiological
studies. RVs belonging to groups A, B, and C are implicated in human and animal
infections, whereas members of groups D-G have been found only in animal hosts.
Group A viruses are certainly the most common in human infections; thus, this
group is considered a relevant target for vaccination. Human group A RVs are
subdivided into three distinct genogroups based on shared features among three
prototype strains named Wa, DS-1, and AU-1. Cross-reactive antigenic epitopes
also present in the VP6 protein confers this subgroup specificity: subgroups I, II,
I/II, and non-I/non-II. G2 and G8 human RVs belong to subgroup I, whereas most
G1, G3, G4, and G9 human RVs belong to subgroup II [52]. At present, this
classification is less used.
The serotype classification is based on the antigenic specificity of the outer capsid
proteins VP7 and VP4. Given that the genes encoding these proteins can
segregate independently, a dual nomenclature system was established in which
the serotypes determined by the VP7 protein (termed G serotypes because VP7 is
a glycoprotein) and the VP4 serotypes (designated P serotypes since VP4 is
protease-sensitive) are considered. There are approximately 27 G and 14 P
serotypes with G1, G2, G3, G4, and G9 comprising more than 90% of all human G
serotypes detected in the world. The P1 serotype accounts for more than 91% of
circulating human RV strains [12].
In addition to serotypes, nucleic acid sequence similarity analyses result in
genotypes. A complete equivalence between G serotypes and genotypes has been
described. In contrast, an absolute association between P serotypes and P
genotypes does not exist. There are at least 35 P genotypes, and the
nomenclature most frequently used denotes them in brackets. Of note, P[8] and
P[4] genotypes correspond to two subtypes (P1A and P1B) of P1 serotype. In
15
summary, the rotavirus classification working group recommended the following
nomenclature: first, the G serotype/genotype, “X” if it is unknown, followed by the P
serotype, and finally the P genotype denoted in brackets [53]. The most common G
and P type associations worldwide related with RV infection in humans are
G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8] and G9P1A[8] [12].
The generation of new antigenic-distinct virus strains depends on point mutations,
gene rearrangements, and reassortments generated when viruses of the same
group co-infect the same cell [54].
2.1.3 Epidemiology
Historically, RV has been the dominant etiological agent of severe GE in children
under 5 years of age worldwide. The United States Food and Drug Administration
approved two live oral RV vaccines, RotarixTM and RotaTeqTM, in 2005 and 2008.
Before the introduction of routine immunization with these vaccines, it was
estimated that RV GE caused 453,000 deaths (CI 95% 420,000 – 494,000) in
children under the age of five, corresponding to 5% of children’s deaths of all
causes and 37% of those attributable to GE yearly worldwide in this age group.
More than 50% of fatal cases attributable to RV occur in only five countries: India
(22%), Nigeria (9%), Pakistan (9%), Democratic Republic of Congo (7%) and
Ethiopia (6%) [1].
In the United States, norovirus has surpassed RV as the main cause of GE in
medical visits after vaccine implementation [55]. Nevertheless, RV is still the most
important etiological agent responsible for severe GE in developing countries that
have not introduced RV vaccines [56]. In Colombia, approximately 1,300 children
younger than 1 year die annually due to acute GE, which accounts for 10% of
deaths in that age group [2]. A study conducted in Bogotá, Cali, and Barranquilla
between December 2003 and November 2004 showed that in children under the
age of 5 years RV infection caused 1 death per 2,000 children; 16 hospitalizations
and 631 medical visits per 1,000 children [57]. Recently, it was estimated that
without vaccination the annual number of medical visits and deaths by RV GE in
children under 2 years would be 105,378 and 470 (CI 95% 295 - 560), respectively
[2].
The pattern of RV transmission depends on the geographic zone considered. In
tropical zones it has a year round pattern, although it seems to be higher in the
cooler months. In temperate zones the rate of infections is higher in autumn and
winter, and in the United States a traditional spatiotemporal dynamic of RV activity
initiating in the southwest in late fall and ending in the northeast 3 months later was
defined. However, it seems that vaccine implementation has changed this pattern,
which will have a probable impact in epidemic dynamics [58, 59].
16
RV strain distribution varies with time, season, and location. As mentioned above,
the most frequent strains worldwide are G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8]
and G9P1A[8], which account for more than 90% of RV disease burden [60]. In
some areas of India, Brazil, and Africa, RV strains G9P[6], G5, and G8,
respectively, are more prevalent than in other parts of the world [61]. However, the
greatest variability is observed in Africa, where additional strains circulate with
unusual G-P compositions and in some cases strains of a probable zoonotic origin
[62].
RV transmission occurs through the fecal-oral route or the respiratory tract, by
person-to-person contact or by contact with contaminated surfaces and objects.
Highly infectious virus is excreted in high quantities in feces of infected people as
early as 2 days before and up to 10 days after the onset of diarrhea. The inoculum
required to infect a homologous host is, indeed, very small: 10 or less from the 1010
RV particles/gram of feces. In contrast, to infect a heterologous host a high
concentration is required; the infection generates small amounts of progeny virus
and is usually asymptomatic. Despite RV infection occurring in other mammals,
transmission of animal RVs to humans is considered to be infrequent, due to hostrange restriction, and likely asymptomatic. Therefore, animals are improbable
reservoirs for human viral strains [49].
2.1.4 Life cycle
RV infection depends on the recognition of specific receptors on the cell surface;
therefore, the receptors define the viral tropism and pathogenesis. In vivo, RV
replicates almost exclusively in mature enterocytes of intestinal villi tips of the small
intestine. In contrast, in vitro RV seems to have a wider tissue tropism: cell lines
from kidney, breast, stomach, bone, and lung, among others, are susceptible to RV
infection [63].
RV strains have different cell-binding requirements. Some strains, usually from
animals, rely on sialic acid for their initial attachment, whereas for most human
strains it is unrequired. Thus, other cell surface proteins have been implicated as
early attachment molecules, including GM1 and GM3 gangliosides, and the integrin
21, which binds to the VP5 fragment of VP4. After recognition of primary
receptors, RVs bind to one or more secondary receptors, such as heat shock
protein HSC70, or through VP7 to the integrins 42, 41, and V1. These
initial recognition steps may occur within specific membrane microdomains
enriched in cholesterol and sphingolipids, called rafts, which seem to be
responsible for the susceptibility of a particular cell to RV infection.
After attachment to the cell surface, RV enters the cell either via direct membrane
penetration or endocytosis. Low Ca++ concentrations in the endosomes promote
the loss of the outer protein layer, and transcriptionally active DLPs are then
17
released into the cytoplasm. This process requires the virus activation by trypsin
cleavage of the VP4 protein into VP5 and VP8 polypeptide domains, which
conduces to a productive infection. Non-trypsin activated RVs are endocytosed
and transported to lysosomes, resulting in an abortive infection. In transcriptionally
active DLPs, the dsRNA is used as template and the VP1 protein produces positive
sense, capped, non-polyadenylated transcripts. These transcripts are used as
templates for translation and for strand synthesis in the replication process to
generate a complete set of eleven dsRNA genome segments. RNA molecules are
ejected from DLPs into the cytoplasm through a series of channels. After
translation, the structural proteins VP1, VP2, VP3, and VP6; the nonstructural
proteins NSP2 and NSP5, and RNA molecules aggregate in viral inclusion bodies
(viroplasms) located in the cytoplasm. In these viroplasms take place replication,
RNA packaging, and DLPs assembly. Viral proteins VP4, VP7, NSP1, NSP3, and
NSP4 are not present in the viroplasm but are necessary for other processes.
NSP4 is attached to the endoplasmic reticulum (ER) membrane and binds VP6 in
DLPs to facilitate its translocation to the intraluminal side of the ER. There, DLPs
are covered with VP7 and VP4 (by an incompletely understood mechanism) to give
rise to TLPs, which will be expulsed from the ER and subsequently from the cell
before or during cell lysis (Figure 2) [64].
Figure 2. Schematic representation of the RV life cycle. During entry into the cell,
the outermost protein layer is lost. Polymerase complexes in the core of the DLP
produce viral mRNAs. Viral proteins and RNAs aggregate in viroplasms, where
particle assembly is carried out. Interaction of newly formed polymerase complexes
with the core capsid protein initiates genome replication, followed by addition of
VP6. DLPs then enter the endoplasmic reticulum via NSP4 and obtain their outer
capsid. After release through lysis or microvesicular trafficking, VP4 must be
18
cleaved by trypsin-like proteases in the intestinal lumen to activate the virus and
start subsequent rounds of infection. Figure obtained from [65].
2.1.5 Clinical manifestations and pathophysiology in humans
After RV uptake, the incubation period lasts around 1 to 3 days prior to the onset of
vomiting and diarrhea, the main symptoms of RV disease. The first clinical
manifestation is usually vomiting, which appears abruptly and causes rapid
dehydration and impedes oral rehydration. Diarrhea appears 1 to 2 days after
vomiting, is usually self-limited, and lasts 4 to 8 days. Other clinical events related
with RV infection include fever (in 30% of cases) and abdominal distress; these
clinical symptoms are generally observed in children rather than in adults. Severe
dehydration and electrolyte alterations are the most serious consequences of RV
infection and occur more frequently in young children. They are responsible for the
majority of fatal cases, which usually occur 1 to 3 days after onset of symptoms in
conditions of poor access to medical attention [64].
Some reports also associate respiratory signs such as cough, pharyngitis, otitis
media, and pneumonia with RV infection, but these findings require further study.
In spite of the frequent RV antigenemia and viremia, extra-intestinal clinical
manifestations are rare but include encephalitis and sterile meningitis, Kawasaki
syndrome, sudden infant death syndrome, hepatic abscesses, exanthema, nonfebrile seizures, and pancreatitis [14]. Under particular conditions, RV infection can
compromise the liver, lung, kidney, spleen, heart, and mesenteric lymph nodes [66,
67]. Recently, it has been reported that in immunocompetent children RV infection
is frequently associated with elevated serum hepatic transaminases [68], pointing
to a clinical significance of the frequently observed antigenemia and viremia [14].
The RV viral capsid structure makes it a very stable virus. This facilitates fecal-oral
transmission and proper delivery into the small intestine, where it infects the nondividing mature enterocytes in the mid and upper part of the villi. Histopathological
disturbances related with RV infection are almost restricted to this site and are
characterized by shortening and atrophy of villi, augmented crypt depth, flattening
of epithelial cells, distended ER, mitochondrial swelling, and increment of
inflammatory cells in the lamina propria [69]. Likewise, postmortem studies on
tissue specimens from children with RV illness showed degenerative changes in
the epithelium, inflammatory infiltrates in the lamina propria, and hyperplasia of
Peyer’s patches. Nevertheless, a direct relationship between histopathological
findings and symptoms of disease is lacking [70].
The physiopathological explanation for RV-induced diarrhea is complex and still
incompletely understood; multiple non-mutually exclusive mechanisms have been
proposed: 1. Virus replication disturbs the metabolic functions of enterocyte
membrane proteins, leading to malabsorptive diarrhea. 2. RV increases
19
intracellular Ca++ concentration, which alters the cytoskeleton and tight junctions
augmenting cell permeability. 3. Virus replication induces NPS4, an enterotoxin
that causes secretory non-cystic fibrosis transmembrane conductance regulator
(CFTR)-mediated diarrhea [69]. NSP4 increases intracellular Ca++ concentration by
activating a Ca++-dependent signaling pathway; this mobilizes Cl- from the
endoplasmic reticulum and consequently induces water and electrolyte loss [49]. 4.
RV stimulates the enteric nervous system (ENS) through NSP4 related serotonin
secretion by enterochromaffin cells, inducing electrolyte secretion and intestinal
fluid loss, in addition to increased intestinal motility [71]. In fact, pharmacological
inhibitors of the ENS reduce RV induced diarrhea [72]. 5. Enterocyte death induces
osmotic diarrhea [69]. Other undiscovered mechanisms of the RV-host interaction
may also contribute to RV pathogenesis.
Finally, the mechanisms of RV induced emesis have not been completely
elucidated. Vomiting may be originated by an axis comprised of NSP4-Ca++ influxserotonin secretion and activation of the solitary tract (considered the center of
vomit in the brain) by the vagus nerve [73].
2.2 IMMUNE RESPONSE AGAINST ROTAVIRUS
2.2.1 Innate immunity
The innate immune response constitutes the first line of host defense during
infection and it plays a central role in the early recognition and subsequent
induction of a proinflammatory response to invading pathogens. Innate immunity to
RV seems to play a very important role: 40% of SCID mice (on a C57BL/6
background), which lack B and T lymphocytes, were able to resolve a primary RV
infection [24]. Likewise, athymic BALB/c mice clear RV infection in the absence of
specific antiviral Abs [74]. Moreover, RV infected children commonly recover during
the first week of illness, before RV- T cells or Abs are entirely established in the
small intestine [75].
The innate immune response relies on the recognition of pathogen-associated
molecular patterns (PAMPs) through a restricted number of pattern recognition
receptors (PRRs). Intestinal epithelial cells (IEC) may recognize RV via PRRs such
as Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like
receptors, and retinoic acid inducible gene 1 (RIG-I)-like receptors (RLRs), which
recognize viral RNA. The engagement of PRRs results in the translocation of NFB and interferon regulatory factors that promote the expression of type I IFN. In
turn, IFN stimulated genes mediate resistance of neighbor cells to viral infection
[76]. RIG-I and melanoma differentiation associated protein-5 (MDA-5) receptors
are also implicated in the recognition of RV replication products, given that the loss
20
of either of them significantly reduces the magnitude of IFN- induction and
facilitates viral replication [77, 78].
RV recognition by PRRs may be cell type-specific. Thus, other membraneassociated or endosomal receptors such as TLR3, TLR7, and TLR9 have been
implicated in triggering innate responses against RV infection [79, 80]. In this
regard, age-dependent TLR3 expression has been inversely associated with
susceptibility to RV infection, viral shedding, and histological damage in mice [80].
In contrast, other studies have shown that TLRs signaling adaptor proteins MyD88
and TRIFF are dispensable for IFN induction in IECs or myeloid dendritic cells
(DC) following infection with murine RV [77, 81]. After MDA-5 engagement by viral
dsRNA, the protein kinase (PKR)-dependent pathway seems important for IFN-
induction. Nevertheless, during RV infection PKR does not appear to be committed
in the early antiviral gene induction [78]. Therefore, the specific role of some PRRs
in the innate response against RV infection is still controversial.
IECs play a crucial role in the host immune response induction by producing
cytokines and chemokines, which result in antigen-specific immunity through B and
T cell responses. Nevertheless, the mechanism of immune modulation exerted by
these cytokines against RV infection is poorly understood. Recently, a study
conducted by our group revealed that RV infected Caco-2 cells released noninflammatory immune response mediators – IL-8, PG-E2, and TGF-1 – but not IL1, IL-6, IL-10, IL-12p70 or TNF- [82]. A subsequent study showed that DC
treated with supernatants from Caco-2 RV infected cells generated a significantly
lower Th1 response, mediated by TGF-1 [83]. These results may explain in part
the poor T-cell immune response observed in blood samples after RV infection.
2.2.2 T cell response
T cells are another important player in the immune response against RV. In mice,
the induction of protective Abs depends on the presence of CD4 T cells [84], and
CD8 T cells provide the first mechanism, although not the only one, to clear a
primary RV infection [24]. In humans, CD4 T cells secrete antiviral cytokines such
as IFN- that are crucial for the response against the pathogen [85, 86]. In fact,
children with an ongoing RV-infection presenting emesis have lower IFN- levels
than those without emetic episodes [87].
The RV-T cell response in humans has been mainly studied through proliferation,
ELISPOT, and intracellular cytokine staining assays, both in children and adults.
RV-lymphoproliferative responses in vitro are variable, but they have been
confirmed to be antigen-specific, with some antigenic epitopes shared by bovine
and human RVs recognized by T cells [88-91]. RV-lymphoproliferative activity
seems to increase with age; in children less than 6 months of age it is almost
21
absent, but it is detected in 80% of individuals older than 5 years [90]. CD4 T cells
seem to predominate over CD8 T cells in proliferation assays, at least 8 days after
the stimulus [89]. Proliferative responses are transitory in children, being almost
undetectable 12 months after an acute RV infection [91, 92]. These results suggest
that children require multiple RV infections to develop a T cell response as the one
observed in adults [92].
In relation to cytokine production profiles against RV, it was shown that RV infected
DC promote a Th1 response [93]. In consequence, circulating RV-T cells in both
healthy and acutely infected adults mostly produce IFN-, but not IL-2, IL-4, IL-13
or IL-17 [85, 94]. In healthy adults, a few CD4 T cells simultaneously produce IFN-
and IL-2, but not solely IL-2 [94]. Considering that the frequencies of RV-T cells
that produce IFN- are similar to those specific for several mucosal respiratory
viruses [94], it seems that the RV-T cell response is not poor in terms of quantity
but may be weak in terms of quality. The described phenotype suggests that the
majority of RV-T cells are terminally differentiated effector cells, not able to provide
long lasting immunity [95]. Likewise, in children RV-CD4 and CD8 T cells that only
produce IFN- can be detected, but at very low frequencies [85, 86, 94].
In acutely infected adults RV-CD4 T cells that produce IL-10 are detectable, but in
the convalescence phase this subset vanishes. This finding, along with the
probably terminally differentiated RV-CD4 T cells and the barely detectable
frequency of RV-T cells in children, suggests that the tolerogenic gut environment
may substantially influence the T cell response against RV. In agreement with this
hypothesis, after depletion of CD25+ T cells and/or the inhibition of the TGF-
signaling pathway, in peripheral blood mononuclear cells (PBMC) from healthy
adults, the frequency of RV-CD4 T cells that produce IFN- increases.
Nevertheless, this mechanism does not seem to play a role in children.
Furthermore, anergic cells could also be part of this population [94]. At present, we
are preparing a manuscript in which the frequencies of RV-T cells producing IL-2,
IFN-, and TNF- are compared to those specific for influenza virus and TT.
Additionally, anergy inhibitors were used to assess the hypothesis of the presence
of anergic T cells in the response against RV.
Notably, regulatory T cells have been involved in mucosal IgA production through
TGF-1 secretion, and in RV infected mice the numbers of FoxP3 + regulatory T
cells are increased. Nevertheless, they do not seem to be essential for the
response against RV, since their absence does not significantly affect RV
clearance or Abs levels. Therefore, FoxP3+ regulatory T cells do not seem to
importantly contribute to the early RV-IgA response in the intestine [96, 97].
Circulating RV-T cells preferentially express the intestinal homing receptor integrin
47 [86]. This and other studies have purified the homing receptors-expressing T
cells before their detection in functional studies. This approach implies the risk to
22
change the T cells’ phenotype due to activation. Therefore, the use of MHC class II
tetramers to characterize CD4 T cells without activating them has appeared as a
new important tool to quantify and characterize the phenotype of antigen-specific T
cells ex vivo. Such an approach has been already used for these purposes in
several settings and viral models [98, 99]. Recently, we identified circulating RV
epitope-specific CD4 T cells in healthy volunteers and vaccinated children, which
expressed intestinal homing receptors (manuscript accepted for publication in
Virology, see appendix 9.6).
2.2.3 B cell response
Bc are probably the most important arm of the immune system in the response
against RV. Bc deficient JHD knockout mice are unable to efficiently clear a
primary RV infection and are susceptible to reinfection. Chronically RV infected
mice are able to clear the virus after passively transferred with B cells that express
intestinal, but not systemic, homing receptors [100]. More recently, it was shown
that RV-Bc are determinant for the removal of the virus from the systemic
circulation [28].
While in the RV mouse model intestinal IgG can protect, it is well known that IgA is
crucial for protection against mucosal pathogens [101]. In agreement with this, it
was recently confirmed in an IgA deficient mouse model that this Ig is very
important for the intestinal primary immune response against RV, and that it has a
fundamental role in the protection against intestinal RV reinfection [23]. In spite of
this, IgA has a minor role in the resolution of systemic primary RV infection and
does not seem to be needed for clearance of antigenemia after reexposure to the
virus. These results suggest that circulating RV- IgG and/or IgM are important in
the clearance of the virus from the systemic compartment [23, 102].
In humans, acute RV infection induces the secretion of RV-IgM in serum, which is
subsequently replaced by RV-IgA and IgG. In the intestine, RV-IgM is also
detected, followed by RV-IgA, and very low levels of IgG [103]. The importance of
RV-specific systemic and intestinal Abs will be described in the next sections in the
context of the correlates of protection after natural infection and vaccination, and in
the mucosal immune system section. The RV-Bc phenotype and characteristics will
also be discussed below.
2.2.4 Correlates of protection after natural infection in children
It is generally accepted that neutralizing Abs against the RV infecting strain present
in the intestine provide protection [18]. Nevertheless, assessment of intestinal fluid
is impractical. In children, after natural RV infection, a serum RV-IgA titer > 1:800
confers a lower risk of infection and completely protects against moderate to
23
severe RV diarrhea. Children with a serum RV-IgG titer > 1:1,600 are protected
against RV infection but not against RV diarrhea. These protective titers were
reached after two successive RV infections either symptomatic or asymptomatic
[11, 25]. In another study, total serum RV-IgA, but not total serum RV-IgG,
correlated with milder disease [104].
It has been proposed that serum RV-IgA correlates with protection because it may
transitorily reflect the intestinal immune response; serum RV-IgA correlates with
serum RV-SIg [104], which in turn, at least one week after an acute RV infection,
correlates with the RV-SIg in duodenal fluid [21]. Notably, serum RV-SIg could not
be detected in serum later than 4 months after infection [104].
There is also evidence to support the importance of serum homotypic and
heterotypic neutralizing Abs in protection against RV reinfection [12]. It seems that
the first RV infections induce homotypic responses in children, but after successive
infections occur, heterotypic Abs against a broader spectrum of G types appear,
even though the real exposure had been to a restricted number of them.
In conclusion, serum neutralizing Abs, and total serum RV-IgA appear to be better
correlates of protection than total serum RV-IgG after natural infection in children
[12].
Fecal RV-IgA has also been explored as a correlate of protection. However, its
measurement is subject to several problems: 1. The intestinal proteolytic
environment poses technical difficulties for its measurement. 2. The interference by
maternal antibodies. 3. Although RV-IgA in stool has been reported to correlate
with protection in studies performed in Australia [105] and the United States [106],
some children that become reinfected have significant levels of RV-IgA in feces.
These reasons explain why stool RV-IgA is an unfit correlate of protection in
humans after natural infection [12].
2.2.5 Rotavirus vaccines
Due to the fact that RV does not induce sterilizing immunity, RV vaccines have
been conceived to prevent severe disease in children, but not to eliminate
infection. In other words, RV vaccines must simulate the effect of two natural RV
infections, which, as mentioned earlier, provide complete protection against
moderate to severe disease of any serotype. Nevertheless, this assumption
diverges from recent results obtained in India, in a region where early infection is
common (56% of children are infected by six months of age) and with high viral
diversity. In that scenario, only after three natural RV infections a 79% protection
against moderate or severe disease was reached [10, 107].
24
In 1998, RotaShieldTM (Wyeth Lederle), a human-simian tetravalent RV vaccine,
was licensed in the United States. But, 9 months after it became commercially
available, it was withdrawn from the market because of a temporal association
between vaccine administration and intestinal intussusception. The incidence of
intussusception was particularly high when the first vaccine dose was administered
in children more than 90 days old [108].
In 2006, two new RV vaccines (RotarixTM [GlaxoSmithKline Biologicals], a
monovalent attenuated human vaccine, and RotaTeqTM [Merk and Co. Inc], a
pentavalent human-bovine reassortant vaccine) were licensed in the United States
after clinical trials with more than 60,000 children (for each vaccine) that assessed
their safety and efficacy in the United States, Europe, and Latin America [109,
110].
RV vaccines were conceived with two different approaches in mind: on the one
hand, Rotarix relies on the assumption that a single RV strain can induce a broadly
cross-reactive neutralizing-Ab response and that a single natural RV infection in
children can avoid a second severe infection [25]. In this case, the 89-12 strain was
isolated from a child with RV GE and attenuated by multiple passages in cell
cultures. This strain was selected because children symptomatically and
asymptomatically infected with similar G1P1A[8] strains were 100% protected
against RV GE in the next season. On the other hand, RotaTeq was formulated
based on the mechanism of host range restriction attenuation and on the premise
that homotypic protection is essential and, in consequence, the most common
circulating human RV serotypes had to be included. Thus, RotaTeq is composed of
five reassortant RV strains made of a parental WC3 strain and genes encoding
VP7 or VP4 from RVs of human origin (G1, G2, G3, G4 and P1A[8]) [13] (Figure
3).
Figure 3. The Rotarix and RotaTeq vaccines. a. Rotarix is an attenuated human
rotavirus vaccine made of a tissue culture-adapted human G1P1A[8] strain. b.
RotaTeq is a bovine (WC3)–human reassortant vaccine composed of five strains:
four of them containing a human RV gene encoding the VP7 neutralizing protein
25
from different serotypes and the last one a human VP4 serotype. Figure obtained
from [13].
Irrespective of their design, the efficacy of the RV vaccines has been similar in the
different scenarios in which they have been tested so far. Therefore, in 2009, the
WHO Strategic Advisory Group of Experts made a global recommendation that RV
vaccines be included in national immunization programs worldwide [5] without any
particular inclination for one of them.
Early adopter countries of the vaccines against RV have shown a significant
decrease in the number of medical visits, hospitalizations, and deaths related to
RV GE. Specifically, studies in eight countries (United States, Australia, Belgium,
Austria, Brazil, Mexico, Panama, and El Salvador) showed a 49% to 80% decline
in hospitalizations of children under the age of five due to RV infection within two
years of vaccine introduction [60]. Of note, hospitalizations for acute GE of any
etiology decreased 17% to 55% after introduction of RV vaccination [60]. This
suggests that the RV disease incidence was underestimated or that RV vaccines
confer non-specific protection against other enteric pathogens. The effect of RV
vaccination on the mortality related to GE of any etiology was particularly assessed
in Mexico and Brazil, where deaths decreased by 35% and 22%, respectively [60].
The majority of countries that have implemented the vaccines against RV are of
middle-to-high-income, and they account for less than 1% of the global deaths from
RV before the introduction of vaccines. Unfortunately, the RV vaccines
effectiveness is directly related to the per capita income of countries; RV
vaccination provided 44% protection against severe RV GE in a low-income
setting, approximately 76% in a low-to-middle-income setting, and greater than
80% in a number of middle-to-high-income settings [60]. These findings are in
agreement with those reported in previous clinical trials.
In Malawi and South Africa, the combined efficacy of Rotarix to prevent severe RV
GE was 61.2% (49.4% in Malawi and 76.9% in South Africa), less than that
observed in Europe (96.4%) and Latin America (84.8%) [111]. The efficacy studies
for RotaTeq performed in Nicaragua (58%) [112], Bangladesh (42.7%), and
Vietnam (63.9%) [113] also concluded that protection against severe RV GE is less
than that reported in industrialized countries. Furthermore, in Africa [114], the
efficacy between the first and second year after vaccination decreased from 64.2%
to 19.6%. This finding also differs from the studies conducted in the United States
and Europe [110, 115].
In conclusion, in spite of the great advance current RV vaccines have meant for the
prevention of GE related mortality in children under the age of five, it is necessary
to develop a new generation of RV vaccines that surpass current limitations
regarding their efficacy in the settings where they are needed the most. To achieve
this goal, research in the following areas need to be further addressed: the
molecular basis of RV virulence and the mechanisms by which it induces GE, the
26
immune response elicited by RV infection, the genetic basis of virulence
attenuation throughout serial cell culture passages and host range restriction, and,
as mentioned earlier, the identification of an appropriate immunological correlate of
protection [13].
2.2.6 Correlates of protection after RV vaccination in children
Two immune markers have been mainly used as correlates of protection after
vaccination in children: RV-IgA, both in stool and serum, and serum RVneutralizing Abs. As mentioned earlier, measurement of stool RV-IgA is subject to
several problems, and, for those reasons, it has been rarely used as a correlate of
protection in vaccine studies. Notably, in the cited RIX4414 trial conducted by our
group, only a minority (32.7%) of vaccinees presented RV-IgA coproconversion,
indicating that this is not an optimal parameter to measure vaccine induced
intestinal antibody responses [17]. Concerning serum RV-neutralizing Abs, in
several studies its utility has been weak, which may be explained in part by the
interference of maternal Abs [116].
Serum RV-IgA has been commonly measured in RV vaccine trials, used as an
indicator of vaccine “take”, and associated with protection in some studies [10].
The rationale for its measurement is the same described above in the correlates of
protection after natural RV infection. Compared with RV-IgG, RV-IgA seems to be
more sensitive to evaluate vaccine immunogenicity [117]. Serum RV-IgA has been
measured in RotaTeq trials and in many cases the RV-IgA seroresponse, a ≥3 fold
rise in RV-IgA units between the preimmune sample and the sample taken after
the last vaccine dose, surpassed the rates of protection against severe RV GE
[12]. Regarding Rotarix, it was concluded that serum RV-IgA responses mirror the
vaccine efficacy and that, in fact, vaccinees negative for RV-IgA are approximately
10 times more prone to suffer a RV GE in the next season than responders [118].
Recently, at the Tenth Rotavirus International Symposium the question if RV-IgA is
really a suitable correlate of protection after vaccination was addressed. A study
conducted in Africa by Dr. Htay Htay Han in vaccinated infants with Rotarix showed
that a RV-IgA titer ≥ 20 U/mL correlated with protection against RV GE and that it
was associated with a lower percentage of individuals with any or severe RV GE.
However, a direct relationship between serum RV-IgA titers and the level of
protection in seropositive children was lacking. Furthermore, vaccinated children
with serum RV-IgA < 20 U/mL showed some level of protection compared to
placebo recipients. Considering this study and the results from a meta-analysis of
eight Rotarix efficacy studies in Europe, Asia, and South America (but not Africa),
Dr. Han concluded that serum RV-IgA can be used as an epidemiological tool at
the population level, but is unsuitable to predict individual protection [119, 120].
27
Dr. Patel and his team showed an inverse relationship between RV-IgA titers and
RV associated disease mortality. Moreover, RV-IgA correlated with vaccine
efficacy for both vaccines; titers ≥ 90 U/mL correlated with higher protection and
less waning of it than did titers < 90 U/mL. Countries with titers above 90 U/mL
exhibit a two-year efficacy of 85% whereas those with titers below 90 U/mL display
only a 44% [119, 121].
In conclusion, and as stated in the introduction, serum RV-IgA is currently the best,
though imperfect, practically measured correlate of protection against RV GE [12,
13].
2.3 THE MUCOSAL IMMUNE SYSTEM
2.3.1 Induction of the mucosal immune response mediated by B cells in the
intestine
In humans, antigen-specific mucosal immune responses initiate in the so-called
inductive sites of the gut-associated lymphoid tissue (GALT), specifically Peyer’s
patches (the principal site for the induction of antigen-specific responses) and
isolated lymphoid follicles. These inductive sites are similar to lymph nodes in that
they possess Bc follicles, T cell zones, and antigen-presenting cells such as
macrophages and DC, but differ in that they lack afferent lymphatics [122].
Consequently, external stimuli must directly come from mucosal surfaces through
the follicle-associated epithelium, which contains specialized epithelial M cells.
These cells transport foreign macromolecules and microorganisms, via a vesicular
transport system, to antigen-presenting cells within and under the epithelial barrier
[123]. Another mechanism for intestinal antigen sampling consists of DC with the
ability to sense the intestinal lumen by penetrating the epithelium with their
processes. Induction of mucosal immunity can also take place in the gut-draining
mesenteric lymph nodes, and, to some extent, at the effector sites (the lamina
propria), home to activated B and T cells [124].
Payer’s patches also differ from lymph nodes in that the proportion of B to T cells is
four to six times higher and are rich in cytokines important for IgA induction, such
as TGF-, IL-4, IL-6, and IL-10. These cytokines are crucial for IgA class-switch
recombination (CSR) of antigen-specific Bc in the germinal centers (GC), for
expansion of IgA+ Bc, and for their differentiation to IgA secreting PC [125].
Once DC are loaded with the antigen, they migrate from the epithelial and
subepithelial areas to the T cell-rich interfollicular regions of Peyer’s patches and
initiate a polarized Th2 response, that in turn releases cytokines with Bc-activating
properties. However, to exert this role, DC must be conditioned by epithelial cells
via TGF- and thymic stromal lymphopoietin (TSLP), which stimulate DC to
28
produce IL-10, an IgA-inducing cytokine that additionally blocks the generation of a
Th1 response by impeding DC’s production of IL-12 [126]. DC present in Peyer’s
patches also secret retinoic acid (that promotes IgA CSR), IL-6 (that helps in the
differentiation towards IgA secreting PC), and inducible nitric-oxide synthase
(iNOS) (that promotes IgA CSR) to induce IgA responses. Additionally, retinoic
acid is essential for the expression of 47 and CCR9 on IgA class-switched Bc,
allowing them to recirculate through the thoracic duct and home to the gut lamina
propria, their final destination as effector cells [127].
In the lamina propria, IgA+ plasmablasts terminally differentiate into IgA secreting
PC. These PC also synthetize the joining (J) chain allowing the formation of IgA
dimers. Particularly in humans, CD4 T cells provoke IgA1 class switching by
activating Peyer’s patch IgM+IgD+ Bc via CD40L and TGF-. The resulting IgA1+ Bc
migrate to the lamina propria and switch to IgA2 in response to a proliferationinducing ligand (APRIL) and IL-10 released by TLR-activated epithelial cells,
especially in the distal intestine. Another IgM+IgD+ Bc may directly switch from IgM
to IgA1 or IgA2 in response to B cell-activating factor of the TNF family (BAFF) or
APRIL and IL-10 [127].
As previously mentioned, induction of an IgA response can also take place in the
lamina propria, where residing DC also present antigens to Bc and may activate
them via BAFF and APRIL. These mediators deliver CD40-independent IgA CSRinducing signals through transmembrane activator and calcium modulating
cyclophilin-ligand interactor (TACI). Recent evidence showed that this could be the
pathway for intestinal IgA production in a T cell-independent manner [128].
It has also been reported that human transitional T2 Bc have a high expression of
the intestinal homing receptor 47 and indeed migrate towards the GALT, where
they seem to be activated by the microbiota and could give rise to IgA secreting PC
or marginal zone Bc with a prediversified repertoire. Another possibility is that
activated immature Bc in the GALT die, thus constituting a checkpoint against
autoimmunity [129].
2.3.2 The polymeric immunoglobulin receptor
Perhaps the most important factor in the mucosal immune system is the polymeric
immunoglobulins (pIgs), particularly polymeric IgA (pIgA). At least 80% of the Ab
production of the body takes place locally in the gut lamina propria. To be able to
exert its protective effect pIgs must be extruded to the intestinal lumen. In fact, in
an adult, it has been estimated that 3 g of dimeric IgA are translocated to the gut
lumen everyday as SIgA, which is more than the total daily production of IgG in the
body. Transcytosis is the process by which pIgs cross the epithelial cells from the
basolateral membrane to the apical surface, and it is mediated by the pIgR [130].
29
Indeed, this is the only identified receptor accounting for the epithelial export of IgA
and IgM in mice deficient in it [131].
The pIgR is a transmembrane glycoprotein of approximately 100 kDa encoded by a
single copy gene localized in chromosome 1, which is constitutively expressed by
secretory epithelial cells in its basolateral membrane, mainly in the intestinal crypts.
The upregulation of pIgR expression involves various proinflammatory cytokines,
such as IFN-, IL-1, and TNF. The IL-4 has also been reported to synergistically
increase the expression of pIgR in conjunction with IFN- [130]. Likely, PAMPs by
means of TLRs can upregulate pIgR [132]. In mice, it was recently shown that
microbiota-specific Th17 cells regulate intestinal pIgR expression and IgA secretion
through IL-17, contributing to intestinal homeostasis [133].
After pIgR has recognized either dimeric IgA or pentameric IgM through the J chain
(required for the high-affinity epithelial binding of pIgs), a clathrin-coated pit
internalizes the complex. Notably, unoccupied pIgR is also continuously
internalized. The molecules are then delivered to basolateral early endosomes and
subsequently to common endosomes, where the complex is separated from other
internalized molecules. The molecules are transported to the apical recycling
endosomes underneath the apical membrane [134]. When the complex arrives at
the apical surface, pIgR-IgA and pIgR-IgM are exocytosed after cleavage of the
receptor, leaving behind the C-terminal segment for intracellular degradation. The
extracellular portion of the pIgR is a piece of approximately 80 kDa particularly rich
in carbohydrates, which is incorporated into the SIg molecules as bound SC in a
modified conformational shape covering most of the J chain (Figure 4). SC endows
particularly SIgA with resistance against proteolytic degradation [135].
Figure 4. Polymeric immunoglobulin receptor (pIgR) mediates the epithelial export
of pIgA (mainly dimers) and pentameric IgM (not shown) to provide secretory
30
antibodies (SIgA and SIgM). SC is covalently attached to pIgA but not to pIgM.
BBE, basolateral early endosome; CE, common endosome; ARE, apical recycling
endosome. Left and right part of the figure obtained from [135, 136], respectively.
Although human pentameric IgM has a higher affinity for free SC in vitro than does
pIgA, the export of pIgA is privileged over that of pentameric IgM by a factor of
approximately 5. This is probably explained by a diffusion restriction of the large
IgM pentamers across the stromal matrix and basement membranes. Remarkably,
it remains unknown how pIgs are directed from its local production site towards
their diffusion through the stromal ground substance to reach the pIgR, instead of
being drained by lymph to the peripheral circulation [135].
As previously stated, unbound pIgR is also internalized and released by proteolytic
cleavage, originating free SC. Actually, around 50% of the exported pIgR ends up
as free SC [137], which has innate immune functions such as neutralization of
bacterial toxins and inhibition of epithelial adhesion of some Gram-negative
bacteria.
2.3.3 Secretory IgA
SIgA is the most abundant class of antibodies found in the human intestine and it is
considered as a first line of defense in protecting the intestinal epithelium from
enteric pathogens and toxins. Accurate differentiation between innocuous and
harmful antigens is critical to guarantee local homeostasis in the gastrointestinal
system.
SIgA
must,
therefore,
induce
the
dual
properties
of
neutralization/protection and regulatory functions [136].
With regard to its protective immune function, SIgA combats microbial infections
via mechanisms different from those used by antibodies in the systemic
compartment because it exerts its functions mainly in an external environment.
SIgA has the ability to block the adherence of pathogens and toxins to the
intestinal epithelium [138]. One of the mechanisms that explain this blockade is
steric hindrance, proposed as a way SIgA that interferes with cholera toxin binding
to epithelial cells [139]. Another method consists in the direct attachment to
receptor-binding domains, as has been shown in the reovirus type I Lang peroral
challenge model in mice [138].
Probably one of the most recognized mechanisms of protection of SIgA is immune
exclusion. It refers to its ability to prevent pathogens and toxins to gain access to
the intestinal epithelium through a series of events: 1. Agglutination: Ab-mediated
cross-linking via polyvalent surface antigens forms macroscopic clumps of bacteria
(or viruses) that, depending on the epitope recognized, may have effects on
bacterial physiology and gene expression, having in some cases direct effects on
the pathogen virulence. 2. Mucus entrapment: SIgA facilitates the entrapment of
bacterial pathogens in the mucus layer overlying the intestinal epithelium, probably
31
taking advantage of the association of oligosaccharide side chains of SC with
mucus. 3. Agglutination and entrapment in mucus promote the clearance of
bacteria through peristalsis. It is important to notice that it remains to be
determined to what extent immune exclusion contributes to protective immunity
against viruses [20]. SIgA protection against these and other pathogens may be
mediated by two additional mechanisms: SIgA is capable of neutralizing pathogens
intracellularly in its way to the apical surface and also can bind antigens in the
lamina propria and promote their excretion, a process termed expulsion [20].
SIgA can also be considered part of the innate immune system. It has been
proposed that given the high degree of similarity between the oligosaccharide side
chains present on the heavy chain and SC of SIgA and those on the luminal side of
the intestinal epithelium, SIgA can serve as a competitive inhibitor of pathogen
attachment to the host cell. As suggested earlier, free SC may act as a decoy
receptor for certain pathogens [140].
With respect to the regulatory role of SIgA, it is important as early as in the
neonatal period. Natural and specific SIgA in breast milk bind commensal bacteria
and are probably involved in the gradual and controlled establishment of the
newborn’s microbiota, which in turn stimulates maturation of the GALT [141]. The
control of commensal microorganisms is another important feature of SIgA. SIgAcommensal bacteria complexes promote their uptake by Peyer’s patch M cells,
which seem to direct the complex towards local DC, that in turn induce local
specific immune responses, preventing a systemic dissemination [142]. SIgA also
has the ability to actively quench the capacity of certain antigens to evoke severe
proinflammatory responses after uptake through Peyer’s patches M cells.
Therefore, the retro-transport of antigen-SIgA complexes seems to be important to
downregulate proinflammatory cytokines and preserve the integrity of the intestinal
barrier. This mechanism also appears to be relevant for the maintenance of
tolerance toward harmless proteins, including allergens [20]. Indeed, it was
recently shown that both human and mouse SIgA induce tolerogenic DC, and that
these DC in turn generate the expansion of regulatory T cells through IL-10
production. Furthermore, SIgA primed DC are capable of inhibiting autoimmune
responses in mouse models of type 1 diabetes and multiple sclerosis [143].
Notably, a considerable proportion of IgA deficient patients are not only prone to
develop mucosal infections, but also intestinal inflammation, allergic, autoimmune,
and mucosal Bc lymphoproliferative disorders [144].
2.3.4 RV secretory IgA (RV-SIgA) in serum
SIgA and SIgM are normally detected in the systemic compartment of healthy
individuals (1 – 91 years old), with a median of 10 mg/L and 14 mg/L, respectively
[145-147]. The mechanism by which SIgA, produced at mucosal surfaces, is
32
transported to the circulation has not been elucidated. This “spillover” could be
explained by leakage of SIgA or by active transport through the epithelial layer by a
specific receptor still to be identified, since no known IgA receptor (pIgR, Fc
receptor, FcRI or asialoglycoprotein receptor) seem to be involved in the process
of uptake of SIgA by Peyer’s patches [148]. The transferrin receptor (CD71) has
been proposed as an IgA receptor [149]. CD71 is abnormally expressed at the
apical pole of enterocytes in patients with active celiac disease and evidence that
SIgA mediates protected transport of pathogenic gliadin peptides through their
binding to CD71 has been obtained [150]. Whether this is the case for pathogenspecific SIgA detected in serum remains to be determined.
The first report of an ELISA assay to detect serum RV-SIg (RV-SIgA and/or RVSIgM) was published thirty years ago and showed that RV-SIg correlated very well
with recent RV infection [19]. Subsequently, using a similar approach, RV-SIg was
not detected in the serum of healthy breast-fed children, even though it was
present in the stool and duodenal fluid of some of them and in their mothers’ milk
and serum [151, 152]. In contrast, RV-SIg increased in serum of children with
naturally acquired RV GE, and about one week after the acute phase of infection it
correlated with the amounts detected in duodenal fluid, supporting the assumption
that the RV-SIg in serum was derived from the local immune system in the
intestine. Serum RV-IgA followed the same pattern, but, contrary to RV-SIg,
remained elevated for at least six months [21]. A more detailed analysis of the RVSIg kinetics in serum showed that it peaks around 10 days (7 – 14 days) after the
onset of GE and approximately a month later becomes undetectable. These results
suggest that serum RV-SIg is frequently observed after RV infection and reflects
intestinal Ig. However, a correlation between the amount of RV-SIg and the
severity of disease was not found [22].
Finally, regarding the mechanism of protection exerted by RV-SIg, in mice anti-VP6
SIgA plays this role via intracellular neutralization, but not via immune exclusion
[153]. In contrast, in humans a critical mechanism of protection is viral exclusion,
since neutralizing Abs against VP4 and VP7 inhibit the infection and function as a
barrier in the apical side of enterocytes. The importance of viral expulsion remains
to be determined [12].
2.4 B CELLS AND THE MAINTENANCE OF SEROLOGICAL MEMORY
2.4.1 Phenotype of circulating B cells and current controversies
Bc in blood comprise three types: transitional Bc, mature naïve Bc, and mBc.
Transitional Bc (approximately 14% of cord blood Bc and 2% of peripheral Bc) are
early emigrants from the bone marrow that will complete their maturation process
in the spleen and/or outside it, and are usually identified as CD19+CD24hiCD38hi
33
[154]. Naïve Bc are defined as cells that have not encountered their cognate
antigen and, therefore, are inactivated and do not secrete antibodies. In contrast,
mBc are antigen-experienced resting cells that can differentiate rapidly into effector
cells after a subsequent encounter with its cognate antigen [155]. In terms of
function, mBc express higher levels of co-stimulatory and activation molecules
such as CD80, CD86, CD95, CD180, and TACI than naïve Bc [156, 157].
Moreover, proliferation and differentiation rates of naïve Bc and mBc in vitro
resemble those of the primary and secondary immune responses in vivo,
respectively, with a higher proportion of mBc that proliferate and differentiate into
PC compared with naïve Bc [158].
The phenotype of the main circulating Bc populations in humans can be described
by the differential expression of three markers: CD27, IgD, and IgM [159]: 1. Naïve
Bc: CD27-IgD+IgM+, represent 60%-70% of the total Bc [160]. 2. IgM+ mBc:
CD27+IgD+IgM+, constitute approximately 15% of circulating Bc [161]. 3. IgM+ only
mBc: CD27+IgD-IgM+, represents around 1%-5% of circulating Bc [161, 162]. 4.
IgD+ only mBc: CD27+IgD+IgM-, constitute less than 1% of Bc [163]. 5. Classswitched mBc: CD27+IgD-IgM-, which can be IgG+, IgA+ or IgE+, and comprise
around 20% of Bc [160]. 6. CD27- mBc: CD27-IgD-, which can express IgM, IgA or
IgG, and represent less than 5% of total Bc [164].
The rationale for the use of these markers comes from the various strategies that
have been used to define mBc. It has been commonly accepted that after antigen
encounter, Bc can further improve antigen-binding capacity by somatic
hypermutation, a process that introduces point mutations in the variable regions of
immunoglobulin loci; the selection of high-affinity mutants results in the affinity
maturation of the Bc response [165]. In addition, the process of CSR permits
adapting the antibody effector functions to the particular antigenic challenge, while
conserving the specificity of the variable region, by changing the constant region
from  and  to , , or . Both processes are accomplished by activation-induced
cytidine deaminase (AID) [166] and the traditional concept was that they occurred
in a sequential manner and exclusively in GCs, where the AID was activated [167].
Therefore, it was initially thought that the absence of IgM and IgD, accompanied by
a considerable mutation load, was indicative of the memory status. However, the
detection of CD27+IgM+ Bc capable of IgM secretion after polyclonal stimulation
[168], and the finding of a significant population of IgM+IgD+ cells harboring
mutations in their Ig genes [169, 170] were at odds with this conception.
Consequently, CD27 was proposed as a universal marker of Bc memory given that
its expression correlated with the capability of Bc to secrete immunoglobulins after
polyclonal stimulation [171, 172], and with somatic hypermutation in IgM+IgD+ cells
[162]. These studies allowed the identification of IgM+ mBc (CD27+IgD+IgM+) and
IgM+ only mBc (CD27+IgD-IgM+). Besides the presence of somatic hypermutations,
other evidences support the notion that these cells are indeed mBc: 1. They are
scarce in children but increase with age [171]. 2. They are long-lived [173]. 3.
34
Similar to switched mBc, they respond to certain polyclonal stimuli generating PC
[171, 174, 175]. 4. They respond in vitro to stimuli conventionally derived from T
cells in a way that resembles switched mBc and is different from the naïve Bc
response [158].
New concepts and controversies have emerged along with the description of IgM +
mBc [176]. Their existence suggests that somatic hypermutation and CSR can
occur independently in vivo, for which there is already some evidence in vitro [177].
Since it is thought that somatic hypermutation precedes CSR, it was proposed that
IgM+ mBc are generated in GCs, but may be early emigrants (before CSR) from a
T cell-dependent response. However, various findings suggest that IgM+ mBc cells
are generated outside GCs: 1. These cells can be found in patients with X-linked
hyper IgM syndrome (Hyper IgM type 1), who do not express a functional CD40L
and therefore lack GCs, whereas IgM+ only and switched mBc are absent [178]. 2.
AID expression has been identified in extrafollicular Bc, implying that the
engagement in a GC reaction is not an absolute requisite for the induction of
somatic hypermutations [179]. Nevertheless, mutations in the BCL6 gene,
considered a genetic footprint of germinal center derivation, identified in IgM + mBc
and IgM+ only mBc argue against a T cell-independent origin and support the
hypothesis that these cells are indeed early emigrants from a GC reaction,
produced by a conventional T cell-dependent response [161].
In mice, IgM+ mBc that belong to the B2 cell lineage seem to be part of two
different subpopulations, defined by the differential expression of IgM and IgD. The
first differentiates at the spleen periarteriolar lymphoid sheath area, expresses the
markers IgMhiIgDlowCD21hiCD23−CD1dhi, and are called marginal zone Bc. The
second expresses IgMlowIgDhiCD21intCD23+CD1dlow and are follicular Bc [180].
Furthermore, in humans it has been proposed that IgM+ mBc are the equivalent of
mice marginal zone Bc, although with two important differences: in humans they
recirculate and carry mutated immunoglobulin receptors, which suggests that these
cells have a pre-diversified immunoglobulin repertoire, generated in the absence of
an immune response against a particular antigen [181, 182].
IgM+ mBc also seem very important in T cell-independent responses, for instance
in the protection against encapsulated bacteria like Streptococcus pneumoniae
[183]. Thus, it is not surprising that Toll-like receptors (TLRs) take part in the
ontogeny and homeostasis of these cells. It was recently shown that TLR9
activation generates somatically mutated mBc from a proportion of transitional Bc
in vitro, and the mutation characteristics found in them resemble those of the same
circulating mBc from Hyper IgM type 1 patients in vivo [184]. Furthermore, in
patients deficient in MyD88 and IRAK4, the IgM+ mBc were significantly reduced, in
the presence of normal switched mBc, and did not increase with age, which
suggests that TLRs play an important role in the homeostasis of IgM+ mBc and not
only in its ontogeny [185].
35
A fraction of IgM+ mBc identified as CD20+CD27+CD43+CD70- has also been
considered as the equivalent of B1 cells in humans because they accomplish three
B1 cell fundamental functions described in mice: 1. Induction of allogeneic T cell
proliferation. 2. Tonic intracellular signaling and 3. Spontaneous IgM secretion
[186]. Nevertheless, some groups claim that these cells are really activated Bc on
their way to PC differentiation [187-190]. Recent evidence supports the notion that
the phenotype of “human B1 cells” actually corresponds to pre-plasmablasts [191].
To add even more debate to the question of what defines a mBc, class-switched
and IgM+ CD27- mBc have been described [164, 192]. Therefore, it is not easy to
clearly establish the origin and function of mBc just by their phenotype. The
analysis of mutations present in the V(D)J exons of the immunoglobulin heavy and
light chains, the study of the repertoire selection based on the V and J gene family
usage, the analysis of clonal relationships, and the quantification of the number of
cell cycles (replication history) have shed some light, but the panorama is still
unclear.
Based on these approaches, it has been recently proposed that CD27-IgG+ and
IgM+ only mBc come from primary GC reactions, and switched mBc (IgA+/IgG+)
arise from subsequent GC reactions. In contrast, IgM+ mBc (CD27+IgD+IgM+,
termed “natural effector cells”) and CD27-IgA+ mBc, present in CD40L-deficient
patients, seem to have a GC-independent origin, but with different tissue origins. At
least in part, IgM+ mBc derive from the splenic marginal zone, whereas CD27 -IgA+
molecular characteristics mirror those of gut lamina propria derived IgA + Bc [193].
Concerning the relationship between circulating CD27- and CD27+ mBc, a recent
study showed that mBc expressing IgM (either IgM+ only, “innate-like”
CD27+IgD+IgM+, or CD27-IgD-IgM+) share unique characteristics, switched CD27+
and CD27- mBc use a similar IGHV repertoire and are distant from IgM + mBc,
although IgG2 and IgA2 mBc have a more “innate-like” repertoire. Moreover, the
expression of CD27 does not seem to be determined by a linear developmental
pathway [194]. A summary of the conclusions of these and other studies using
genetic approaches can be found in Table 1. In conclusion, multiple IgM+ mBc
populations may exist in humans, each of them performing different functions
related with diverse origins and in response to particular antigens.
The road for further research on human mBc may have been opened by recent
studies concerning different pathways of mBc generation in the mouse model. One
of them showed that GC- dependent and independent mBc might proceed from a
single CD38+GL7+ Bc precursor [195]. This multipotent cell produces GCindependent mBc at a very early time point in the primary immune response.
These mBc are predominantly IgM+ and CD73-. GC-dependent mBc are generated
later and are mainly switched mBc that, in contrast to GC-independent mBc,
express CD73 [195]. In agreement with these findings, it was recently shown that
CD73 induces CSR in an autonomous manner through the hydrolysis of ATP to
adenosine [196]. A contemporary study utilizing a conditional ablation of BCL6 also
36
added convincing evidence that GC- dependent and independent mBc indeed
coexist. Furthermore, GC-independent mBc are unmutated, long lived, IgM+ or
IgG+, and, surprisingly, generated in a T-cell dependent manner in the absence of
follicular T helper cells [197]. Furthermore, both mutated and unmutated mBc can
optimally adapt to a secondary antigenic challenge throughout the accumulation of
mutations and the development of a new Ab repertoire [198].
2.4.2 RV-specific B cells and how they are studied
There are few studies concerning antigen-specific Bc that use flow cytometry due
to a paucity of model antigens that permit conducting them [199]. Our group and
others developed a flow cytometry assay to characterize the phenotype of Bc that
express surface RV-specific Ig (RV-sIg), based on an assay previously validated in
mice [200, 201], in which a fluorescent RV antigen is specifically recognized by
RV-sIg expressed on Bc. This antigen consists of recombinant virus-like particles
(VLP) made up of VP6 and a fusion protein comprised of green fluorescent protein
(GFP) coupled to the N-terminal end of VP2 devoid of its first 92 amino acids [202].
This construct was chosen as a RV model antigen given that an important
proportion of cross-reactive RV-Abs recognize VP2 and VP6 and because VP6, in
addition to being the most abundant structural RV protein, is recognized by the
majority of Abs against RV in infected animals and humans [201, 203].
As mentioned earlier, RV-Abs are of critical importance to prevent reinfection in
mice [23, 24], and correlate with protection in humans [11, 25-27]. This implies that
Bc are very important for the immune response against RV infection; Bc are
determinant for the clearance of a primary RV infection in mice [24] and for the
removal of the virus from the systemic circulation [28].
One particularity of RV-Bc is that there is a fairly high frequency (around 1-2%) of
RV-naïve Bc in newborns [41], which secrete low-affinity Abs against VP6 [42].
Curiously, children 2 months old have a high frequency of CD27+IgD+IgM+ RV-mBc
regardless of the serum RV-IgA status, indicative of a previous RV infection [17]. It
is still unclear whether these cells have been induced by symptomatic or
asymptomatic RV infections and why they can be present in the absence of serum
RV-IgA.
RV infects almost every child from 2-6 months (when transplacentally transferred
maternal IgG start waning) and 3 years of age, 50% of whom will have at least one
symptomatic infection [7]. After an acute RV infection, RV-sIg Bc and RV-PC
predominantly express the intestinal homing receptor 47 [204] and in
consequence have the predicted phenotype of Bc stimulated in the Peyer’s
patches [205]. The phenotype of RV-Bc changes from the acute to the
convalescent phase of infection. Plasmablasts and PC characterize the primary
RV-Bc response accompanied by the expression of intestinal homing receptors in
37
the great majority of them: CD38hiCD27hiCD138+/-CCR6-47+CCR9+CCR10+CLAL-selectinint/- and IgM+, IgG-, IgA+/-. In the convalescence phase, between one and
two weeks after infection, the RV-Bc phenotype changes and corresponds to RVmBc with or without an intestinal homing migration pattern: CD38 int/-CD27int/CCR6+47+/-CCR9+/-CCR10-. Concurrently, RV-IgA and RV-IgM appear in serum
[206] and stool [105]. One third of RV-mBc are most likely targeted to the small
intestine because they express both 47 and CCR9, another third may be
directed to other mucosal surfaces given that only express 47, and the last third
is probably targeted to the systemic compartment [16]. This compartmentalized
RV-mBc response in humans reflects the fact that, although RV replication in both
animals and humans overall takes place in the intestinal tract, antigenemia and
viremia are common features of RV infection [14, 15].
In an attempt to identify useful correlates of protection, circulating RV-mBc were
characterized in healthy adults and its relation with serological memory was
assessed as well [43]. Compared to TT-mBc, used as a model antigen, RV-Bc
seem to be a distinctive subset, because they are enriched in the CD27+IgM+
(which includes the CD27+IgD+IgM+ subset) and in the CD27-IgG+ mBc subsets
[43, 44]. Recently, this was confirmed and it was determined that RV-IgM+ mBc are
significantly enriched in the IgMhiIgDlow subset. Moreover, human CD27+IgM+ RVmBc have a substantial ability to switch to IgG in vitro and in vivo, and to mediate a
significant reduction of RV antigenemia and viremia in an adoptive transfer
immunodeficient mice model [102]. Regarding the relationship between RV-mBc
and serological memory, CD27+IgA+ RV-mBc correlated positively with RV-IgA
plasma levels, but CD27+IgG+ RV-mBc did not correlate with RV-IgG in plasma. In
contrast, CD27+IgG+ TT-mBc correlated with TT-IgG plasma levels. Therefore, the
relation of RV-mBc with serological memory appears to be different from that of
TT-mBc [43].
The Ig gene expression patterns in circulating RV-Bc have been analyzed as well.
One of the first reports concluded that infant and adult human RV-Bc have
common immunodominant variable gene repertoires since they share similar VH,
D, JH, VL, and JL segment usage, degree of junctional diversity, and length of H
chain CDR3 region [207]. This suggests that the Bc repertoire is not responsible for
the poor quality of RV-Ab responses in children. Nevertheless, RV-Bc VH
sequences from children with acute RV GE have a lower mutation load than the
corresponding sequences in adults [208]. Furthermore, the mutations detected in
the VH1-46 gene segment in adults, which is an immunodominant gene in RV-Bc
of both children and adults, conferred functional advantages to VP6-specific Abs
[42].
The Ig gene repertoire of VP6-Bc has been evaluated in circulating naïve Bc, IgM+
mBc, and switched mBc (including IgM+ only mBc given that this population was
defined as CD27+IgD-) from healthy adults [44]. IgM+ RV-mBc had a shorter CD3
38
length compared to RV-naïve Bc and switched RV-mBc, probably explained by an
increased TdT exonuclease activity. This study also reported that IgM+ and
switched mBc share a predominant usage of the VH3 family, whereas the VH1
family is the most frequently used by naïve RV-Bc. In contrast, the same study
reported that both naïve and the two subpopulations of mBc in total non-antigenspecific Bc predominantly use the VH3 family. Altogether, these results suggest
that the dominant VH1-expressing naïve RV-Bc do not have an advantage in the
selection processes that lead to IgM+ and switched RV-mBc [44].
Considering that RV-Bc are concentrated in the intestine, the genes used by these
cells in healthy adults have been recently assessed in PC that bound RV-VLP and
were isolated from the small bowel. The VH genes analyzed were highly mutated
and preferentially expressed the VH4 family, which is not dominant among
circulating RV-Bc [209]. Nevertheless, it was previously shown that both RV-IgDsystemic Bc and RV-IgD-47+ intestinal homing Bc preferentially use the VH1-46
gene segment, and that circulating RV-Bc 47+ were unmutated, as opposed to
systemic RV-Bc [210]. In agreement with this, around 30% of IgA and IgG
plasmablasts derived from the terminal ileum of healthy adults have been shown to
secrete polyreactive Abs, and the majority of them recognize RV VLPs [211].
These apparent contradictions may reflect the lack of a direct comparison between
circulating and intestinal derived RV-Bc.
2.4.3 The serological memory and the theories of how it is maintained
Pathogen-specific protective IgG levels following natural infection or vaccination
can persist for decades, or in some cases for a lifetime, in the apparent absence of
the antigen [30]. This serological memory provides the host with a first line of
defense against reinfection by many microorganisms [212], and critical pathogenspecific Ab titers that correlate with protection have been identified for several
vaccines [213]. Additionally, in autoimmune diseases, auto-Abs of different
isotypes have been associated with disease activity and pathogenesis [214] and in
some cases predict disease severity [215-217]. The mechanisms that contribute to
the maintenance of serological memory are still unclear and three main theories
have been proposed, one dependent on mBc and the other two unrelated to them
[218].
The first theory considers that serum Abs are secreted by short-lived PC, which are
continuously replenished by mBc that become activated (proliferate and
differentiate into Ab-secreting descendant cells) by non-antigen-specific polyclonal
stimulation through TLR or bystander T-cells [35]. Thus, numbers of circulating
antigen-specific mBc and levels of antigen-specific serological memory in healthy
individuals should correlate [36]. However, this is not always the case. At present,
it is considered that there is substantial evidence, at least in healthy volunteers, to
discard this theory as the main mechanism to explain the maintenance of
39
serological memory [34, 218]. Furthermore, the existence of correlations between
mBc and serological memory are thought to be an epiphenomenon in which both
parts are equally stable but independently maintained, without implying a direct
cause-and-effect relationship [30]. Nevertheless, in determined circumstances
such as autoimmunity, given the existence of conditions that facilitate the rapid
production of short-lived PC, this mechanism could have an important role [219221].
Therefore, in healthy adults, IgG serological memory seems to be maintained by
long-lived PC, independently of mBc [30, 33]. Two non-mutually exclusive theories
have been proposed to explain the survival of long-lived PC [218]: 1. Long-lived PC
reside in a limited number of survival niches in the bone marrow or secondary
lymphoid organs, and consequently do not require repopulation by mBc; however,
there may be a competition for such niches between newly generated
plasmablasts and older PC. Notably, this theory does not exclude the existence of
short-lived PC, instead, it proposes that plasmablasts may or may not gain the
competence to respond to survival signals, which will finally determine its lifespan
[33, 222]. 2. PC and mBc represent independently regulated populations [34]. The
lifespan of PC is related to the integrated signals through the B-cell receptor, which
largely depend on the antigen’s repetitive nature, and signals obtained through
CD4 T-cell help and, therefore, is imprinted at the time of the immune response
induction [218].
2.4.4 Rituximab and its effect on memory B cells and serological memory
Autoimmune diseases are clinical syndromes caused by the activation of T or B
cells, or both, in the absence of an ongoing infection or other discernible cause.
They have a multifactorial origin and can cause organ-specific or systemic injury
[223]. Auto-Abs are a typical feature in the majority of autoimmune diseases that
can be used as autoimmunity diagnostic markers, which may or may not be
implicated in disease pathogenesis. However, auto-Abs are only one of the many
factors involved; it is considered that complex mechanisms including cells and
molecules from both the innate and adaptive immune response play an important
role. Thus, autoimmunity may be just the result from an over-activated immune
system [224].
Recent evidence has shown that Bc have a central role in autoimmunity mediated
by Ab-dependent or independent mechanisms. These include: antigen
presentation, induction of CD4 helper T cells (Th 1, Th2, and Th17) and CD8 effector
T cells, maintenance of T cell memory, inhibition of regulatory T cells, immune
complexes formation, complement activation, pro-inflammatory cytokines
secretion, chemokine production, and orchestration of tertiary lymphoid tissue
generation [225]. Therefore, a reasonable therapeutic target in these patients
40
consists in the reduction of the number and activity of Bc, which can be
accomplished by the use of biologic therapeutic agents such as RTX [226].
RTX is a chimeric monoclonal Ab directed against the transmembrane glycoprotein
CD20 on Bc. The CD20 molecule is thought to be a Ca++ channel and to participate
in Bc activation and proliferation. It appears from the pre-B maturation stage and
disappears in terminally differentiated plasmablasts and PC. As a therapeutic
target, CD20 has many advantages: it is not eliminated from the cell surface, it
does not have a soluble form, and it is poorly internalized after Ab recognition
[227]. The mechanism through which RTX diminishes or depletes mature Bc is not
completely elucidated, but the most probable one depends on the expression of Fc
receptors (FcRI y FcRIII), with monocytes as the principal effector cells, whereas
T and natural killer cells do not seem to participate [228]. The activation of the
complement system also seems to contribute [229].
RTX effectiveness has been demonstrated in many autoimmune diseases such as
thrombocytopenic purpura [230], severe pemphigus [231], RA [232], SLE [233],
and Sjögren’s syndrome [234], with an increasing use in pathologies for which its
use was not initially conceived [235]. RTX causes a rapid and very significant
reduction of circulating Bc subpopulations during 6 – 9 months after one cycle of
therapy. Nevertheless, the variability of depletion seen in different individuals under
the same conditions is a consistent finding [236]. The factors that influence the
time at which repopulation initiates are not clearly established, but the magnitude
of earlier depletion, drug clearance rate, and the capacity of the bone marrow to
regenerate most probably determine it. Repopulation is comprised mainly of naïve
Bc, with an increased frequency of transitional Bc, which resembles bone marrow
transplantation [237]. Regarding Bc depletion in secondary lymphoid organs and
other solid tissues in humans, the information is scarce. Studies in primates
showed that higher doses are required to deplete bone marrow, spleen, and lymph
nodes in this sequence [236].
With respect to the effect of RTX on serological memory, after Bc depletion with
one cycle total IgA, IgG, IgM, and IgE levels significantly decrease, but within
normal ranges [37]. In contrast, IgG Ab titers against pathogens such as measles
[37], tetanus [38], and pneumococcal capsular polysaccharide [40] remain
constant. In regard to auto-Abs, results differ: on the one hand, it has been
reported that anti-double-stranded DNA (dsDNA) and anti-C1q [40], both of IgG
isotype, and IgA-, IgG-, and IgM-class rheumatoid factors (RF) diminish
significantly after RTX therapy [39]. On the other hand, auto-Abs against Ro52,
Ro60, and La44, also of IgG isotype, remain unchanged after RTX therapy [37].
These results suggest that the mechanism of maintenance of serological memory
could depend on the type of antigen and/or Ab isotype evaluated, particularly in
patients with autoimmune diseases. Nevertheless, simultaneous assessment of
antigen-specific mBc and antigen-specific Abs of different isotypes in patients that
received B-cell depletion therapy with RTX is lacking.
41
Table 1. Genetic characteristics and proposed origin of different human B cell subsets
Bc subset
Phenotype
Transitional
Naïve
CD38hiCD24hi
CD38dimCD24dim
CD27-IgM+
CD27+IgD+IgM+
IgM+ mBc
IgM+
mBc
Only
CD27+IgDloIgMhi
[180]
CD27+IgDhiIgMlo
[180]
CD27+IgD-IgM+
IgD+
mBc
Only
CD27+IgD+IgM-
Switched
mBc
CD27+IgG+
CD27+IgA+
CD27- mBc
GC generation and
possible tissue of
origin [193]
-
IGH and/or IGL usage of V or J gene
segments
Number of cell cycles
[193]
SHM and subclass usage
Clonal relationships
IGHV4-34, IGHV4-59 [193]
None
2
No
No
-
GC/GCi. At least in
part originate from
the
splenic
marginal
zone
(GCi).
>IGHJ4 and <IGHJ6 than naïve.
>IGHV3 (IGHV3-23) and <IGHV1
usage than naïve. The opposite to Sw
CD27+ mBc [194].
7 (<GC)
= GC, enriched for mutated
IGKV3-20 [193]. <Sw CD27and CD27+.
??
??
??
??
These cells seem not to be clonally
related to Sw mBc [194, 238]. There
is also evidence in both human GC
and peripheral blood to support that
Sw mBc are in part derived from
IgM+ mBc [161, 239].
??
??
??
??
??
??
9 (= GC)
= GC in IGHV, higher in
IGKV3-20 [193]. = CD27- mBc.
GC,
response
probably driven by
superantigens
[240].
Consecutive
GC
responses.
>IGHJ4 and <IGHJ6 than naïve Bc.
>IGHV3 (IGHV3-23) and <IGHV1
usage than naïve [194].
Preferential Igλ usage. >IGHV3-23
and to a lesser extent >IGHJ6 than
naïve and Sw CD27+ mBc [240].
?? Due to the high
SHM load it is
expected to be high.
High in IGH and IGL. >Sw
CD27+ mBc [240].
They make large clones, unrelated
to other populations [240].
>IGHJ4 and <IGHJ6 than naïve Bc.
>IGHV1 than IgM+ mBc and naïve.
However, IgG2 resembles the IGHV
usage of IgM+ mBc. [194].
Ig+ (55%) [193]. >IGHJ4 and <IGHJ6
than naïve. >IGHV1 than IgM+ mBc
and naïve [194].
10 (>GC)
High (IGHV and IGKV3) [193].
>CD27- mBc [194]. IGHG2
(51%) and IGHG1 (40%) [193].
10 (>GC)
= IGHJ4 and <IGHJ6 than naïve.
>IGHV3 (IGHV3-23) and <IGHV1
usage than naïve [194].
>IGHJ4 and <IGHJ6 than naïve Bc.
IGHV1 > IgM+ mBc and naïve [194].
??
High (IGHV and IGKV3),
>CD27+IgG+ [193]. >CD27mBc [194]. IGHA2 (19%)
[193].
Similar to the other IgM+
subsets [194].
IgG1, IgG3, and IgA1 use more
IGHV1 and lower IGHV3 than IgM+
mBc. IgG2 and IgA2 have a similar
usage of IGHV1 and IGHV3
compared to IgM+ mBc, particularly
IgG2 [194].
Primary
responses.
Consecutive
responses.
GC
GC
CD27-IgD-IgM+
CD27-IgD-IgG+
Primary
responses.
GC
CD27-IgD-IgA+
9 (= GC)
GCi.
Probably Ig+ (80%) [193]. >IGHJ4 and < 4 (<GC)
generated locally at IGHJ6 than naïve Bc. IGHV1 > IgM+
the intestine.
mBc and naïve [194].
All mBc populations had more transitions than transversion mutations and strand bias in the AT mutations [194].
GC: generated in a germinal center, GCi: generated independently from a germinal center.
42
= GC in IGHV, higher in
IGKV3-20 [193]. >Naïve and
IgM+ mBc [194]. IGHG1 (63%)
and IGHG3 (31%) [193].
>GC in IGHV, = in IGKV3-20
[193]. >Naïve and IgM+ mBc
[194]. IGHA2 (33%) [193].
All IgM+ mBc have a similar usage
of IGHJ and IGHV families [194].
CD27- can precede CD27+ and vice
–versa [194].
3. HYPOTHESES
3.1 HYPOTHESIS 1: Circulating RV-SIg reflects more precisely the intestinal
protective immune response induced by the vaccine RIX4414 and is, thus, a better
correlate of protection than circulating RV-IgA.
3.2 HYPOTHESIS 2: RV-mBc are distributed in a different manner in patients with
autoimmune diseases and contribute to RV-serological memory in a unique way.
4. OBJECTIVES
General objectives are presented according to the two hypotheses assessed in this
work, accompanied by their specific objectives.
3.1 To confirm that plasma RV-SIg can be detected in children after natural RV
infection and to compare it to plasma RV-IgA as a correlate of protection in children
vaccinated with RIX4414.

3.1.1 To determine if plasma RV-SIg is detected in children with RV-GE or GE
from other etiology and in children vaccinated with RIX4414 and placebo
recipients.

3.1.2 To assess the correlation of RV-SIg titers with circulating RV-mBc after
vaccination and with the protection conferred by the vaccine against RV.
3.2 To explore the distribution of RV-mBc and their relationship with serological
memory in patients with autoimmune diseases before and after treatment with
RTX.

3.2.1 To compare circulating RV- and TT- mBc in healthy volunteers and
patients with autoimmune diseases before and after RTX treatment.

3.2.2 To assess the relationship between RV-specific mBc and the
corresponding plasma Abs in healthy volunteers and patients with autoimmune
diseases.

3.2.3. To understand the contribution of some mBc subsets to the maintenance
of serological memory, depending on the type of antigen and Ab isotype
studied.
43
5. MATERIALS AND METHODS
Materials and methods are described in each article. A more detailed version can
be found in the appendices.
6. JOURNAL ARTICLES
6.1 Introduction to the article: Rotavirus specific plasma secretory
immunoglobulin in children with acute gastroenteritis and children
vaccinated with an attenuated human rotavirus vaccine
Despite the great advance current RV vaccines have meant for the prevention of
GE related mortality in children under the age of five, it is necessary to develop a
new generation of RV vaccines that overcome current limitations regarding its
efficacy, particularly in low-income countries. To achieve this goal, one of the
definitive areas that must be addressed is the lack of an appropriate immunological
correlate of protection [13].
At present, serum RV-IgA is the best, though imperfect, practically measured
correlate of protection against RV GE [12, 13]. Recently, two cut-off points have
been established for serum RV-IgA after vaccination, with different implications. On
the one hand, after Rotarix vaccination a RV-IgA titer ≥ 20 U/mL correlates with
protection against RV GE and is associated with a lower percentage of individuals
with any or severe RV GE. Vaccinees with serum RV-IgA < 20 U/mL also show
some level of protection compared to placebo recipients [119]. On the other hand,
for both Rotarix and RotaTeq, serum RV-IgA titers ≥ 90 U/mL correlate with higher
protection and less waning of it than titers < 90 U/mL [119, 121]. Nevertheless,
serum RV-IgA can be used as an epidemiological tool at the population level, but
does not permit predicting individual protection. Certainly, some vaccinees with
serum RV-IgA develop mild RV GE, and protection provided by the vaccines can
be higher or lower than the levels predicted by serum RV-IgA detected in
vaccinees [12, 13].
Given that RV preferentially replicates in the intestine and local mucosal immunity
is considered to be essential in human RV immunity [13], an adequate correlate of
protection could be a marker able to precisely reflect the intestinal immune
response induction. SIg in serum has been proposed as a method for indirectly
measuring intestinal Ig [19]. RV-SIg has been detected in serum of children with
acute RV infection [19, 21], and it correlated with the amounts detected in
duodenal fluid one week after the infection [22]. Based on these precedents, we
sought to confirm the presence of plasma RV-SIg in children with natural RV
infection and to determine if circulating RV-SIg could reflect more precisely the
intestinal protective immune response induced by the attenuated RIX4414 human
44
RV vaccine, and be a better correlate of protection than circulating RV-IgA after
vaccination.
We then evaluated total plasma SIgA and plasma RV-SIg by ELISA in children with
RV-GE or GE from other etiology and in 50 vaccinated children and 62 placebo
recipients. RV-SIg was only detected in children with evidence of previous RV
infection or with acute RV gastroenteritis. We report, for the first time, that
vaccinees had higher RV-SIg titers than placebo recipients after each applied
dose, and that RV-SIg titers increased after the second vaccine dose. RV-SIg
measured after the second dose correlated with protection when vaccinees and
placebo recipients were analyzed jointly. We propose that plasma RV-SIg may
serve as a valuable correlate of protection for RV vaccines.
45
Research Paper
Research Paper
Human Vaccines & Immunotherapeutics 9:11, 2409–2417; November 2013; © 2013 Landes Bioscience
Rotavirus specific plasma secretory
immunoglobulin in children with acute
gastroenteritis and children vaccinated with
an attenuated human rotavirus vaccine
Instituto de Genética Humana; Facultad de Medicina; Pontificia Universidad Javeriana; Bogotá, Colombia;
2
R&D Laboratory; Division of Immunology and Allergy; University State Hospital; Lausanne, Switzerland
1
Keywords: rotavirus, vaccine, correlate of protection, secretory immunoglobulin
Abbreviations: RV, rotavirus; SIg, secretory immunoglobulin; SIgA, secretory immunoglobulin A; SC, secretory
component; GE, gastroenteritis; D1, dose 1; D2, dose 2; rhSC, recombinant human secretory component
Rotavirus (RV)–specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV
infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were
evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the
attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of
previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and
RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection
when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for
RV vaccines.
Introduction
Rotavirus (RV) is the principal cause of severe gastroenteritis (GE) in young children, being responsible, before the introduction of routine immunization, for approximately 453,000
deaths annually worldwide.1 Two RV vaccines are available and
recommended for infants worldwide by the WHO2 : Rotarix
(GlaxoSmithKline Biologicals), an attenuated human RV vaccine, and Rotateq (Merck and Co. Inc.), a bovine-human reassortant vaccine. Both vaccines are less efficacious (39% to 77%)
in some low-income countries in Africa and Asia,3 where 85%
of worldwide mortality occurs.4 The improvement of these vaccines or the development of new RV vaccines is hindered by
the lack of a widely accepted immunological correlate of protection. At present, serum RV-specific IgA (RV-IgA) measured
shortly after natural infection or vaccination represents the best
practically measured correlate of protection against RV GE.5
However, some vaccinees with serum RV-IgA develop mild RV
GE, and protection provided by the vaccines can be higher or
lower than the levels predicted by serum RV-IgA detected in
vaccinees.6,7
RV preferentially replicates in the intestine, and local mucosal
immunity is thought to be key in human RV immunity.7 During
an acute RV infection in children, circulating IgD- RV-specific B
cells express intestinal-homing receptors (α4β7+, CCR9+), and
thus probably reflect mucosal immunity.8 In agreement with
this finding, in our previous double blind trial of the attenuated RIX4414 human RV vaccine, correlations between protection from disease and frequencies of RV-memory IgD-, CD27+,
α4β7+, CCR9+ circulating B cells measured after dose 1 (D1) and
plasma RV-IgA after dose 2 (D2) were found. However, the correlation coefficients for both tests were low, suggesting that other
factors are important in explaining protection from disease.9 In
this trial, only a minority (32.7%) of vaccinees presented RV-IgA
coproconversion, indicating that this is not an optimal parameter
to measure vaccine-induced intestinal antibody responses.9
Secretory Ig (SIg) in serum has been proposed as an alternate
method for indirectly measuring intestinal Ig.10 Polymeric IgA
and IgM are transported across mucosal epithelial cells by the
polymeric Ig receptor.11 At the epithelial surface the receptor is
cleaved and part of it (the secretory component [SC]) remains
attached to the Ig, forming SIg, which may retro-transcytose
*Correspondence to: Juana Angel; Email: [email protected]
Submitted: 03/08/2013; Revised: 05/30/2013; Accepted: 07/02/2013
http://dx.doi.org/10.4161/hv.25610
www.landesbioscience.comHuman Vaccines & Immunotherapeutics2409
©2013 Landes Bioscience. Do not distribute.
Daniel Herrera1, Camilo Vásquez1, Blaise Corthésy2, Manuel A Franco1, and Juana Angel1,*
across epithelial cells and eventually enter the circulation.11
RV-SIg has been detected in serum of children with recent RV
infection,10,12 but not in the serum of healthy breast-fed children,
even though it was present in the stool and duodenal fluid of some
of them and in their mothers’ milk and serum.13,14 Moreover,
serum RV-SIg correlated with the amounts detected in duodenal
fluid one week after the acute infection.15 These results suggest
that serum RV-SIg is frequently observed after RV infection and
reflects intestinal Ig.
It is generally accepted that neutralizing antibodies against
the RV infecting strain present in the intestine provide protection.16 However, assessment of intestinal fluid after RV vaccination is impractical and measurement of stool antibodies is subject
to technical problems, including interference by maternal antibodies.9,17 Hence, circulating RV-SIg could reflect more precisely
the intestinal protective immune response induced by the vaccine
and be a better correlate of protection than circulating RV-IgA
after vaccination.
We here confirm the presence of plasma RV-SIg in children
with natural RV infection, and further addressed its occurrence
in children vaccinated with the attenuated human RV vaccine
RIX4414. We report, for the first time, that vaccinees have
higher RV-SIg titers than placebo recipients after each of the two
administered doses, and that RV-SIg titers increased after D2.
Furthermore, RV-SIg measured after D2 correlated with protection when vaccinees and placebo recipients were analyzed jointly.
We propose that plasma RV-SIg may be a valuable correlate of
protection for RV vaccines.
Results
Total plasma SIgA, RV-SIg and RV-IgM in children with
acute GE
Based on the presence of RV antigen or RNA in stools and
RV-IgA in plasma, children with acute GE from prior studies
(Table S1)18,19 were classified in 3 groups: group A: children without evidence of previous RV infection (RV-IgA-) and without
RV GE (n = 5); group B: children with evidence of previous RV
infection (RV-IgA+) but without RV GE (n = 20) and group C:
children with acute RV GE with primary infection (RV-IgA-)
(n = 7) or secondary infection (RV-IgA+) (n = 4). Umbilical cord
blood samples taken from healthy full-term newborn infants,
group D (n = 4), were used as controls. Plasma samples from 10
adult healthy volunteers were assessed in parallel.
The mean concentration of total plasma SIgA for adult
healthy volunteers was 12.27 μg/ml (6.73–25.8 μg/ml), which
is comparable to values previously reported,20,21 and there was
no significant difference when serum or plasma samples from
these volunteers were evaluated (data not shown). Moreover, as
previously shown,22,23 umbilical cord blood (group D) had significantly less total SIgA than any of the groups with acute GE
(Fig. 1A). Group C (children with RV GE) had significantly less
total plasma SIgA than group A (Fig. 1A) and than groups A and
B analyzed jointly (children without RV GE, data not shown).
Plasma RV-SIg was detected in children with previous RV
infection and with current primary or secondary RV infection
(groups B and C, respectively), but not in children without previous RV infection (group A) or in umbilical cord blood samples
(group D, Fig. 1B). As expected,12 children with acute RV GE
had significantly higher titers of plasma RV-SIg than children
with previous RV infection but without an ongoing RV GE
(group C vs. group B, Fig. 1B), and none of the 10 adult healthy
volunteers had plasma RV-SIg (data not shown).24
The anti-human SC monoclonal antibody (mAb) used as a
capture antibody in the ELISAs can recognize both SIgA and
SIgM (Fig. 1A and data not shown). The ELISA protocol for total
SIgA includes an anti-IgA antibody, and does not identify SIgM
(data not shown); in contrast, the RV-SIg ELISA can detect both
RV-SIgA and RV-SIgM. Therefore, we next explored RV-IgM
responses in all groups of children (Fig. 1C) and potential correlations between RV-SIg and RV-IgA (Fig. 2A) and RV-IgM
(Fig. 2B). Plasma RV-IgM was only detected in children with
evidence of previous RV infection and those with acute RV GE,
and was higher in the latter than the former (Fig. 1C).
2410Human Vaccines & Immunotherapeutics
Volume 9 Issue 11
©2013 Landes Bioscience. Do not distribute.
Figure 1. Total plasma SIgA, RV-SIg and RV-IgM in children with acute GE. (A) The concentration of SIgA for each sample was determined based on a
standard curve of plasma with a known SIgA concentration (14.6 μg/ml). The limit of detection was 4.8 ng/ml. (B) and (C) The reported values correspond
to the log10 of the inverse titer measured by ELISA. The plasma RV-IgM for groups A and D is below the limit of detection. Lines and error bars denote
the mean and SEM, respectively. Differences between groups were evaluated with the nonparametric Mann–Whitney test and all p values reported are
1-tailed. Groups of children: Group A: RV-IgA-, RV-GE-; group B: RV-IgA+, RV-GE-; group C: RV-IgA- (triangles) and RV-IgA+ (open squares) RV-GE+ and group
D: umbilical cord blood samples taken from healthy full-term newborn infants.
Correlation tests between RV-SIg and RV-IgA (group B and
children from group C with secondary infection) or RV-IgM
(groups B and C) were performed either separately or jointly.
No correlation between RV-SIg and RV-IgA was observed in any
case (Fig. 2A, and data not shown). In contrast, there was a significant correlation between RV-SIg and RV-IgM when groups B
and C were analyzed jointly (children with evidence of RV infection) although with a low correlation coefficient (Fig. 2B). Thus,
plasma RV-SIg seems to be induced independently of plasma
RV-IgA and to a certain degree separately of plasma RV-IgM in
children with natural RV infection.
Competitive binding assays with rhSC
To confirm anti-human SC mAb-mediated detection of total
SIgA and RV-SIg in ELISAs, competitions with recombinant
human secretory component (rhSC) were performed. As shown
in Figure 3A, a 50% reduction in binding of colostral SIgA was
obtained with 0.38 μg/ml rhSC, which corresponds to the naturally found 1:1 molar ratio occurring between rhSC and SIgA.
As previously stated, the RV-SIg ELISA does not distinguish
between RV-SIgA and RV-SIgM. Therefore, a plasma sample
with presumably only RV-SIgA (RV-SIg+, RV-IgA+, RV-IgM-)
and a plasma sample with presumably only RV-SIgM (RV-SIg+,
RV-IgA-, RV-IgM+) were selected to compete with rhSC for the
binding to the anti-human SC mAb. As shown in Figure 3B
and C, the same concentration of rhSC (0.38 μg/ml) inhibited
approximately 50% of the maximum signal observed for both
types of samples. Thus, detection of either total SIgA or RV-SIg
strictly relies on the binding of SIg to the anti-human SC mAb.
Total plasma SIgA and RV-SIg in children vaccinated with
the attenuated RIX4414 human RV vaccine
In our previous trial of the RIX4414 human RV vaccine, children received the “all-in-one” vaccine formulation, in which the
calcium carbonate buffer is lyophilized with the virus and the
powder is reconstituted with water before vaccination. This formulation induced lower RV-IgA seroconversion rates25 ; however,
it contains the same vaccine strain virus found in the Rotarix
formulation and protected at similar levels against any RV GE
and severe RV GE.9
There were no significant differences for total plasma SIgA
among study groups (Fig. 4A). As shown in Figure 4B, vaccinees
had higher RV-SIg titers than placebo recipients, both after D1
and D2. Titers of RV-SIg in vaccinees were significantly higher
after D2 than after D1 (Fig. 4B), suggesting a boost effect of the
second dose.
Figure 3. Effect of competing rhSC on measurement of purified colostral SIgA and RV-SIg. Reported values are optical density units (OD, 450 nm) (Y axis)
and the log10 of the rhSC concentration in μg/μl (X Axis) used to compete the binding of: (A) purified colostral SIgA (0.076 μg/ml); (B) a plasma sample
from a child in which RV-SIg was presumably only RV-SIgA (RV-IgA+, RV-IgM-); and (C) a plasma sample from a child in which RV-SIg was presumably only
RV-SIgM (RV-IgA-, RV-IgM+). Open dots in graphics show the concentration of rhSC inducing approximately 50% of inhibition and the dashed lines correspond to the signal observed when samples were competed with albumin at the same concentrations as for rhSC.
www.landesbioscience.comHuman Vaccines & Immunotherapeutics2411
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Figure 2. Correlations of RV-SIg with RV-IgA and RV-IgM. (A) Correlation (Spearman one-tailed test) between plasma RV-SIg and plasma RV-IgA titers of
children from group B (RV-IgA+, RV-GE-) and children from group C with secondary infection (RV-IgA+, RV-GE+) analyzed jointly. (B) Correlation (Spearman
one-tailed test) between plasma RV-SIg and plasma RV-IgM titers of children from groups B and C analyzed jointly.
In an attempt to establish the relationship between RV-IgA
and RV-SIg after vaccination, the distribution of RV-SIg was
analyzed in children with and without RV-IgA (RV-IgA + vs.
RV-IgA-, Table 1). The frequency of RV-SIg+ children was significantly higher in RV-IgA+ vaccinees than in RV-IgA- vaccinees after D2 (16/25 vs. 8/25, chi-square test p < 0.05, Table 1).
Moreover, the frequency of placebo recipients with RV-SIg+ was
significantly higher in children RV-IgA+ than in those RV-IgAafter D2 (4/5 vs. 12/57, Fisher exact probability test p < 0.05,
Table 1). In addition, a significant correlation was found between
RV-IgA and RV-SIg in all study groups, except for vaccinees after
D1 (data not shown); however, in all cases the correlation coefficients were low (Spearman test rho < 0.4). The frequency of
RV-SIg+ children was significantly higher in RV-IgA- vaccinees
than in RV-IgA- placebo recipients after D1 and/or D2 (20/23
vs. 21/54, chi-square test p < 0.05, Table 1). These results suggest
that plasma RV-IgA and RV-SIg partially overlap, but depict different antibody responses.
Next, the relationship between RV-SIg titers and protection
was assessed. First, as shown in Table 2, the protection rates for
vaccinees, as well as for placebo recipients, increased as a function of RV-SIg titers detected after D2. Second, when vaccinees
and placebo recipients were analyzed jointly there was a correlation between protection and RV-SIg titers measured after D2
(Spearman test p < 0.05, rho = 0.22). Third, the frequency of
protected children was significantly higher in RV-SIg+ children
(titers ≥ 1:100) than in those RV-SIg- (titer < 1:100) (37/40 vs.
55/72, chi-square test p < 0.05) and the presence of RV-SIg
conferred an almost four times increase in the probability to be
protected against any RV GE (OR: 3.81, CI 95%: 1.04–13.93).
Finally, protected children had significantly higher RV-SIg titers
than non-protected children after D2 (Fig. 4C). In contrast,
analysis of samples after D1 did not show any statistically significant correlation or difference between study groups. Altogether,
these results suggest that RV-SIg is related to protection both
after vaccination and natural RV infection.
Additionally, no correlations were found between any
RV-specific B cells subset previously studied, including RV-specific
IgD + CD27+α4β7+ CCR9+ and IgD-CD27+α4β7+ CCR9+, and
plasma RV-SIg (data not shown).9
Finally, we addressed the possibility that plasma RV-IgG
could correlate with protection after vaccination with RIX4414.
Although vaccinees had higher RV-IgG titers than placebo recipients after D2 (Fig. S1A), RV-IgG did not correlate with protection in any case (Table S3, Spearman test p = 0.38, rho = 0.026,
when vaccinees and placebo recipients were analyzed jointly).
Furthermore, there was no difference in RV-IgG titers between
protected and non-protected children (Fig. S1B).
Discussion
We confirmed12,15 that RV-SIg can be detected in blood of
naturally infected children (Fig. 1B), and showed that children
vaccinated with the attenuated RIX4414 human RV vaccine have
higher RV-SIg titers than placebo recipients, both after D1 and
D2, and in vaccinees higher titers were observed after D2 than
after D1 (Fig. 4B). Furthermore, RV-SIg measured after D2 correlated with protection in vaccinees and placebo recipients analyzed jointly (Table 2). The lack of correlation of RV-SIg with
protection in vaccinees is probably related to the low number
(five) of vaccine failures in these children.9 To our knowledge,
this is the first study in which plasma antigen specific SIg has
been evaluated as a correlate of protection after vaccination.
Unexpectedly,12 children with acute RV GE (group C) had
less total SIgA than children with acute GE of a different etiology
(groups A and B analyzed jointly). Considering that plasma SIgA
may be short-lived, similar to circulating IgA (4–6 d),26 and that
the mean time of blood drawing after onset of diarrhea was 4.2
d, this result suggests that acute RV GE may disrupt the intestine
epithelial barrier to a greater extent than other pathogenic conditions, affecting the mechanism by which total SIgA is selectively
retro-transcytosed from the intestinal lumen.
2412Human Vaccines & Immunotherapeutics
Volume 9 Issue 11
©2013 Landes Bioscience. Do not distribute.
Figure 4. Total plasma SIgA and RV-SIg in vaccinees and placebo recipients. Data for 50 vaccinees and 62 placebo recipients after dose 1 (D1) or dose 2
(D2) are shown. Lines and error bars denote the mean and SEM, respectively. Differences between vaccinees and placebo recipients, as well as between
protected and non-protected children, were evaluated with the Mann–Whitney test and between vaccinees after D1 and D2 with the Wilcoxon test. All
p values reported are 1-tailed. (A) The reported values correspond to μg/ml interpolated from a plasma pool with a known SIgA concentration (14.6 μg/
ml). (B and C) The reported values correspond to the log10 of the inverse titer measured by ELISA.
Table 1. Number of vaccinees and placebo recipients with/without (+/−) plasma RV-IgA with plasma RV-Sig.
Vaccinees n = 50
Placebo recipients n = 62
RV-IgA
RV-IgA
RV-IgA+
RV-IgA-
(RV-SIg+)
(RV-SIg+)
(RV-SIg+)
(RV-SIg+)
After D1
13 (6)
37 (14)
4 (2)
58 (13)
After D2
25 (16)
25 (8)
5 (4)
57 (12)b
After D1 and/or D2c
27 (18)
23 (20)
8 (6)
54 (21)
+
-
a
Statistically significant difference between the subgroups with RV-IgA and without RV-IgA for vaccine and placebo recipients (chi-square test and Fischer
exact probability test p < 0.05, respectively). cStatistically significant difference between the subgroups of vaccinees and placebo recipients without RV-IgA
(chi-square test p < 0.05).
RV-SIg has been reported to appear as early as 3–4 d after
the onset of RV diarrhea, with the number of individuals positive for serum RV-SIg increasing significantly around day 10,
and becoming undetectable approximately a month later.15 The
transient nature of RV-SIg is probably one of the reasons why its
measurement has not been implemented for evaluating vaccine
immunogenicity.27 We used a labeled avidin-biotin ELISA protocol, which is expected to be more sensitive than the one available
in previous reports, and detected RV-SIg in 17 out of 20 children with evidence of previous RV infection without an ongoing
RV GE. This result challenges the notion that plasma RV-SIg
can only be transitorily detected. Nonetheless, RV-SIg was transiently observed in some vaccinated children, since only half of
vaccinees with RV-SIg after D1 also had RV-SIg after D2. Of 15
placebo recipients with RV-SIg after D1 only 4 had it after D2.
This potential divergence in plasma RV-SIg persistence between
naturally infected and vaccinated and placebo recipient children
could be explained by differences in the mean age (14.2 vs. 4 mo,
respectively), by the number of RV infections in the children, and
by differences between the effects of wild type virus and the vaccine virus on the plasma RV-SIg positivity rates. Further studies
are necessary to determine the kinetics of RV-SIg in blood after
RV vaccination.
Both RV-SIgA and RV-SIgM are probably measured in our
RV-SIg ELISA, because RV-SIg can be identified both in children without RV-IgA (children with primary RV infection from
group C) and in children with low levels or no RV-IgM (group
B). Moreover, plasma RV-SIg is probably produced with a different kinetics than plasma RV-IgA, since there was no significant correlation between RV-SIg and RV-IgA in children with
evidence of previous RV infection (Fig. 2A) that were analyzed
2–10 d after the onset of diarrhea. Likewise, RV-SIg is probably
produced independently of RV-IgM, because in children with
primary RV infection RV-SIg and RV-IgM exhibit no correlation
(data not shown). Nonetheless, some of the RV-SIg may include
RV-SIgM, due to the fact that there is a significant correlation
between RV-SIg and RV-IgM when groups B and C are analyzed
jointly, although the correlation coefficient was low (Spearman
test rho = 0.33). Thus, RV-SIg induced after natural infection is
most probably composed of both RV-SIgA and RV-SIgM, and
these Igs are independently produced from RV-IgA and RV-IgM,
supporting the hypothesis that plasma RV-SIg is generated in the
intestinal compartment.
Correlations were found between RV-SIg and RV-IgA in vaccinees after D2 and placebo recipients after D1 and D2 (data
not shown), but not in vaccinees after D1, or in children with
evidence of previous RV natural infection (Fig. 2A). This may
be explained by differences between these groups: children with
evidence of previous RV infection were older (mean age 14.2 mo)
than vaccinees (4 mo old) and may have been more exposed to
RV infections. Thus, age and history of RV infections seem to be
important determinants of RV-SIg responses and will need to be
further investigated.
In children that received Rotarix, the main effect of the second dose was to provide a “catch-up’ immunization.7,9,28 Our
results for RV-IgA and RV-SIg are in agreement with this finding: RV-IgA was detected in 13 vaccinees after D1 and it was
detected in 14 more children after D2. RV-SIg was detected in
20 vaccinees after D1, and it was detected in 14 more children
after D2. However, a detectable boost (4-fold increase in preexisting plasma RV-SIg) for RV-SIg was observed in three children whereas for RV-IgA it was detected only in one child. In
addition, a significant difference between vaccinees and placebo
recipients’ titers was observed for plasma RV-SIg (Fig. 4B) but
not for RV-IgA.9
In our trial plasma RV-SIg seems to be a better correlate of
protection than plasma RV-IgA, because although RV-IgA also
correlated with protection when vaccinees and placebo recipients were analyzed jointly after D2 (Table S2, Spearman test p =
0.05, rho = 0.152),9 in contrast to RV-SIg (Table 2), the frequency
of protected children was not significantly higher in RV-IgA +
children (titers ≥ 1:100) than in those RV-IgA- (titer < 1:100)
(Table S2, Chi-square test p > 0.05,). Moreover, the frequency
of protected children was significantly higher in RV-IgA- (titer
< 1:100) vaccinees compared with RV-IgA- placebo recipients
(Table S2), which implies that other factors, aside from RV-IgA,
are important in explaining protection from disease in vaccinees.
The lack of correlation between RV-SIg detected after vaccination and any RV-memory B cells subsets analyzed may be
explained by the fact that RV-SIg detected is probably a mixture
of SIgM and SIgA.
Since naturally acquired serum RV-IgG has been correlated
with protection against RV infection in children,29 and passively
transferred serum RV-IgG mediated mucosal immunity against
RV infection in a macaque model,30 we assessed RV-IgG titers
in plasma samples of vaccinees and placebo recipients after D2.
www.landesbioscience.comHuman Vaccines & Immunotherapeutics2413
©2013 Landes Bioscience. Do not distribute.
a, b
Table 2. Correlation between RV-SIg titers after D2 and protection against any RV GE
RV-SIg Titers after D2
Vaccinees* (%)
Placebo recipients* (%)
< 100
22/26 (84.61)
33/46 (71.73)
100
10/11 (90.90)
8/10 (80)
18/21 (85.71)b
≥ 200
13/13 (100)
6/6 (100)
19/19 (100)c
a
Vaccinees+placebo recipients* (%)
55/72 (76.39)
Although RV-IgG titers were significantly higher in vaccinees
than placebo recipients (Fig. S1A), RV-IgG did not correlate
with protection (Table S3). This lack of correlation is most probably due to the presence of variable amounts of maternal RV-IgG
antibodies, transferred via the placenta, when the second vaccine
dose was administered (4 mo old).
Human antigen-specific SIg has been quantified in secretions,31-33 as well as in serum,34-36 both after natural infection
or vaccination with multiple pathogens and antigens. Mucosal
priming seems to be required for induction of SIg, but immunization at a given mucosal surface may induce a response at
multiple mucosal sites.37 Although SIg in serum may potentially
come from colostrum,38 this is unlikely in our study, because the
children studied were older than two months of age, and very
few (7%) of the placebo recipients had RV-SIg while almost all of
them (> 96%) were breast fed.9
The mechanism by which SIgA produced at mucosal surfaces is transported to the circulation has not been clearly established: passive leakage of SIgA or active transport mediated by
an unknown receptor could be involved.11,39 The transferrin
receptor (CD71) has been proposed as an IgA receptor.40 CD71
is abnormally expressed at the apical pole of enterocytes in
patients with active celiac disease and evidence that polymeric/
secretory IgA mediates protected transport of pathogenic gliadin
peptides through their binding to CD71 has been obtained.41
Whether this is the case for pathogen-specific SIg detected in
serum remains to be determined. Although some RV-SIg could
be retro-transcytosed bound to the viral antigen during acute
infection, it seems that several months after the virus has disappeared RV-SIg continues to be retro-transcytosed (Fig. 1B).
Frequent RV infections have been associated with celiac disease
in genetically predisposed children.42 Thus, further studies associating RV infection with celiac disease and IgA nephropathy (in
which abnormally localized SIg may play a pathogenic role)36,43
are required.
Since present RV vaccines are less efficacious in countries
where they are most needed, new vaccine formulations are being
tested. For this reason, the RV’s field would greatly benefit of the
use of a correlate of protection to accelerate this process. Our
results suggest that RV-SIg could be complementary to RV-IgA
in evaluating vaccine immunogenicity and could be a valuable correlate of protection for RV vaccines. However, because
of our lack of preimmune plasma samples, our study does not
demonstrate vaccine induced RV-SIg. In addition, due to the fact
that children received an “all-in-one” vaccine formulation, which
is less immunogenic than the final RIX4414 formulation, the
correlation of RV-SIg with protection may be underestimated. In
fact, the Rotarix commercial formulation induces higher RV-IgA
seroconversion rates than the RIX4414 formulation used in the
present study, and this may also be the case for RV-SIg.28 Hence,
further studies are needed to assess plasma RV-SIg kinetics in
vaccinees, and to properly determine its value as a RV correlate of
protection for existing vaccines.
Materials and Methods
Ethics statement
Written informed consent was obtained from each volunteer
or infant’s parents or legal guardian. Studies were approved by
the Ethics Committee of the San Ignacio Hospital and Pontificia
Universidad Javeriana and conducted in accordance with the
guidelines of the Helsinki Declaration.
Subjects and sample processing
We studied plasma samples from 36 children with acute GE
from prior published studies.18,19 These children (14 females and
22 males; mean age: 13.5 mo, range: 6–22; 20 breast fed and 16
not breast fed) were admitted with gastroenteritis to the pediatric emergency service or were hospitalized at the moment of
sample collection. Demographic and clinical data of the children
are presented in Table S1 arranged in the 3 groups described in
the results section. The mean time of blood drawing after onset
of diarrhea was 5 d (range: 1–12).
Plasma and serum samples were obtained from 10 healthy
adult volunteers (7 females and 3 males; mean age: 28 y, range:
25–38), without any gastrointestinal symptoms, during the
month previous to the blood drawing. Additionally, 4 umbilical cord blood samples taken from healthy full-term newborn
infants were also included.
In our previous double-blind randomized controlled study,9
children received two doses of either placebo (n = 160) or 106.7
focus-forming units of the attenuated RIX4414 human RV vaccine (n = 159). Vaccine and placebo groups were very similar
in terms of the median age at the time of the first and second
vaccination (60 and 122 d, respectively), gender (M/F 85/74
and 84/76, respectively), and percentage of breast fed at dose 1
and dose 2 (96.9% and 95%, 88.6% and 88.1%, respectively).
2414Human Vaccines & Immunotherapeutics
Volume 9 Issue 11
©2013 Landes Bioscience. Do not distribute.
*Shown is the number of protected vaccinees, placebo recipients or vaccinees and placebo recipients analyzed jointly / the total number of children in each
population for each RV-SIg titer. aThe frequency of protected children was similar in RV-SIg- (titer < 1:100) vaccinees and placebo recipients (chi-square test
p > 0.05). bThe frequency of protected children was significantly higher in RV-SIg+ children (titers ≥ 1:100) than in those RV-SIg- (titer < 1:100) when vaccinees
and placebo recipients were analyzed jointly (chi-square test p < 0.05). cCorrelation between RV-SIg titers and protection (Spearman test p < 0.05, rho =
0.22) when vaccinees and placebo recipients were analyzed jointly.
No. 16-10-01) (70 μl of 1/1,000 dilution), SP, and plates were
developed and analyzed as described above. The concentration
of total SIgA in the plasma pool, used as a positive control, was
interpolated from a standard curve generated with purified SIgA
from human colostrum (AbD Serotec, Cat. No. PHP133). The
corresponding concentration for each plasma sample tested was
in turn interpolated from the plasma pool curve using a fourparameter logistic-log function.46
ELISA for measuring plasma RV-IgM
RV-IgM ELISA was performed as previously described,24
with minor modifications. Briefly, 96-well Immulon 2 microtiter ELISA plates (Dynex Technologies) were coated overnight at
4°C with Goat F(ab’)2 anti-human IgM (Invitrogen, Cat. No.
AHI1601) (70 μl of 1/500 dilution). The plates were then blocked
and incubated with plasma samples diluted in 5% blotto. The
remaining steps of the assay are as described for plasma RV-SIg
ELISA. Samples were considered as positive using the same criteria previously described.
ELISA for measuring plasma RV-IgG
Briefly, and as previously described with minor modifications,24 plates were coated with either a supernatant from RF
virus-infected MA104 cells or the supernatant of mock-infected
cells (negative control) and incubated overnight at 4°C. After
blocking, serial dilutions of plasma samples were deposited in
each well. After incubation, the following sequence of reagents
was added: biotin-labeled goat anti-human IgG (KPL, Cat. No.
16-10-06) (70 μl of 1/1000 dilution), SP, and plates were developed and analyzed as described. Serial dilutions of a pool of plasmas from children with RV-IgG was used as a positive control
and a plasma from a child without evidence of previous RV infection (RV-IgA-) and without RV-IgG from placental transfer of
maternal IgG antibodies was used as a negative control.
Recombinant human SC and competitive binding assays
Recombinant human secretory component (rhSC) was
obtained as previously described,47 and was used to ensure the
activity of the capture antibody in the SIg ELISAS. Bovine serum
albumin (Merck, 1120180100) (used as a negative control) or
rhSC was added in 1/2 serial dilutions (starting from 6.1 μg/ml
onwards) after the blocking step, and incubated for 10 min at
37°C. Next, purified SIgA was added at a concentration of 0.076
μg/ml (the concentration of SIgA present in a 1/200 dilution of
the positive control plasma pool), plates were incubated for 2 h
and the assay continued as described above. A similar strategy
was used for RV-SIg, using a dilution of plasma (1/200) that gave
a sub-saturating signal in the ELISA.
Statistical analyses
Analysis was performed with SPSS software version 20.0 (IBM
Inc.) and with GraphPad Prism version 6 for Mac. Differences
between groups were evaluated with nonparametric Wilcoxon,
Mann–Whitney, Fisher exact probability or chi-square tests, as
required. Correlations were evaluated using Spearman’s test.
Significance was established if p ≤ 0.05, in 1 tailed tests.
Disclosure of Potential Conflicts of Interest
JA and MF were co-principal investigators for a trial of the
RIX4414 rotavirus vaccine (the precursor to the Rotarix vaccine)
www.landesbioscience.comHuman Vaccines & Immunotherapeutics2415
©2013 Landes Bioscience. Do not distribute.
Details about the vaccine trial and the strategy for clinical evaluation of protection were previously published.9 Briefly, from the
moment infants received their second dose of vaccine/placebo,
they were contacted every 2 weeks until they were 13 mo old to
identify cases of GE. Of the 319 children who received two doses
of vaccine/placebo, a subgroup of 119 was randomly selected for
immunological assessments (50 vaccinees and 69 placebo recipients). Plasma samples from all these children, except seven placebo recipients (children in whom the informed consent form
did not authorize further studies), were included in the present
studies. Plasma samples were collected 14–16 d after receiving
each dose of RIX4414 or placebo.
All plasma samples from previous studies,9,18,19 which had
been stored at -80°C, were thawed, diluted in 50% glycerol and
preserved at -20°C for use. All assays, except competitive binding
assays, were blinded experiments.
ELISA for measuring plasma RV secretory immunoglobulin
96-well vinyl microtiter ELISA plates (Thermo Electron
Corporation, Cat. No. 2401) were coated with an anti-human
SC mAb (clone GA-1) (Sigma-Aldrich, Cat. No. I 6635) (70
μl of 1/10,000 dilution) and incubated overnight at 4°C. After
blocking with 5% blotto, serial dilutions of plasma samples in
2.5% blotto were deposited in each well. After incubation, samples were discarded and 1/10 dilutions of a supernatant from
RF (Bovine RV strain P6[1]G6, 107 focus forming units/ml)
virus-infected MA104 cells or the supernatant of mock-infected
cells (negative control) were added. Of note, most antibodies detected with this antigen are specific for the major capsid protein VP6, which contains group- and subgroup-specific
antigenic determinants and exhibits a high level of sequence
conservation.44,45 Then, the following sequence of reagents was
added: guinea pig anti-rhesus RV hyperimmune serum (70 μl
of 1/4000 dilution); biotinylated goat anti-guinea pig serum
(Vector Laboratories, Cat. No. BA-7000) (70 μl of 1/2,000
dilution); peroxidase-labeled streptavidin (SP) (Kirkegaard
and Perry Laboratories [KPL], Cat. No. 14-30-00) (70 μl of
1/1,000 dilution) and tetramethyl benzidine substrate (SigmaAldrich, Cat. No. 50-76-00). The reaction was stopped by the
addition of 17.5 μl 2 M sulfuric acid. Absorbance was read at
a wavelength of 450 nm on an ELISA plate reader (Multiskan
EX; Thermo Labsystems). Serial dilutions of a pool of plasmas
from children with RV-SIg was used as a positive control and a
plasma from a child without evidence of previous RV infection
(RV-IgA-) was used as a negative control in each plate. Samples
were considered positive if the optical density in the experimental wells was > 0.1 units and 2-fold greater than the optical density in the corresponding negative control wells. To be accepted
for analysis, the titer of the positive control plasma could not
differ by more than one dilution from plate to plate.
ELISA for measuring total plasma secretory IgA
To detect total SIgA, a sandwich ELISA was developed using
a previously described approach.43 Briefly, plates were coated with
the anti-human SC mAb or PBS (Gibco, Cat. No. 21600-069)
(negative control). After blocking, serial dilutions of plasma samples were applied in each well. The following sequence of reagents
was then added: biotin-labeled goat anti-human IgA (KPL, Cat.
Acknowledgments
This publication was financed by the Pontificia Universidad
Javeriana (ID4215). We thank the subjects who participated in
the previous studies for their generosity, the personnel of the
Pediatrics Department of the San Ignacio Hospital for their help
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36. Eijgenraam JW, Oortwijn BD, Kamerling SWA, de
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Landes Bioscience
www.landesbioscience.com
Supplemental Material to:
Daniel Herrera, Camilo Vásquez, Blaise Corthésy, Manuel A
Franco, and Juana Angel
Rotavirus specific plasma secretory immunoglobulin
in children with acute gastroenteritis and children
vaccinated with an attenuated human rotavirus vaccine
2013; 9(11)
http://dx.doi.org/10.4161/hv.25610
www.landesbioscience.com/journals/vaccines/article/25610
Supplementary Figure 1. RV-IgG in vaccinees and placebo recipients
Data for 50 vaccinees and 62 placebo recipients after dose 2 (D2) are shown.
Lines and error bars denote the mean and SEM, respectively. Differences
between vaccinees and placebo recipients, as well as between protected and
non-protected children, were evaluated with the Mann–Whitney test. All p
values reported are 1-tailed. A. and B. The reported values correspond to the
log10 of the inverse titer measured by ELISA.
1 Table S1. Demographic and clinical data from children with acute GE (n = 36).
Group Gender Agea Breast
Diarrheab RV
feeding
- RV Agd
RV
IgAc
Feces Plasma Genotype
A
M
9
No
7
Neg
Neg
Neg
Neg
A
F
6
Yes
5
Neg
Neg
Neg
Neg
A
F
13
Yes
3
Neg
Neg
Neg
Neg
A
M
14
Yes
5
Neg
Neg
Neg
Neg
A
M
7
No
8
Neg
Neg
Neg
Neg
B
M
13
Yes
8
100
Neg
Neg
Neg
B
M
10
No
10
100
Neg
Neg
Neg
B
F
21
No
5
100
Neg
Neg
Neg
B
F
22
Yes
4
400
Neg
Neg
Neg
B
M
16
No
5
800
Neg
Neg
Neg
B
F
8
No
4
200
Neg
Neg
Neg
B
M
16
Yes
4
800
Neg
Neg
Neg
B
F
10
Yes
4
100
Neg
Neg
Neg
B
M
14
Yes
8
3200
Neg
Neg
Neg
B
M
15
No
5
400
Neg
Neg
Neg
B
M
15
No
5
200
Neg
Neg
Neg
B
M
11
Yes
12
3200
Neg
Neg
Neg
B
M
14
Yes
7
51200 Neg
Neg
Neg
B
M
20
No
4
50
Neg
Neg
Neg
B
F
11
No
3
3200
Neg
Neg
Neg
P
1 B
F
15
Yes
4
800
Neg
Neg
Neg
B
M
17
No
2
3200
Neg
Neg
Neg
B
F
14
Yes
10
800
Neg
Neg
Neg
B
M
14
Yes
3
400
Neg
Neg
Neg
B
M
8
Yes
4
200
Neg
Neg
Neg
C
M
11
No
3
Neg
39
Neg
P[6]
C
F
15
Yes
2
Neg
166.5
73.9
P[8]
C
F
21
No
2
Neg
87.8
90
P[4]
C
F
20
No
10
Neg
108.2
112.6
P[4]
C
M
6
Yes
3
Neg
104
23.8
P[8]
C
M
11
Yes
2
Neg
100
48.3
P[8]
C
M
14
Yes
7
Neg
103.4
33
P[8]
C
F
12
Yes
1.25
800
19
Neg
P[8]
C
M
14
No
10
1600
87
Neg
P[8]
C
M
21
Yes
3
200
94.4
Neg
P[4]
Ce
F
10
No
3
100
87.4
Neg
P[8]
2 a
3 respectively: 9.8 (1.59); 14.2 (0.87); 14.09 (1.46)]. b Days after onset of diarrhea at
4 the time of blood drawing [Mean and (SEM) for groups A, B and, C, respectively:
5 5.6 (0.87); 5.55 (0.6); 4.2 (0.97)]. c Inverse of RV-IgA titer. d % of positive control.
6 Neg: negative.
7 RV infection (RV-IgA+).
Age of the children in months [Mean and (SEM) for groups A, B and, C,
e
The last four children of group C are individuals with secondary
8 9 2 10 Table S2. Correlation between RV-IgA titers after D2 and protection against
11 any RV GE
RV-IgA Titers
Vaccinees* (%)
after D2
Placebo
Vaccinees+placebo
recipients* (%)
recipients* (%)
<100
23/25 (92)
44/64 (68.75)a
67/89 (75.28)
100
9/10 (90)
1/1 (100)
10/11 (90.90)b
200
7/8 (87.5)
1/1 (100)
8/9 (88.88)
400
3/3 (100)
1/1 (100)
4/4 (100)
800
2/3 (66.66)
1/1 (100)
3/4 (75)
≥1600
1/1 (100)
1/1 (100)
2/2 (100)c
12 *Shown is the number of protected vaccinees, placebo recipients or vaccinees and
13 placebo recipients analyzed jointly / the total number of children in each population
14 for each RV-IgA titer.
15 a
16 vaccinees than in RV-IgA- placebo recipients (Fischer exact probability test p <
17 0.05).
18 b
19 children (titers ≥ 1:100) than in those RV-IgA- (titer < 1:100) when vaccinees and
20 placebo recipients were analyzed jointly (chi-square test p > 0.05).
21 c
22 0.152) when vaccinees and placebo recipients were analyzed jointly.
23 Data are from reference 8 and include the seven placebo recipient children not
24 analyzed for RV-SIg. Similar results are obtained if they are excluded.
The frequency of protected children is significantly higher in RV-IgA- (titer <100)
The frequency of protected children was not significantly higher in RV-IgA+
Correlation between RV-IgA titers and protection (Spearman test p = 0.05, rho =
3 25 Table S3. Correlation between RV-IgG titers after D2 and protection against
26 any RV GE
RV-IgG Titers
Vaccinees* (%)
after D2
Placebo
Vaccinees+placebo
recipients* (%)
recipients* (%)
800
0/0
2/3 (66.66)
2/3 (66.66)
1600
3/3 (100)
12/14 (85.71)
15/17 (88.23)
3200
7/7 (100)
8/12 (66.66)
15/19 (78.94)
6400
19/21 (90.47)
12/18 (66.66)
31/39 (79.48)
12800
13/15 (86.66)
10/12 (83.33)
23/27 (85.18)
51200
2/3 (66.66)
2/2 (100)
4/5 (80)
102400
0/0
1/1 (100)
1/1 (100)
204800
1/1 (100)
0/0
1/1 (100)
27 *Shown is the number of protected vaccinees, placebo recipients or vaccinees and
28 placebo recipients analyzed jointly / the total number of children in each population
29 for each RV-IgG titer.
30 Plasma samples are the same analyzed for RV-SIg.
4 6.2 Introduction to the article: Simultaneous Assessment of RotavirusSpecific Memory B Cells and Serological Memory after B Cell Depletion
Therapy with Rituximab
RV viremia is directly related to antigenemia, which is a common finding in acutely
infected children with RV, independent from the existence of diarrhea. This
appears to be very important because antigen load is inversely related to serum
RV-IgA and RV-IgG titers [15]. Moreover, it was recently shown that RV infected
children with RV RNA and RV antigen in both stool and serum were more
susceptible to have severe symptoms [241]. Also, it was concluded that RV
infection is occasionally accompanied by mild hepatitis [67] and frequently by a
detectable increase of hepatic transaminases [68].
Immune mediators of RV clearance from the systemic compartment in humans
have not been thoroughly identified. In mice, both T and B cells are important, but
none of them is absolutely required. Nevertheless, serum RV-Abs alone are
effective to delay RV antigenemia appearance, and they are sufficient to
temporarily reduce antigenemia in chronically infected SCID mice [28].
Therefore, although local mucosal immunity is crucial in the human immune
response against RV [13], the involvement of the systemic compartment by RV
infection suggests that the systemic immune response could also be very
important. In this regard, the study of circulating RV-mBc and their relation with
serological memory seems relevant to better understand the immune response
against RV, and thus improve the basis for finding better correlates of protection
after vaccination [31, 32].
Patients with autoimmune diseases treated with RTX constitute a unique
opportunity to evaluate the relation of antigen-specific mBc with antigen-specific
serological memory in humans. It provides the case in which one of the
components, circulating mBc, is temporarily absent, so its effect on the other
component, serological memory, can be assessed. Additionally, autoimmunity is a
condition in which short-lived PC seem to be important for the maintenance of
serological memory [35], in contrast to what is considered in healthy adults [30,
33]. Even though pathogen-specific IgG Abs and auto-Abs of different isotypes
have been evaluated in patients with autoimmunity treated with RTX [37-40],
antigen-specific mBc and antigen-specific Abs of different isotypes have not been
simultaneously assessed.
Furthermore, RV has several distinguishing features that make it a relevant model
to study the relationship between mBc and serological memory: 1. It is one of the
few models with tools that permit the characterization of antigen-specific mBc in
humans. 2. Circulating RV-specific Bc seem to be peculiar: a group of naïve Bc
bind RV- VLPs [41, 42], and RV-mBc are enriched in the CD27+IgM+ and in the
CD27-IgG+ mBc subsets [43, 44], subpopulations important in autoimmune
46
diseases pathogenesis [45-48]. 3. The relationship between RV-mBc and
serological memory appears to be somewhat different from that of TT-mBc, one of
the most studied antigens concerning the maintenance serological memory [43].
We, thus, hypothesized that RV-mBc are distributed in a distinct manner in patients
with autoimmune diseases and are, therefore, related to serological memory in a
peculiar way, dissimilar from the proposed manner for pathogens studied thus far.
To evaluate this hypothesis, circulating total, RV- and TT-mBc and Abs, and some
auto-Abs, were evaluated in patients with autoimmunity before and after RTX
therapy, and in sex and age-matched healthy adults.
We found that the relative contribution of each mBc subset to RV-mBc was,
overall, comparable between healthy volunteers and patients before RTX
treatment. We also present direct evidence that in the absence of circulating TTand RV-mBc the corresponding antigen-specific serological memory remains
steady, irrespective of the Ab isotype and subclass studied here and, therefore,
seems to be maintained by long-lived PC unaffected by RTX. In contrast, shortlived PC seem to maintain at least a fraction of IgM-RF, IgG-anti-CCP, and IgGanti-dsDNA auto-Abs, as well as a high proportion of the total IgM pool, which are
probably in equilibrium with auto-Ag-specific mBc depleted by RTX.
47
PLOS ONE
Simultaneous Assessment of Rotavirus-Specific Memory B Cells and Serological
Memory after B Cell Depletion Therapy with Rituximab
--Manuscript Draft-Manuscript Number:
PONE-D-13-48893
Article Type:
Research Article
Full Title:
Simultaneous Assessment of Rotavirus-Specific Memory B Cells and Serological
Memory after B Cell Depletion Therapy with Rituximab
Short Title:
RV-Serological Memory after Rituximab Theraphy
Corresponding Author:
Manuel Antonio Franco, M.D. Ph.D.
Pontificia Universidad Javeriana
Bogota, COLOMBIA
Keywords:
serological memory; memory B cells; Rituximab; autoimmune diseases; rotavirus
Abstract:
The mechanisms that contribute to the maintenance of serological memory are still
unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM+ and CD27subpopulations, which are associated with autoimmune diseases pathogenesis. In
patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies
(auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs
remain unchanged. Thus, maintenance of autoimmune and pathogen-specific
serological memory may depend on the type of antigen and/or Ab isotype evaluated.
Antigen-specific mBc and antigen-specific Abs of different isotypes have not been
simultaneously assessed in patients after RTX treatment. To study the relationship
between mBc subpopulations and serological memory we characterized total, RV- and
tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune
diseases before and after treatment with RTX. We also measured total, RV- and TTAbs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively.
Minor differences were observed between the relative frequencies of RV-mBc in
healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc
and total, RV- and TT-specific mBc [IgM+, switched (IgA+/IgG+), IgM+ only, IgD+ only,
and CD27- (IgA+/IgG+/IgM+)] were significantly diminished. An important decrease in
total plasma IgM and minor decreases in total IgG and IgA levels were also observed.
IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly
diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained
stable.
In conclusion, serological memory against RV and TT seem to be maintained by longlived plasma cells, unaffected by RTX, and an important proportion of total IgM and
serological memory against some auto-antigens seem to be maintained by short-lived
plasma cells, dependent on mBc precursors depleted by RTX.
Order of Authors:
Daniel Herrera
Olga Lucía Rojas
Carolina Duarte
Rubén Darío Mantilla
Juana Ángel
Manuel Antonio Franco, M.D. Ph.D.
Suggested Reviewers:
Jennifer H Anolik, MD, PhD
University of Rochester Medical Center, School of Medicine and Dentistry
[email protected]
Her research interests include the role of B cells in the pathophysiology of human
systemic lupus. She has been one of the pioneers in the use of B-cell depletion for the
therapy of autoimmune diseases and investigation of the effects of B cell depletion on
immune function in SLE patients.
Baoming Jiang, D.V.M, PhD
Centers for Disease Control and Prevention
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Simultaneous Assessment of Rotavirus-Specific Memory B Cells and
1 2 Serological Memory after B Cell Depletion Therapy with Rituximab
3 4 Daniel Herrera1, Olga L. Rojas2, Carolina Duarte-Rey3, Rubén D. Mantilla3,
5 Juana Ángel1 and Manuel A. Franco1*.
7 1
6 8 9 Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad
Javeriana, Bogotá, Colombia. 2Unidad de Inmunología, Escuela de Medicina y
Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia. 3Riesgo de
10 Fractura S.A. - CAYRE I.P.S, Bogotá, Colombia.
12 *Correspondence to: Dr. Manuel A. Franco, Instituto de Genética Humana,
14 Phone: 57-1-3208320 Ext 4077. Fax: 57-1-2850356.
11 13 Pontificia Universidad Javeriana, Carrera 7 # 40-62, Bogotá, Colombia.
15 1 16 ABSTRACT
18 still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM+ and
17 The mechanisms that contribute to the maintenance of serological memory are
19 CD27- subpopulations, which are associated with autoimmune diseases
21 (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other
23 maintenance of autoimmune and pathogen-specific serological memory may
20 pathogenesis. In patients with autoimmune diseases treated with Rituximab
22 auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus,
24 depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific
26 simultaneously assessed in patients after RTX treatment. To study the
25 mBc and antigen-specific Abs of different isotypes have not been
27 relationship between mBc subpopulations and serological memory we
29 in patients with autoimmune diseases before and after treatment with RTX. We
31 nephelometry, ELISA, and EliA tests, respectively. Minor differences were
33 patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV-
35 CD27- (IgA+/IgG+/IgM+)] were significantly diminished. An important decrease in
37 observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were
39 titers remained stable.
28 characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry
30 also measured total, RV- and TT-Abs, and some auto-Abs by kinetic
32 34 observed between the relative frequencies of RV-mBc in healthy controls and
and TT-specific mBc [IgM+, switched (IgA+/IgG+), IgM+ only, IgD+ only, and
36 total plasma IgM and minor decreases in total IgG and IgA levels were also
38 significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG
2 40 In conclusion, serological memory against RV and TT seem to be maintained
42 total IgM and serological memory against some auto-antigens seem to be
44 by RTX.
41 43 by long-lived plasma cells, unaffected by RTX, and an important proportion of
maintained by short-lived plasma cells, dependent on mBc precursors depleted
45 3 46 Introduction
48 can persist for decades, or in some cases for a lifetime, in the apparent
50 first line of defense against reinfection by many microorganisms [2], and critical
52 identified for several vaccines [3]. Additionally, in autoimmune diseases,
54 activity and pathogenesis [4] and in some cases predict disease severity [5-7].
47 Pathogen-specific protective IgG levels following natural infection or vaccination
49 absence of the antigen [1]. This serological memory provides the host with a
51 pathogen-specific antibody (Ab) titers that correlate with protection have been
53 55 autoantibodies (auto-Abs) of different isotypes are associated with disease
The mechanisms that contribute to the maintenance of serological memory in
56 healthy individuals are still unclear and, in general, have been studied only with
58 adults, IgG serological memory seems to be maintained by long-lived plasma
57 respect to the IgG isotype and for a limited number of antigens. In healthy
59 cells (PC), independently of memory B cells (mBc) [1,8]. Two non-mutually
61 [9]: 1) long-lived PC reside in a limited number of survival niches in the bone
63 competence to respond to survival signals of these niches, which will finally
65 lifespan of PC is related to the integrated signals through the B-cell receptor,
67 through CD4 T-cell help, and therefore, is imprinted at the time of the immune
69 independently regulated populations [11].
60 exclusive theories have been proposed to explain the survival of long-lived PC
62 marrow or secondary lymphoid organs. Plasmablasts may or may not gain the
64 determine their lifespan as long-lived PC or short-lived PC [8,10]. 2) The
66 which largely depend on the antigen repetitive nature, and signals obtained
68 response induction [9]. This theory proposes that PC and mBc represent
4 70 However, under certain circumstances, such as autoimmunity, short-lived PC,
71 which need to be continuously replenished by mBc, may also contribute to
73 PC contribute to serological memory, a correlation is expected between
75 serological memory [13].
77 treated with B-cell depletion therapy using Rituximab® (RTX), an anti-CD20
72 maintain serological memory (see below) [12]. In conditions where short-lived
74 numbers of circulating antigen-specific mBc and levels of antigen-specific
76 Serological memory has been evaluated in patients with autoimmune diseases
78 monoclonal Ab that depletes circulating Bc and leaves PC unaffected. After Bc
80 decrease, but within normal ranges [14]. In contrast, IgG Ab titers against
82 polysaccharide [16] remain constant. In regard to auto-Abs results differ: on the
84 anti-C1q [16], both of IgG isotype, and IgA-, IgG-, and IgM-class rheumatoid
86 auto-Abs against Ro52, Ro60, and La44, also of IgG isotype, remain
88 of maintenance of serological memory could depend on the type of antigen
79 depletion with one RTX cycle total IgA, IgG, IgM, and IgE levels significantly
81 pathogens such as measles [14], tetanus [15], and pneumococcal capsular
83 one hand, it has been reported that anti-double-stranded DNA (dsDNA) and
85 factors (RF) diminish significantly after RTX therapy [17]. On the other hand,
87 unchanged after RTX therapy [14]. These results suggest that the mechanism
89 and/or Ab isotype evaluated, particularly in patients with autoimmune disease.
91 have not been simultaneously assessed in patients that received B-cell
93 In a recent study, we characterized circulating RV-specific B cells (RV-Bc
90 However, antigen-specific mBc and antigen-specific Abs of different isotypes
92 depletion therapy with RTX.
94 identified by their capacity to bind GFP-coupled virus like particles [VLPs]) and
5 95 tetanus toxoid-specific B cells (TT-Bc identified by their capacity to bind biotin
97 volunteers [18]. Compared with TT-Bc, RV-Bc seem to be peculiar because a
99 CD27+IgM+ (which includes the CD27+IgD+IgM+ subset) and in the CD27-IgG+
101 RV-IgA plasma levels, but a correlation between CD27+IgG+ RV-mBc and RV-
96 labeled TT) and assessed their relation with serological memory in healthy
98 group of naïve Bc bind RV-VLPs [19,20], and RV-mBc are enriched in the
100 mBc subsets [18,21]. In addition, CD27+IgA+ RV-mBc correlated positively with
102 IgG was absent. In contrast, CD27+IgG+ TT-mBc correlated with TT-IgG plasma
104 seems to be somewhat different from that of TT-mBc, making it a relevant
106 CD27+IgD+IgM+ mBc (IgM+ mBc) and CD27- mBc, the mBc subsets in which
108 mBc have been shown to be decreased in patients with systemic lupus
110 Syndrome [24]. Moreover, there is a negative correlation between the
112 patients [22]. In RA patients IgM+ mBc seem to migrate to the synovial
103 levels. Therefore, the association between RV-mBc and serological memory
105 model to study the relationship between mBc and serological memory.
107 RV-mBc are enriched, are relevant in autoimmune diseases pathogenesis. IgM+
109 erythematosus (SLE) [22], rheumatoid arthritis (RA) [23], and Sjögren’s
111 circulating number of IgM+ mBc and auto-Abs levels and disease activity in SLE
113 membrane in a tumour necrosis factor dependent manner [23]. In contrast,
115 with disease activity [25]. Since RV-mBc are enriched in these subsets, such
114 circulating CD27- mBc are increased in SLE patients and positively correlate
116 cells could be related to serological memory in a unique manner and we
118 autoimmunity.
117 hypothesized they could be distributed in a peculiar manner in patients with
6 119 Here, total, RV- and TT-specific B-cell subsets were characterized in patients
121 and in healthy individuals. Total immunoglobulins (Igs), RV- and TT-Abs, and
120 with autoimmune diseases before and after B-cell depletion therapy with RTX,
122 some auto-Abs were also measured in the same timeframes. Titers of RV-IgA,
124 In contrast, IgM RF, IgG against cyclic cytrullinated peptide (CCP), and IgG
123 RV-IgG, and RV-IgG1, as well as TT-IgG, remained stable after RTX treatment.
125 against dsDNA were significantly diminished, as well as total Igs, especially
127 by long-lived PC, unaffected by RTX, and an important proportion of total IgM
129 short-lived PC, dependent on mBc depleted by RTX.
131 Materials and Methods
133 Written informed consent was obtained from each adult volunteer. Studies were
135 Universidad Javeriana and conducted in accordance with the guidelines of the
137 Pools of plasma samples from children enrolled in prior published studies [26-
126 128 130 total IgM. Thus, serological memory against RV and TT seem to be maintained
and serological memory against some auto-antigens seem to be maintained by
132 Ethics Statement
134 approved by the Ethics Committee of the San Ignacio Hospital and Pontificia
136 Helsinki Declaration.
138 28] in whom the informed consent form (also approved by the Ethics Committee
140 their use in unrelated research studies were used as positive controls in some
139 of the San Ignacio Hospital and Pontificia Universidad Javeriana) authorized
141 experiments.
143 Subjects
142 7 144 Fourteen patients, twelve females and two males, nine of them diagnosed with
145 RA and five with SLE according to the American College of Rheumatology
147 disease activity, despite treatment with disease-modifying anti-rheumatic drugs
149 score of 28 joint counts (DAS28) or SLE Disease Activity Index (SLEDAI),
151 selected to receive RTX by their treating rheumatologist. Additional clinical
153 included: lupus nephritis (n = 2), autoimmune thrombocytopenia (n = 1), overlap
155 regimen included two infusions of intravenous RTX (1,000 mg), 14 days apart,
157 median age at RTX treatment was 46 years (range 26 – 69) and the median
159 patients had been diagnosed (within three years or less). Clinical follow up
146 international criteria [29,30] were included. All patients had moderate or high
148 or standard immunosuppressive therapy, measured by the disease activity
150 respectively. Given the failure to standard treatment regimens, they were
152 manifestations considered to use RTX as the treatment of choice in patients
154 of RA and SLE (n = 4), and antiphospholipid syndrome (n = 2). The treatment
156 in combination with intravenous methylprednisolone (100-250 mg) [31,32]. The
158 disease duration to the time of RTX treatment was 3 years (range 1 – 30); eight
160 could be done in eleven patients within the following six months post B-cell
162 clinical improvement. Supplementary Table S1 describes accompanying
164 treatment, baseline disease activity, and clinical follow up. Ten age and sex
161 depletion therapy. Out of these, seven patients showed subjective and objective
163 autoimmune diagnoses, concomitant and relevant previous pharmacologic
165 matched healthy volunteers were used as controls.
167 Sample collection and processing
166 8 168 Peripheral blood mononuclear cells (PBMC) were isolated by LymphoSep (MP
170 samples from ten of the fourteen patients, described above, immediately before
172 samples were only available after RTX treatment), and from ten age and sex
174 volunteer after each blood draw, and plasma was collected and stored at −80
176 measurement.
178 Production of fluorescent virus like particles (VLPs)
180 using baculovirus expression vectors, as previously described [33]. Briefly, Sf9
182 infection greater than 5 PFU/cell. One baculovirus expressed RF (bovine RV)
184 fused to the N terminus of RF VP2 deleted in the first 92 amino acids. Infected
186 gradient centrifugation in CsCl. The optimal concentration of the RV VLPs for
188 Of note, RV VP6 is an immunodominant protein, and the majority of human RV-
190 infected animals and humans recognize the VP6 protein on the outer shell of
169 Biomedicals, Solon, OH) density-gradient centrifugation from heparinized
171 and four to six months after RTX infusion (in the remaining four patients PBMC
173 matched healthy controls. A complete blood count test was performed for each
175 °C for subsequent total and antigen-specific Igs assessment and auto-Abs
177 179 Fluorescent RV VLPs were a kind gift of Annie Charpilienne and were produced
181 cells were co-infected with 2 recombinant baculoviruses at a multiplicity of
183 VP6 and the other a fusion protein consisting of green fluorescent protein (GFP)
185 cultures were collected five to seven days post infection and purified by density
187 labeling of specific mBc was determined using PBMC from healthy volunteers.
189 specific B cells bind to VP6 [34]. Additionally, the majority of RV antibodies in
191 the VLPs [35].
192 9 193 Biotinylated - TT antigen
195 biotinylated using the EZ-Link Photoactivatable Biotin kit (Pierce Biotechnology,
197 [18], with minor modifications. A total of 750 µg of TT protein was incubated
199 under a UV (365 nm) lamp, and then dialyzed against PBS for 18 hours, to
201 Pierce, Illkirch, France). The optimal concentration of the biotinylated TT for
203 TT vaccinated volunteers, and the specificity of binding was evaluated with a
194 Tetanus Toxoid (Statens Serum Institute, Denmark) for in vitro tests was
196 Rockford, IL) according to manufacturer's instructions, as previously described
198 with biotin at a molar ratio of 20 mol of dye per mole of protein, for 20 min on ice
200 remove excess biotin, using a Slide-A-Lyzer dialysis cassette (Thermo Scientific
202 labeling of specific mBc was determined using PBMC from healthy and recently
204 competition assay using non-biotinylated TT (data not shown).
206 Flow cytometry assays
208 Gaithersburg, MD) and incubated with the GFP labeled VLPs (0.9 µg/test) or
210 temperature (RT). The cells were then washed with PBS – 1% bovine serum
212 Chemicals, Paris, KY) (staining buffer), and surface stained with Abs against
214 Jose, CA), IgD-HorizonTM V450 (IA6-2 clone; BD), CD27- phycoerythrin (PE)-
205 207 Fresh PBMC, 4 to 6 x 106, were washed twice with PBS (Gibco-BRL,
209 without this reagent (negative control) for 45 minutes in the dark, at room
211 albumin (Merck, Darmstadt, Germany), 0.02% sodium azide (Mallinckrodt
213 CD19-allophycocyanin (APC)-H7 (SJ25C1 clone; Becton Dickinson [BD], San
215 Cy7 (M-T271 clone; BD), goat anti-IgA-R-PE (Jackson ImmunoResearch, West
217 700 (145-8 clone; BD). Biotinylated-TT, or no reagent, was also added at this
216 Grove, PA), IgG-APC (G18-145 clone; BD) and custom-made IgM-Alexa Fluor
10 218 step and incubated for 30 minutes in the dark, at RT. Cells were then washed
220 peridinin chlorophyll protein (PerCP) (BD). Streptavidin-PerCP was also added
222 this reagent (negative control). After staining, the cells were washed and
224 window, were acquired on a FACSAria (BD) or LSRFortessa (BD) flow
226 intensity values among experiments run on different days and regardless of the
228 the cut-off between positive and negative cell populations for each marker [36].
219 with staining buffer and the biotinylated-TT was detected using streptavidin-
221 to the PBMCs without biotinylated-TT to assess the background generated by
223 resuspended in staining buffer. At least 200,000 B cells, gated on a CD19+
225 cytometer. Application settings were used to obtain constant fluorescence
227 flow cytometer used. Fluorescence minus one controls were used to determine
229 Of note, Abs against CD3/CD14-HorizonTM V500 (UCHT1 and M5E2 clones,
230 respectively; BD) were used as a dump channel in samples taken from patients
232 use, the dump channel was disregarded in the final analyses.
234 of CD19+ B cells/mL, calculated after background subtraction, based on the
236 Evaluable total B cell subpopulations were defined as those with ≥10 acquired
238 calculated considering the median CD19+ events acquired after RTX treatment,
231 after RTX treatment. However, since conclusions were similar with or without its
233 Results for total and antigen-specific mBc were expressed as absolute numbers
235 total lymphocyte numbers in the patient's complete blood count test results.
237 events [37]. The frequency, in terms of CD19+ B cells, of those 10 events was
239 and multiplied by the median absolute B cell count after RTX treatment. This
240 241 242 value, 3.5 CD19+ B cells/mL, corresponds to the dotted lines present in each
total B cell subpopulation of Figure 2 and represents the flow cytometry
detection limit for total B cell subpopulations. A common detection limit could
11 243 not be determined for antigen-specific mBc subsets due to the variable
245 mBc subsets with ≥10 acquired events, and at least two-fold greater than the
247 Flow cytometry analysis was performed using FlowJo software version 9.6.2 for
244 background. Nevertheless, the reported values correspond to antigen-specific
246 observed background.
248 Mac (Treestar, Ashland, OR).
250 Measurement of total immunoglobulins (IgA, IgG and IgM) and IgM
252 Plasma samples were thawed and simultaneously assessed by kinetic
254 Fullerton, CA), following the manufacturer’s instructions. Measuring ranges are
256 mg/dL); IgG: 200 – 3600 mg/dL (normal reference range values: 751 – 1560
249 251 Rheumatoid Factor
253 nephelometry on an IMMAGE® immunochemistry system (Beckman Coulter,
255 as follows: IgA: 40 – 700 mg/dL (normal reference range values: 82 – 453
257 mg/dL); IgM: 25 – 400 mg/dL (normal reference range values: 46 – 304 mg/dL);
258 and IgM rheumatoid factor (RF): 20 – 800 IU/mL (negative cut off value: <20
260 70-75%.
262 ELISAs for detection of RV-specific IgA, IgG, and IgG1 and TT-specific IgG
259 261 IU/mL), with a sensitivity level in RA patients reported by the manufacturer of
263 in plasma
265 modifications [18]: 96-well vinyl microtiter ELISA plates (Thermo Electron
267 infected MA104 cells or the supernatant of mock-infected cells (negative
264 RV and TT antibodies were assessed as previously described with minor
266 Corporation, Milford, MA) were coated with either a supernatant from RF virus-
12 268 control), for RV-specific ELISAs, or 0.5 µg/mL of TT or PBS (negative control),
270 dilutions of plasma samples were deposited in each well. After incubation, the
272 or IgG (Kirkegaard & Perry Laboratories [KPL], Gaithersburg, MD) or, biotin-
274 peroxidase (KPL) and tetramethyl benzidine substrate (KPL). Pools of plasma
276 were used as positive controls. Plasmas from a child without evidence of
278 used as negative controls. Samples were considered positive if the optical
280 optical density in the corresponding negative control wells. To be accepted for
282 one dilution from plate to plate.
284 Measurement of anti-CCP and anti-dsDNA autoantibodies (IgG isotype)
286 immunoassay (EliA test), following the manufacturer’s instructions, on a Phadia
288 has a measuring range of 0.4 to at least 340 U/mL, a negative cut off value of <
290 U/mL; the manufacturer’s reported clinical sensitivity and specificity are 87.8%
292 range of 0.5 to at least 400 IU/mL, a negative cut off value of < 10 IU/mL, an
269 for TT-specific IgG ELISA, and incubated overnight at 4°C. After blocking, serial
271 following sequence of reagents was added: biotin-labeled goat anti-human IgA
273 labeled mouse anti-human IgG1 (Sigma-Aldrich, St. Louis, MO); streptavidin-
275 samples from children with RV-IgA, or RV-IgG, and from adults with TT-IgG
277 previous RV infection (RV-IgA-) and from an adult negative for TT-IgG were
279 density in the experimental wells was > 0.1 units and two-fold greater than the
281 analysis, the titer of the positive control plasma could not differ by more than
283 285 Plasma samples were simultaneously assessed by a fluorescence enzyme
287 ImmunoCAP 100 system (Phadia AB, Uppsala, Sweden). The anti-CCP test
289 7 U/mL, an equivocal range of 7 – 10 U/mL, and a positive cut off value of > 10
291 and 96.7%, respectively, in RA patients. The anti-dsDNA test has a measuring
13 293 equivocal range of 10 – 15 IU/mL, and a positive cut off value of > 15 IU/mL; the
295 specificity of 93.2%.
297 Statistical analyses
299 GraphPad Prism version 6. Differences between groups were evaluated with
301 were evaluated with Spearman's test. When data followed a normal distribution,
302 303 tailed tests.
304 305 Results
307 and patients
309 CD27- mBc subpopulations [22,23,25], in which RV-mBc are enriched, we
311 volunteers (HV) and patients prior RTX treatment (prior-RTX) by flow cytometry
313 enrichment of RV-mBc was found in the IgMhiIgDlow subset in HV and patients
294 296 manufacturer’s reported sensitivity for active SLE is 70.8% and clinical
298 Analysis was performed with SPSS software version 20.0 (IBM Inc.) and with
300 nonparametric Mann–Whitney and Wilcoxon tests, as required. Correlations
306 a Student’s t-test was applied. Significance was established if p < 0.05, in 2
RV-mBc are enriched in the circulating IgM+ mBc subsets in both controls
308 Since patients with RA and SLE show altered frequencies in the IgM+ mBc and
310 compared the relative frequencies of total and RV-Bc subsets in healthy adult
312 (see analysis strategy in Supplementary Figure S1A). As expected [34], an
314 prior-RTX (Figure 1A and 1B, respectively). Additionally, an enrichment of RV-
315 mBc was also found in the IgMlowIgDhi mBc subset in both HV and patients
317 enrichment of RV-naïve Bc was observed in patients prior-RTX (Figure 1B).
316 prior-RTX, and in the IgM+ only mBc subset in HV (Figure 1A). An apparent
14 318 An enrichment of RV-mBc was not detected in the CD27- mBc subset in HV and
319 patients prior-RTX (Figure 1A and 1B, respectively). However, in agreement
321 mBc, RV-mBc were enriched in the CD27-IgG+ subset in HV, but not in patients
320 with previous results [18], when the analysis was done in terms of the CD27-
322 prior-RTX (data not shown).
323 324 similar between HV and patients prior-RTX.
325 326 Total and antigen-specific Bc subsets are significantly decreased after B-
328 Patients with autoimmune diseases treated with RTX represent a unique
330 humans, because it provides a scenario in which one of the components,
332 serological memory, can be evaluated. Therefore, we compared the absolute
334 and ten patients before and after RTX treatment (see analysis strategy in
Thus, the relative contribution of each mBc subset to RV-mBc was, in general,
327 cell depletion therapy with RTX
329 opportunity to assess the relation between mBc and serological memory in
331 circulating mBc, is temporarily absent, so its effect on the other component,
333 numbers (CD19+ B cells/mL) of total, RV- and TT-specific Bc subsets in ten HV
335 Supplementary Figure S1B).
337 prior-RTX. As shown in Figure 2, IgM+ mBc, IgM+ only mBc, and switched IgG+
339 same was observed for IgM+ mBc IgMhiIgDlow and IgMlowIgDhi, IgD+ only mBc,
336 First, we compared circulating total mBc subsets present in HV and patients
338 mBc were significantly decreased in patients prior-RTX, compared with HV. The
340 and switched IgA+ mBc (see Supplementary Figure S2).
342 and after-RTX. After RTX treatment, six of the ten patients studied had clinical
341 Second, we compared circulating total mBc subsets present in patients before
15 343 peripheral Bc depletion (less than 5,000 CD19+ cells/mL) (Figure 2). The four
345 peripheral Bc depletion (data not shown). In patients after-RTX a significant
347 Figure S2). The median decrease in all subsets, with respect to the
349 IgG+ mBc, which had a median decrease of 87% (data not shown).
351 patients before and after-RTX. RV-IgM+ mBc, RV-IgM+ only mBc, RV-switched
344 patients assessed by flow cytometry only after RTX treatment also had clinical
346 decrease in all total Bc subsets was observed (Figure 2 and Supplementary
348 pretreatment values, was above 96%, except for switched IgA+ and switched
350 Third, we compared circulating antigen-specific mBc subsets present in HV and
352 IgA+ mBc, and RV-switched IgG+ mBc were significantly decreased in patients
354 and IgMlowIgDhi, and RV-IgD+ only mBc were also significantly diminished (see
353 prior-RTX, compared with HV (Figure 3). In addition, RV-IgM+ mBc IgMhiIgDlow
355 Supplementary Figure S3). In patients after-RTX a significant decrease in all
357 Figure S3). The median decrease in all subsets, respect to the baseline values,
359 which had a median decrease of 92% and 93%, respectively.
361 antigen-specific-mBc subsets decreased in similar proportions. Of note, we
363 switched mBc [22,38]. In patients after-RTX both total and antigen-specific Bc
356 RV- and TT-specific Bc subsets was observed (Figure 3 and Supplementary
358 was above 98%, except for RV-switched IgA+ and RV-switched IgG+ mBc,
360 Taken together, these results showed that in patients prior-RTX total and
362 confirmed that patients prior-RTX have decreased IgM+ mBc subsets and
364 subsets are significantly diminished at similar levels.
366 Total plasma immunoglobulins and auto-Abs, unlike RV- and TT-Abs, are
365 367 significantly decreased after B-cell depletion therapy with RTX
16 368 To determine the effect of B-cell depletion therapy with RTX on the total
370 by kinetic nephelometry (Figure 4). Levels of total plasma Igs in patients prior-
372 patients prior-RTX (2-tailed Mann-Whitney’s test, p = 0.0578). As expected [14],
374 remained within normal ranges (Figure 4). Two patients had an increase in the
376 increase in the IgG level. Of note, excluding the patients who showed an
378 respect to the pretreatment values, was significantly greater than the
380 and p = 0.0093, respectively) (data not shown).
382 serological memory, we assessed IgM-RF and IgG-anti-CCP levels (relevant
369 serological memory, we measured the levels of total plasma IgA, IgG, and IgM
371 RTX and HV were similar; nevertheless, total IgM tended to be higher in
373 after RTX treatment total plasma Igs were significantly decreased, although
375 IgA level, one of them also in the IgM level, and another patient showed an
377 increase after RTX treatment, the total plasma IgM percentage decrease,
379 percentage decrease for total IgA and IgG (2-tailed Student’s t-test, p = 0.0031
381 To evaluate the effect of B-cell depletion therapy with RTX on autoimmune
383 auto-Abs in RA) and IgG-anti-dsDNA levels (relevant auto-Ab in SLE) in
385 in HV as controls. In four HV the levels of RF were just above the negative cut
387 (Figure 5B), and in two levels of anti-dsDNA were positive (Figure 5C).
389 dsDNA auto-Abs levels (Figure 5A, 5B, and 5C, respectively). Following RTX
391 In order to contrast the effect of B-cell depletion therapy on the levels of
384 patients with the corresponding diagnoses before and after RTX treatment, and
386 off value (Figure 5A), in all of them levels of anti-CCP auto-Abs were negative
388 Compared with HV, patients prior-RTX had higher RF, anti-CCP, and anti-
390 treatment, all auto-Abs levels were significantly diminished (Figure 5).
392 individual auto-Abs and their respective isotype of total plasma Ig, data after
17 393 RTX treatment were expressed as the percentage decrease respect to the
395 analyzed. The percentage drop in IgG-anti-dsDNA and IgG-anti-CCP was
397 0.0001 and p = 0.0378, respectively). There was no significant difference
399 As expected [15], and in contrast to the effect of B-cell depletion therapy on
401 constant after RTX treatment (Figure 6A). Similarly, RV-IgA (Figure 6B) and
403 titers because CD27-IgG+ mBc predominantly express the IgG1 and IgG3
405 seems to be a predominant RV-IgG subclass [40]. Plasma RV-IgG1 titers in
407 In conclusion, B-cell depletion therapy with RTX did not have an effect on
409 significantly decreased the auto-Abs assessed and the total plasma Igs,
394 pretreatment values. Only patients with an abnormal pretreatment value were
396 significantly higher than that in total plasma IgG (2-tailed Student’s t-test, p <
398 between the percentage drop in IgM-RF and total plasma IgM (data not shown).
400 total plasma Igs and the auto-Abs evaluated, plasma TT-IgG titers remained
402 RV-IgG (Figure 6C) remained unchanged. We further assessed the RV-IgG1
404 subclasses [39], RV-mBc are enriched in the CD27-IgG+ subset, and RV-IgG1
406 patients before and after RTX treatment were similar (Figure 6D).
408 pathogen-specific serological memory, irrespective of the isotype evaluated, but
410 especially total IgM.
412 Correlations between mBc subsets and serological memory
414 auto-Abs or Ag-specific Abs. All IgM+ mBc subsets correlated with total plasma
411 413 We next set to determine if selected mBc subsets could correlate with total Igs,
415 IgM (Table 1) only when data before and after RTX treatment in all patients
417 total switched IgG+ and CD27-IgG+ mBc correlated with anti-dsDNA levels.
416 were analyzed jointly. Likewise, in patients with SLE as their principal diagnosis,
18 418 TT-switched IgG+ mBc correlated with plasma TT-IgG titers in HV; in contrast,
420 plasma RV-IgA or RV-IgG titers were absent in HV and in patients (Table 2 and
419 correlations between RV-switched IgA+ and IgG+ mBc and their corresponding
421 data not shown).
423 Discussion
425 specific mBc after B-cell depletion therapy with RTX (Figure 3 and
427 6). In contrast, the auto-Abs measured were significantly diminished (Figure 5).
429 IgG (Figure 4 and data not shown). Moreover, when data before and after RTX
431 correlated (Table 1). To the best of our knowledge, this is the first time that
433 isotypes are determined in patients receiving B-cell depletion therapy with RTX,
435 plasma IgM is described in patients with autoimmunity.
422 424 Here, we show that despite a significant decrease of circulating RV- and TT-
426 Supplementary Figure S3), plasma RV- and TT- Abs remained constant (Figure
428 Similarly, total IgM decreased at significantly higher levels than total IgA and
430 treatment were analyzed jointly, IgM+ mBc subsets and total plasma IgM
432 circulating antigen-specific mBc and antigen-specific Abs of the IgA and IgG1
434 and the first time that a correlation between several IgM+ mBc subsets and total
436 The results presented here advance our knowledge of RV-mBc. We confirmed
438 that RV-mBc were also significantly enriched in the IgMlowIgDhi and IgM+ only
437 [18,21,34] that in HV RV-mBc are enriched in the IgMhiIgDlow subset and found
439 mBc subsets (Figure 1A), a tendency previously observed [34]. In contrast, in
441 1B) was absent, possibly because this subset was significantly decreased
440 patients prior-RTX enrichment of RV-mBc in the IgM+ only mBc subset (Figure
442 compared with HV (Figure 3). An enrichment of RV-naïve Bc was observed in
19 443 patients prior-RTX (Figure 1B). However, this may be simply because total and
445 compared with HV (Figure 2), or because naïve autoreactive B cells in patients
447 antigens (due to a deficiency in negative selection) [41].
449 mBc are heterogeneous: the IgMhiIgDlow subset resembles the spleen marginal
451 “innate” Ig responses to pathogens [42]. However, other IgM+ mBc subsets
444 446 448 RV-IgM+ mBc subsets were significantly diminished in patients prior-RTX
with autoimmune diseases may be more susceptible to cross-react with other
The enrichment of RV-mBc in all IgM+ mBc subsets in HV is intriguing. IgM+
450 zone B cells, may use a prediversified subset of Ig genes and participates in
452 seem to behave as “true” antigen selected mBc [43]. Similarly, the enrichment
453 of RV-mBc in the IgM+ only subset (at the expense of switched IgA+ or IgG+
455 mBc have undergone a less extensive maturation in germinal centers than
457 mice model, human CD27+IgM+ RV-mBc switch to IgG in vitro and in vivo, and
454 mBc), which has a lower frequency of somatic mutations [21], suggests that RV-
456 other mBc subpopulations. Besides, in an adoptive transfer immunodeficient
458 significantly reduce RV antigenemia and viremia [34]. Thus, further studies are
460 The response to RTX treatment is variable; it usually induces a very significant
462 of therapy [44]. Ten of the fourteen patients studied had clinical peripheral Bc
464 other four patients were also significantly diminished (Figure 2). Blood samples
466 respectively. Therefore, it is possible that RTX therapy had failed to completely
459 required to understand the repertoire of IgM+ RV-mBc.
461 reduction of circulating Bc subpopulations for six to nine months after one cycle
463 depletion by RTX (Figure 2 and data not shown). Levels of circulating Bc in the
465 from two of these four patients were taken 5 and 6 months after RTX treatment,
467 deplete them of circulating Bc or that they were already in the repopulation
20 468 phase post treatment. Regardless of the case, our conclusions are unaffected
469 by these findings.
471 subset of IgM-RF, IgG-anti-CCP, and IgG-anti-dsDNA auto-Abs are mainly
473 specific mBc depleted by RTX. In contrast, long-lived PC, unaffected by RTX
475 and RV, regardless of the antigen-specific isotype and subclass studied here.
477 probably reflects its differential effect on both auto-Abs and Abs against
479 Here, we present direct evidence that in the absence of circulating TT- and RV-
481 and therefore seems to be maintained by long-lived PC unaffected by RTX. In
483 memory is maintained by long-lived PC [45-47]. These and other findings
485 maintenance of serological memory in healthy individuals [12]. The existence of
487 been put forward to support this model [12]. However, antigen-specific mBc
489 these correlations do not necessarily imply a cause-and-effect relationship
491 switched IgG+ mBc correlated with plasma TT-IgG titers in HV (Table 2), these
470 Concerning serological memory, altogether our results suggest that at least a
472 maintained by short-lived PC, which are probably in equilibrium with auto-Ag-
474 treatment, probably maintain serological memory against pathogens, like TT
476 The small effect of B-cell depletion therapy on total IgA and IgG (Figure 4)
478 pathogens.
480 mBc the corresponding antigen-specific serological memory remains steady,
482 agreement with this finding, in some mouse models antigen-specific serological
484 [11,48,49] are at odds with the mBc bystander activation model of the
486 correlations between mBc and serological memory in healthy individuals has
488 correlate with serum antigen-specific Ab levels only in some cases [1], and
490 between the mBc and the corresponding Abs. Accordingly, although TT-
21 492 titers were stable after RTX treatment; thus, the latter correlation may simply
494 The lack of correlation between RV-Abs and RV-mBc may be related to the fact
496 repetitive nature and high avidity, can detect low affinity Bc [21] that produce
493 reflect equally stable but independently maintained parameters [1,11].
495 that the GFP-VLPs used to measure the RV-mBc, because of their antigen
497 antibodies undetected in the RV-ELISAs. Furthermore, a lack of correlation
499 maintenance of serological memory by long-lived PC. In our patients with
501 PC, but only a correlation between switched IgG+ mBc and CD27- IgG+ mBc
503 last correlation, and the lack of other expected correlations, must be analyzed
505 corresponding auto-Abs is lacking.
507 that the percentage drops in IgG-anti-dsDNA and IgG-anti-CCP were
509 suggest a differential effect of B-cell depletion therapy with RTX on the various
498 between mBc and serological memory does not necessarily indicate
500 autoimmunity, all auto-Abs studied seem to be mainly maintained by short-lived
502 with anti-ds-DNA auto-Abs in SLE patients was found (Table 2). However, this
504 with caution, because direct assessment of auto-antigen-specific mBc and the
506 As previously shown for IgG-RF and IgG-anti-CCP auto-Abs [17], we observed
508 significantly higher than the percentage drops in total plasma IgG. These results
510 types of IgG serological memory. Similarly, when IgA-RF and total IgA were
512 was significantly greater than that in total serum IgA [17].
514 pool may depend on short-lived PC, because the decrease effect of B-cell
511 assessed after B-cell depletion therapy with RTX, the decrease of the IgA-RF
513 In contrast to circulating total IgG and IgA, a high proportion of the total IgM
515 depletion therapy with RTX on IgM-RF and total plasma IgM was similar in this
516 and prior studies [17]. Accordingly, all IgM+ mBc subsets correlated with total
22 517 plasma IgM when data before and after RTX treatment were analyzed jointly
519 rapidly than total IgA and IgG after B-cell depletion therapy with RTX [14]. Of
518 (Table 1). Furthermore, total IgM levels have been reported to decrease more
520 note, in patients with autoimmune diseases the total IgM pool may be enriched
522 CD154, detected in some patients, have been associated to a rapid PC
524 in all volunteers (data not shown); thus, the effect of B-cell depletion therapy on
521 in Abs produced by short-lived PC, since the overexpression of IL-10 and
523 differentiation and auto-Ab production [50-52]. Plasma RV-IgM was undetected
525 pathogen-specific plasma IgM remains undetermined.
527 Mann-Whitney’s test, p = 0.0578) (Figure 4) all IgM+ mBc subsets were
529 S2). Even so, IgM+ mBc subsets correlated with total plasma IgM when data
526 Although total plasma IgM tended to be higher in patients prior-RTX (2-tailed
528 significantly diminished compared with HV (Figure 2 and Supplementary Figure
530 before and after RTX treatment were analyzed jointly (Table 1). Thus, it is
532 structures, may be related to circulating IgM+ mBc subsets and account for an
531 possible that PC producing autoimmune IgM, localized in ectopic lymphoid
533 important proportion of the total circulating IgM Abs. In support of this
535 patients [23], and its decreased frequency negatively correlates with auto-Abs
537 transgenic mouse model of RA, pathogenic B cells accumulate in lymph nodes
534 hypothesis, IgM+ mBc (CD27+IgD+IgM+) seem to migrate to the synovium in RA
536 levels and disease activity in SLE patients [22]. Moreover, in a human TNF
538 draining arthritic joints and are eliminated after B-cell depletion therapy with
539 RTX, with a concomitant disease improvement [53].
540 541 Conclusions
23 542 Serological memory against TT and RV, irrespective of the isotype and
544 B-cell depletion therapy with RTX. In contrast, short-lived PC, continuously
546 proportion of IgM Abs.
548 Acknowledgements
550 generosity, Elisa Navas for her help in contacting the patients, Maria Consuelo
552 Camilo Vásquez for assistance with Figure 1, and Marlee Neel for reviewing the
554 (ID3867).
543 545 547 subclass studied here, seems to be maintained by long-lived PC, unaffected by
replenished by mBc, seem to maintain some auto-Abs and an important
549 We thank the patients and healthy volunteers who participated for their
551 Romero for her assistance with the measurement of total Igs and auto-Abs,
553 manuscript. This work was supported by Pontificia Universidad Javeriana
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730 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 32 751 FIGURE LEGENDS
753 only mBc subsets.
755 multiparametric flow cytometry. All p values reported are 2-tailed (p < 0.05,
757 respectively. A. Healthy volunteers (n = 10). B. Patients (n = 10) with
759 Figure 2. Comparison of selected total B cell subpopulations among the
761 Healthy volunteers (HV) (n = 10), patients before RTX treatment (prior-RTX) (n
763 cells, the dashed line represents the clinical depletion limit after RTX treatment
765 cytometry detection limit of 3.5 CD19+ B cells/mL. Solid lines and error bars
767 HV and patients prior-RTX were evaluated with Mann–Whitney tests and
769 values reported are 2-tailed.
771 study groups.
773 respectively. Differences between HV (n = 10) and patients prior-RTX (n = 10)
752 Figure 1. RV-mBc are enriched in the IgMhiIgDlow, IgMlowIgDhi, and IgM+
754 Summary of the frequencies of seven subsets of total and RV-mBc assessed by
756 Wilcoxon test). Lines and error bars denote the median and interquartile range,
758 autoimmune diseases before RTX treatment.
760 study groups.
762 = 10) and patients after RTX treatment (after-RTX) (n = 10). For total CD19+ B
764 (less than 5,000 CD19+ cells/mL). The dotted lines represent the estimated flow
766 denote the median and interquartile range, respectively. Differences between
768 between patients prior-RTX and patients after-RTX with Wilcoxon tests. All p
770 Figure 3. Comparison of RV-specific B cell subpopulations among the
772 Solid lines and error bars denote the median and interquartile range,
774 were evaluated with Mann–Whitney tests and between patients prior-RTX and
33 775 patients after-RTX (n = 10) with Wilcoxon tests. All p values reported are 2-
777 Figure 4. Comparison of total immunoglobulins in plasma among the
779 Levels of total IgA (A), IgG (B) and IgM (C) in plasma determined by kinetic
781 normal reference range (IgA: 82 – 453 mg/dL; IgG: 751 – 1560 mg/dL; IgM: 46
783 range, respectively. Differences between HV (n = 10) and patients prior-RTX (n
776 tailed.
778 study groups.
780 nephelometry. The dotted lines represent the lower and upper values of the
782 – 304 mg/dL). Solid lines and error bars denote the median and interquartile
784 = 14) were evaluated with Mann–Whitney tests and between patients prior-RTX
786 tailed.
788 levels in plasma among the study groups.
785 and patients after-RTX (n = 14) with Wilcoxon tests. All p values reported are 2-
787 Figure 5. Comparison of RF, anti-CCP and anti-dsDNA autoantibodies
789 Levels of rheumatoid factor (RF) determined by kinetic nephelometry (A), anti-
791 dsDNA) (C) determined by a fluorescence enzyme immunoassay (EliA test) in
793 patients had both diagnoses). Solid lines and error bars denote the median and
795 limits below which a sample is considered negative according to the technique
797 IU/mL). Differences between HV and patients prior-RTX were evaluated with
799 with Wilcoxon tests. All p values reported are 2-tailed.
790 cyclic citrullinated peptide (anti-CCP) (B) and anti-doubled stranded DNA (anti-
792 patients diagnosed with RA (A and B) (n = 11) or with SLE (C) (n = 7) (some
794 interquartile range, respectively. The dotted lines correspond to the clinical
796 used for its detection (RF: <20 IU/mL, anti-CCP: <7 U/mL and anti-dsDNA: <10
798 Mann–Whitney tests and between patients prior-RTX and patients after-RTX
34 800 Figure 6. Comparison of RV- and TT-specific immunoglobulins titers in
802 Titers of TT-IgG (A), RV-IgA (B), RV-IgG (C) and RV-IgG1 (D) in plasma
804 interquartile range, respectively. Differences between HV (n = 10) and patients
801 plasma among the study groups.
803 determined by ELISA. Solid lines and error bars denote the median and
805 prior-RTX (n = 14) were evaluated with Mann–Whitney tests and between
806 patients prior-RTX and patients after-RTX (n = 14) with Wilcoxon tests.
807 35 808 809 810 Table 1. Memory B cell subsets that correlate with total immunoglobulins.
Cell subseta
P Valueb
Rho
Pt. IgM+ Only mBc with total plasma IgM
0.0013
0.6687
Pt. IgM+ mBc with total plasma IgM
0.0305
0.4842
Pt. IgM+ mBc IgMhi IgDlow with total plasma IgM
0.0148
0.5361
Pt. IgM+ mBc IgMlow IgDhi with total plasma IgM
0.0306
0.484
Pt. CD27- IgM+ mBc with total plasma IgM
0.0364
0.4702
a
All correlations for patients (Pt.) were established when data before and after
RTX treatment were analyzed jointly (n = 10).
811 b
813 Table 2. Memory B cell subsets that correlate with antigen-specific
812 814 815 816 817 Spearman’s two-tailed test.
immunoglobulins or autoantibodies
Cell subseta
P Valueb
Rho
HV. TT-Switched IgG+ mBc with TT-IgG
0.0242
0.7184
SLE Pt. Switched IgG+ mBc with anti-dsDNA
0.0438
0.6606
SLE Pt. CD27- IgG+ mBc with anti-dsDNA
0.0234
0.7212
a
All correlations for patients (Pt.) were established when data before and after
RTX treatment were analyzed jointly (n = 10).
b
Spearman’s two-tailed test.
818 36 Table S1. Clinical features of patients with autoimmune diseases
Patient
Pt. 1
Age
33
Gender
F
Principal
Concomitant
Disease
Concomitant
Relevant
Disease activity
Clinical follow up
diagnosis
autoimmune
duration
immunosuppressive
previous
before
approximately
disease
(years)
pharmacologic
pharmacologic
treatment
treatment
treatment
RA
SLE
nephritis
(Lupus
IV
6
AI
PDN,
HCQ,
AZA,
SSZ, CQ, HCQ,
MMF
MTX
AZA, DFZ
AZA
RTX
6
months after RTX
treatment
DAS28: moderate
DAS28: low
SLE
60%
9/24)
Pt. 2
69
F
SLE
Autoimmune
14
associated
clinical
thromobocytopenia,
steroid-refractory
improvement,
Sjögrens’
thrombocytopenia
platelet
syndrome,
count
improvement
antiphospholipid
syndrome,
hypothyroidism
Pt. 3
26
F
SLE
2
PDN, HCQ, MMF
MTX
SLEDAI
8:
Without
clinical
moderate activity,
improvement.
renal compromise
Cyclophosphamide
treatment.
Pt. 4
33
F
RA
Pt. 5
35
F
SLE
Antiphospholipid
1
SSZ, CQ, MTX
DAS28: high
1
PDN, AZA
SLEDAI
DAS28: moderate
6:
Clinical
syndrome,
moderate activity,
improvement with
hypothyroidism
great
regard to articular
Pt. 6
54
F
RA
Hypothyroidism
8
PDN, MTX
Pt. 7
46
F
SLE
RA
3
MTX, PDN, AZA
Pt. 8
42
F
RA
SLE,
7
PDN, MTX, D-Pen
6
PDN, AZA, CQ
CREST,
Infliximab
CQ
articular
compromise
compromise
DAS28: high
DAS28: moderate
ND
ND
DAS28: high
DAS28: moderate
hypothyroidism
Pt. 9
29
F
SLE
RA
SLE
without
changes
Pt. 10
47
M
RA
2
MTX, SSZ, CQ
DAS28: high
Without
clinical
improvement
Pt. 11
a
49
F
RA
3
PDN,
HCQ,
MTX,
DAS28: moderate
SSZ
60%
clinical
improvement
Pt. 12
58
F
RA
30
PDN, HCQ, LEF
Etanercept, MTX
DAS28: moderate
ND
Pt. 13
50
F
RA
3
DFZ, HCQ, SSZ
Etanercept, LEF
DAS28: moderate
Without
clinical
improvement
Pt. 14
a
58
M
RA
3
PDN, HCQ, MTX
DAS28: high
ND
A complete flow cytometry assay could not be done for patients RTX 1, RTX 13, RTX 20, and RTX 22 at both time frames (before and after RTX treatment);
therefore they were not included in the total and Ag-specific Bc analyses.
MTX: Methotrexate, SSZ: Sulfasalazine, HCQ: Hydroxychloroquine, CQ: Chloroquine, PDN: Prednisolone, AZA: Azathioprine, LEF: Leflunomide, MMF:
Mycophenolate, Deflazacort: DFZ, D-Penicillamine: D-Pen, ND: not done.
Supplementary Figure legends.
Supplementary Figure 1. RV-memory B cells gating strategies.
Two comparable analysis strategies were used to dissect total and RV-Bc
subsets (CD19+) based on their expression of surface markers. A
representative result is shown for a HV. Naïve and three main subsets of mBc
can be identified based on IgD and CD27 expression: naïve Bc (IgD+CD27-),
IgM+ mBc (IgD+CD27+), switched mBc (IgD-CD27+), and CD27- mBc (IgD-CD27). A. If only CD27+ mBc are considered (naïve and CD27- mBc are excluded)
five mBc subsets can be defined in terms of IgM and IgD expression: IgM+ only
mBc, IgM+ mBc IgMhiIgDlow, IgM+ mBc IgMlowIgDhi, IgD+ only mBc, and switched
mBc (IgM-IgD-). Total (top row plots) and RV-mBc (VLPs-GFP+) (lower row
plots) were gated. B. When isotype expression is considered, naïve B cells and
IgM+ mBc express IgD and IgM; switched mBc express IgG and IgA, but a
subset which only expresses IgM can also be identified on the IgD-CD27+ gate:
IgM+ only mBc; and CD27- mBc express IgA, IgG or IgM.
Supplementary Figure 2. Comparison of other total B cell subpopulations
among the study groups.
Other total B cell subpopulations studied are shown for healthy volunteers (HV),
patients before RTX treatment (prior-RTX) and patients after RTX treatment
(after-RTX). The dotted lines represent the estimated flow cytometry detection
limit of 3.5 CD19+ B cells/mL. Solid lines and error bars denote the median and
interquartile range, respectively. Differences between HV (n = 10) and patients
prior-RTX (n = 10) were evaluated with Mann–Whitney tests and between
patients prior-RTX and patients after-RTX (n = 10) with Wilcoxon tests. All p
values reported are 2-tailed.
Supplementary Figure 3. Comparison of other RV and TT-specific B cell
subpopulations among the study groups.
Other RV- and TT-specific B cell subpopulations studied are shown for HV (n =
10), patients prior-RTX (n = 10) and patients after-RTX (n = 10). Solid lines and
error bars denote the median and interquartile range, respectively. Differences
between HV and patients prior-RTX were evaluated with Mann–Whitney tests
and between patients prior-RTX and patients after-RTX with Wilcoxon tests. All
p values reported are 2-tailed.
7. GENERAL DISCUSSION AND CONCLUSIONS
RV vaccines have been introduced in the national immunization programs of
approximately 40 countries by 2012 [242]. This has meant a breakthrough in the
prevention of RV disease burden. In middle and high-income countries, significant
decreases of 49-89% in hospitalizations due to RV GE among children under the
age of five, and 17-55% reduction in all-cause GE hospitalizations, have been
observed within two years after RV vaccine introduction [60]. Notably, additional
benefits have been observed in older unvaccinated children and adults with an 829% reduction in GE hospitalizations in the RV season following vaccine
introduction in the United States [243].
Unfortunately, and similar to what has happened with other enteric vaccines (oral
polio, cholera, and typhoid), there is an important disparity in the efficacy of RV
vaccines between middle (72%-83%) to high-income countries (>90%) and lowincome countries (39%-49%), where mortality is greatest [9, 10]. Many factors may
contribute for the lower efficacy rates in low socio-economic settings, irrespective
of the geographical zone considered: higher titers of maternal antibodies (either
transplacentally transferred or those present in breast milk), oral polio vaccine
interference, nutritional deficiencies (zinc, vitamin A, and vitamin D), concomitant
infections with other gut microbes (viruses, tropical enteropathy, and helminthes),
and differences in the gut microbiota, among others [244]. These factors are in turn
associated with an impaired immune response to live oral vaccines and natural
infection.
Recently, a mathematical model of RV transmission and disease was developed to
understand the reduced vaccine efficacy in low-income settings [245]. The model
predicted 93%, 86%, and 51% prevention against severe RV GE in high, middle,
and low socio-economic settings. The most important factors responsible for the
lower efficacy in low-income settings were reduced immunogenicity of vaccination,
probably the most modifiable factor, and reduced protection conferred by natural
infection. If vaccine doses resemble the effect of primary and/or secondary natural
infections, the observed higher rates of subsequent infections and persistence of
severity in low-income settings are consistent with the lower vaccine efficacy and
suggested decreased duration of protection [245].
Many strategies are considered to improve the immunogenicity of current vaccines
[9, 245]: delaying the administration schedule (to allow waning of maternal
antibodies), adding a third dose to the Rotarix scheme (it was estimated that a third
dose may improve vaccine efficacy by 9% in low-income settings), and
supplementing with micronutrients (to enhance immune function). Moreover, there
are various candidate vaccines ongoing different clinical phases of assessment
[244]. Nevertheless, an adequate immunological correlate of protection to easily
48
evaluate the effect of the mentioned interventions to the current RV vaccines, and
to test the vaccines under development, has not been identified to date [242].
This work advances the RV field mainly in three ways: 1. We proposed that plasma
RV-SIg might serve as a valuable correlate of protection for RV vaccines (first
article). 2. Circulating RV-Bc subpopulations were thoroughly assessed in healthy
adults and patients with autoimmunity and a similar distribution was found among
them (second article). 3. We presented direct evidence that RV-serological
memory seems to be maintained by long-lived PC, in agreement with the proposed
model of independent functions and importance of mBc and PC in the immune
response and protection from reinfection [34, 218] (second article). In the following
pages, these conclusions will be discussed in as much as they provide starting
points for further research.
We confirmed [21, 22] that plasma RV-SIg can be detected in naturally infected
children, but with a noticeable difference compared to the literature. It has been
reported that plasma RV-SIg, in contrast to plasma RV-IgA, can be detected in the
acute phase of the infection and early in the convalescence, but no later than one
month after the onset of diarrhea [22]. Notwithstanding, RV-SIg was detected in the
great majority of children with evidence of previous RV infection without an ongoing
RV GE, which is at odds with the aforementioned result. It is possible that our
ELISA assay is sensitive enough to detect very low concentrations of RV-SIg,
previously undetected by other assays. Even though, in some vaccinees RV-SIg
was only transiently observed. These differences between our results and those
reported in the literature, and between vaccinees and children with previous natural
RV infection (using the same technique) require further assessment. Therefore, to
better define the best time frame for the detection of plasma RV-SIg, particularly if
it is going to be used as a correlate of protection, it is necessary to determine the
kinetics of RV-SIg in blood after RV vaccination.
Children vaccinated with the attenuated RIX4414 human RV vaccine presented
higher RV-SIg titers than placebo recipients, both after D1 and D2, and in
vaccinees higher titers were observed after D2 than after D1. These significant
differences between vaccinees and placebo recipients were unobserved for
plasma RV-IgA [17]. In addition, a detectable boost for RV-SIg was observed in
three children whereas for RV-IgA it was detected only in one child. Moreover, the
frequency of protected children was significantly higher in RV-IgA- (titer < 1:100)
vaccinees compared to RV-IgA- placebo recipients, which suggests that other
factors are important for conferring protection from disease in vaccinees [17]. In
agreement with these findings, in our trial, plasma RV-SIg seemed to be a better
correlate of protection than plasma RV-IgA. RV-SIg measured after D2 also
correlated with protection in vaccinees and placebo recipients analyzed jointly and,
in contrast to plasma RV-IgA, the frequency of protected children was significantly
higher in RV-SIg+ children (titers ≥ 1:100) than in those RV-SIg- (titer < 1:100).
Furthermore, the presence of RV-SIg conferred an almost four times increase in
49
the probability to be protected against any RV GE. These results, and those
reported in naturally infected children, suggest that RV-SIg is associated with
protection both after vaccination and natural infection.
Our studies are the first in which plasma antigen-specific SIg is evaluated as a
correlate of protection after vaccination. However, given that preimmune plasma
samples were unavailable, the level of vaccine induced RV-SIg is unknown. In
addition, the correlation of RV-SIg with protection may be underestimated because
the Rotarix commercial formulation induces higher RV-IgA seroconversion rates
than the RIX4414 formulation used in the present study [246], and this may also be
the case for RV-SIg. Therefore, the value of plasma RV-SIg as a correlate of
protection needs to be thoroughly assessed with current RV vaccines, and RV-SIg
induction by the vaccines must be addressed to add evidence for a probable
mechanistic role in protection.
The ELISA assay used to detect plasma RV-SIg is time-consuming and laborintensive due to its multiple steps, which increase its complexity. Additionally, it
requires a relative high amount of plasma, and uses a bovine viral lysate as the RV
antigen, the production of which adds labor to the process. Therefore, one of our
purposes for the near future is to optimize this assay to make it adequate for largescale studies. Recently, the use of recombinant VP6 in a DELFIA assay [247],
which employs lanthanide chelate europium (Eu3+)-labeled secondary antibodies,
to assess the RV-IgG response showed comparable results to the conventional
virus-capture ELISA. This assay is less labor intensive, uses lower sample volume,
and only requires a single test dilution [247]. Furthermore, the assessment of Ab
responses to recombinant viral proteins, instead of using the intact virus capsid,
may be another strategy to find useful correlates of protection, as suggested for
NSP4 [248].
In our previous study with the RIX4414 RV vaccine [17], circulating RV-IgDCD27+47+CCR9+ mBc measured after D1 correlated with protection from
disease, although with a low correlation coefficient. Since these cells expressed
intestinal homing receptors and it is considered that plasma RV-SIg reflects local
mucosal immunity, a correlation of RV-SIg titers with circulating RV-mBc after
vaccination was expected, but it was undetected. This can be explained, at least in
part, by the fact that RV-SIg is probably a mixture of SIgM and SIgA. Another
possibility is that a direct cause-and-effect relationship between RV-mBc
(irrespective of their phenotype) and Abs, in this case plasma RV-SIg, is inexistent.
SIgs (SIgA and SIgM) are normally detected in the systemic compartment of
healthy individuals (1 – 91 years old) [145-147]. However, the mechanism by which
SIgA, produced at mucosal surfaces, is transported to the circulation is
undetermined [20]. Additionally, the physiological function of SIgs (structurally
equipped to perform its functions mainly in the luminal side of the mucosae) in the
systemic compartment remains to be established.
50
SIgA has been implicated in the pathophysiology of some autoimmune diseases,
particularly IgA nephropathy and celiac disease. In patients with IgA nephropathy,
a clinical association between mucosal infections and worsening of the renal
condition has been reported, which suggested a role of SIgA in the disease
pathogenesis [147]. Consistent with this association, SIgA levels in blood are
higher in patients with IgA nephropathy than in healthy controls, and the level of
serum SIgA correlates with the severity of the pathological phenotype and with
clinical parameters like proteinuria and serum creatinine levels [147]. Additionally,
deposits of SIgA are found in the glomerular mesangium of some patients [147,
249].
The transferrin receptor (CD71) was characterized as a novel IgA receptor in
patients with IgA nephropathy [149]. It is upregulated on glomerular mesangial
cells in these patients and is involved in the deposit of IgA 1 [149]. Recently, it was
reported in a mouse model that IgA1, soluble CD89 (another described IgA
receptor), transferrin receptor, and transglutaminase-2, as a critical factor, are
needed for mesangial cell activation and disease development [250].
In patients with active celiac disease, CD71 is abnormally expressed at the apical
pole of enterocytes and serves as a receptor for SIgA [150]. It is proposed that
CD71 mediates the retro transport of SIgA-gliadin peptides complexes from the
apical side of the enterocyte to the lamina propria. This abnormal transport would
protect gliadin peptides from being normally degraded by lysosomal acid proteases
[150]. Recently, direct evidence was provided in this regard: CD71-SIgA-gliadin
complexes, or CD71-SIgA, are endocytosed and directed to the recycling pathway,
thus, avoiding lysosomal degradation [251]. Furthermore, transglutaminase-2
seems to control CD71 endocytosis [252]. Notably, transglutaminase-2 seems
essential both for IgA nephropathy and celiac disease pathogenesis.
The role of antigen-specific SIgA in the induction of autoimmunity is intriguing. On
the one hand, as mentioned earlier, an association between mucosal infections
and worsening of renal compromise in patients with IgA nephropathy has been
reported. Nevertheless, a study that directly assessed this relationship, through the
challenge of IgA nephropathy patients and controls with cholera toxin subunit B via
intranasal vaccination (as a neoantigen), concluded that the levels of antigenspecific SIgA were equivalent in both patients and controls [253]. On the other
hand, frequent RV infections have been associated with an increased risk for
developing celiac disease in genetically predisposed children [254]. Furthermore,
in one of the few prospective studies concerning reactive arthritis [255], total serum
SIgA and serum Yersinia-specific SIgA levels were higher in patients who
developed joint symptoms compared with those who did not develop them.
These precedents prompted us to explore the possibility of serum SIg as an
important factor in the pathogenesis of reactive arthritis and undifferentiated
51
spondyloarthritis. Preliminary results show that patients with these diagnoses have
significantly higher total SIgA concentrations than healthy controls. This constitutes
one of our interests for further research, along with the role that RV infections may
play in the induction of autoimmune diseases, as it is suggested by the mentioned
findings in celiac disease.
As mentioned before, although RV preferentially replicates in the intestine,
antigenemia and viremia are frequently observed in infected children and animals,
and its clinical significance has started to become evident [15, 241]. Therefore, the
systemic immune response may be as important as the one originated in the
intestine. Moreover, Abs are crucial for protecting infants against RV reinfection
[12]. Nevertheless, how the Bc that produce these antibodies are related to the
virus itself and how RV-mBc contribute to the maintenance of serological memory
is still poorly understood. Thus, characterization of RV-specific mBc and the
understanding of their relation with serological memory are probably crucial to
identify useful correlates of protection for vaccines [31, 256].
We thoroughly assessed circulating RV-Bc subpopulations in healthy adults and
patients with autoimmunity, advancing our knowledge of RV-mBc (second article).
We confirmed [43, 44, 102] that in HV RV-mBc are enriched in the IgMhiIgDlow
subset and endorsed that RV-mBc are also enriched in the IgMlowIgDhi and IgM+
only mBc subsets [102]. Moreover, patients with autoimmunity had a similar
distribution of IgM+ mBc subpopulations.
The enrichment of RV-mBc in all IgM+ mBc subsets in HV is puzzling. The IgM+
mBc population (CD27+IgD+IgM+), which has provoked so much controversy, in
turn comprises IgMhiIgDlow and IgMlowIgDhi mBc. IgM+ mBc have been termed by
some as “natural effector” or “innate-like” cells [181, 193, 194], since they are
detected in the absence of GC [178, 181]. In contrast, there is also evidence to
consider these cells as “true” mBc derived from a GC reaction [161]. These
apparent contradictions may be explained by the fact that IgM+ mBc are indeed
heterogeneous and may have very different origins and functions. This is fairly
clear in the mouse model, in which IgMhiIgDlow cells are termed spleen marginal
zone Bc, and IgMlowIgDhi are follicular Bc. In humans, this remains to be
determined; however, it is thought that IgMhiIgDlow cells resemble the spleen
marginal zone B cells, may use a prediversified subset of Ig genes, and participate
in “innate” Ig responses to pathogens [257].
A comprehensive genetic characterization of the IgMhiIgDlow and IgMlowIgDhi
subsets is lacking both in non-antigen specific mBc and antigen-specific mBc. RV
seems an adequate model to study these cells, particularly because RV-mBc are
enriched in them, and the VLP-GFP construct used to identify RV-Bc allows to
perform fluorescence activated cell sorting of these populations. Since human
CD27+IgM+ RV-mBc switch to IgG in vitro and in vivo, and significantly reduce RV
antigenemia and viremia in an adoptive transfer immunodeficient mice model [102],
52
to deepen the understanding of these subsets becomes even more important; it is
of primary interest for us to identify the phenotype of the cells capable of classswitching. Furthermore, given that the RV-immune response is compartmentalized,
it would also be important to address and compare the genetic profile of circulating
and intestinal RV-mBc subsets.
Concerning IgM+ only mBc, since they are absent in X-linked hyper IgM patients,
like switched mBc, it is thought that they arise from an independent diversification
pathway [178], compared to IgM+ mBc cells, and that they are the bona fide IgM+
mBc [159]. In fact, IgM+ only mBc seem to come from primary GC reactions [193],
then, the enrichment of RV-mBc in this subset (at the expense of switched IgA+ or
IgG+ mBc) suggests that RV-mBc have undergone a less extensive maturation in
GC than other mBc subpopulations. Remarkably, regardless of the described
origins and phenotypic differences, to date, all IgM+ mBc subsets seem to have a
similar usage of IGHJ and IGHV gene families [194].
The role of RV-IgM+ mBc subsets in the immune response against RV is
incompletely understood. It is important to determine if these cells increase in an
antigen-specific manner after RV vaccination –supporting their status as mBc– and
to establish if they express the intestinal homing receptors 47 and CCR9–
confirming that they are the product of an immune response induced in the
intestine [205].
Recently, the phenotype of pneumococcus-specific Bc was analyzed in healthy
adults using fluorescently labeled pneumococcal polysaccharides [258]. PPS14and PPS23F-specific Bc were significantly enriched in CD27+IgM+ mBc, like RVmBc. Unfortunately, pneumococcus-IgM+ mBc were not further characterized in
terms of the other IgM+ subsets described for RV-mBc (IgM+ mBc IgMhiIgDlow and
IgMlowIgDhi, and IgM+ only mBc). Notably, in splenectomized patients, circulating
IgM+ mBc are absent and remain permanently depleted, in contrast to switched
mBc that can be regenerated in the lymph nodes and increase with time [259].
These findings support the assumption that a great proportion of IgM+ mBc are
circulating marginal zone Bc from the spleen [183]. Since RV-mBc are enriched in
these cells, it would be interesting to assess RV-Bc in splenectomized patients and
to evaluate the immune response to RV vaccines in such patients.
Finally, regarding the relation of mBc with serological memory, we present direct
evidence that despite the absence of circulating RV- and TT-mBc, after RTX
treatment, the corresponding antigen-specific Abs levels remain constant,
regardless of the antigen-specific isotype and subclass evaluated here (RV-IgA,
RV-IgG, RV-IgG1, and TT-IgG). To our knowledge, the simultaneous study of
circulating antigen-specific mBc and antigen-specific Abs of the IgA and IgG1
isotypes in patients receiving RTX has no precedents. We conclude that RV- and
TT-serological memory seems to be maintained by long-lived PC unaffected by
RTX.
53
Consequently, in contrast to our hypothesis, the enrichment of RV-mBc in IgM+
mBc and CD27- mBc, relevant mBc subsets in the pathogenesis of various
autoimmune diseases, appears irrelevant for the relation of RV-mBc with RVserological memory. In other words, even for a pathogen like RV, with the
mentioned mBc peculiarities and an apparently different relationship with
serological memory (compared to that of TT-mBc and TT-IgG) [43], long-lived PC
seem to be committed to the maintenance of Abs levels in resting conditions.
Consistent with these findings, long-lived PC maintain antigen-specific serological
memory in some mouse models [260-262] and there is a whole body of evidence
[34, 263, 264] at odds with the mBc bystander activation model of the maintenance
of serological memory in healthy individuals. Therefore, under normal physiological
conditions, mBc and long-lived PC would accomplish different and separated, but
complementary, protective functions [34, 218].
Long-lived PC are unable to sense antigens, but are derived from previous
immunological encounters in which somatic hypermutation improved the Ig
antigen-binding capacity. Thus, long-lived PC that produce high affinity Abs
constitute a first line of defense against reinfection. Meanwhile, mBc constitute a
backup measure in case Abs, and the effector mechanisms triggered by them, fail
to control the antigenic challenge. Furthermore, mBc are equipped with antigenselected matured surface Igs, which allow them to launch rapid recall responses
[265]. In secondary responses, mBc can accumulate mutations and develop a new
Ab repertoire [198], from which new optimally adapted PC may derive, thus,
improving the PC compartment and increasing the level and quality of specific Abs.
Concerning autoimmune serological memory, the auto-Abs measured were
significantly diminished, in contrast to pathogen-specific Abs. This suggests that, at
least, an important proportion of IgM-RF, IgG-anti-CCP, and IgG-anti-dsDNA autoAbs are maintained by short-lived PC, which are probably in equilibrium with autoAg-specific mBc depleted by RTX. Notably, auto-Abs that decrease after Bc
depletion therapy with RTX are still detectable [236]. It is considered that long-lived
PC may be responsible for this, as well as for some auto-Abs that remain steady
after treatment with RTX [266]. Therefore, it would be important to simultaneously
assess auto-antigen-specific mBc and the corresponding auto-Abs. At present, this
could be done making use of a peptide mimetope of DNA that allows the
identification of potentially dsDNA-specific autoreactive Bc [267].
With respect to total Abs levels, RTX treatment had a small effect on total IgA and
IgG levels, which probably reflects its differential effect on both auto-Abs and Abs
against pathogens, and the proportion of Abs depending on long-lived and shortlived PC in the corresponding total pool. Compared to total IgA and IgG, total IgM
decreased at significantly higher levels, suggesting that a high proportion of the
total IgM pool may depend on short-lived PC. In support of these findings, after
RTX treatment, the percentage drops in IgG-anti-dsDNA and IgG-anti-CCP were
54
significantly higher than the percentage drops in total plasma IgG (second article),
as well as the decrease of IgA-RF compared to that of total IgA [39], whereas the
decrease of IgM-RF and total plasma IgM was similar in this and prior studies [39].
In agreement with these results, all IgM+ mBc subsets correlated with total plasma
IgM when data before and after RTX treatment were analyzed jointly. This is the
first time that such correlation is described in patients with autoimmunity. The
overexpression of IL-10 and CD154 detected in some patients, which is associated
with a rapid PC differentiation and auto-Ab production [219-221], would explain the
apparent enrichment of the total IgM pool in Abs produced by short-lived PC.
However, further studies are required to address the effect of B-cell depletion
therapy on pathogen-specific plasma IgM.
55
8. PERSPECTIVES
8.1 To evaluate the plasma RV-SIg kinetics in vaccinated children and to
determine its value as a RV correlate of protection using the commercial
formulation of current RV vaccines.
8.2 To optimize the plasma RV-SIg ELISA assay throughout the assessment of
other RV antigens such as double-layered particles, and the use of peroxidase
substrates that increase sensitivity.
8.3 To explore the role of SIgA and RV-SIg in the pathophysiology of reactive
arthritis and other autoimmune diseases, given the importance of SIgA in the
pathogenesis of celiac disease and IgA nephropathy.
8.4 To assess the genes (IGH and IGL gene segment usage, mutation load,
clonality, germinal center dependence or independence status, and probable tissue
of origin) used by circulating, particularly IgM+ mBc IgMhiIgDlow versus IgMlowIgDhi,
and intestinal RV-mBc subsets.
8.5 To deepen the understanding of the immunological function of the different RVIgM+ mBc subsets in comparison to other antigens, such as Streptococcus
pneumoniae, for which enrichment in IgM+ mBc has also been reported, in
splenectomized patients.
56
9. APPENDIX
9.1 DETAILED MATERIALS AND METHODS
Written informed consent was obtained from each volunteer or infant’s parents or
legal guardian. Studies were approved by the Ethics Committee of the San Ignacio
Hospital and Pontificia Universidad Javeriana and conducted in accordance with
the guidelines of the Helsinki Declaration.
9.1.1 Subjects and sample processing: children with gastroenteritis, children
vaccinated with RIX4414, and placebo recipient children
Plasma samples from 36 children with acute GE from prior published studies were
assessed [94, 268]. These children (14 females and 22 males; mean age: 13.5
months, range: 6 – 22; 20 breast fed and 16 not breast fed) were admitted with
gastroenteritis to the pediatric emergency service or were hospitalized at the
moment of sample collection. Demographic and clinical data of the children are
presented in supplemental material Table S1 of the first article. The mean time of
blood drawing after onset of diarrhea was 5 d (range: 1–12).
Plasma and serum samples were obtained from 10 healthy adult volunteers (7
females and 3 males; mean age: 28 years, range: 25–38), without any
gastrointestinal symptoms during the month previous to the blood drawing.
Additionally, 4 umbilical cord blood samples taken from healthy full-term newborn
infants were also included.
In our previous double-blind randomized controlled study [17], children received
two doses of either placebo (n = 160) or 10 6.7 focus-forming units of the attenuated
RIX4414 human RV vaccine (n = 159). Vaccine and placebo groups were very
similar in terms of the median age at the time of the first and second vaccination
(60 and 122 days, respectively), gender (M/F 85/74 and 84/76, respectively), and
percentage of breast fed at dose 1 and dose 2 (96.9% and 95%, 88.6% and
88.1%, respectively). Details about the vaccine trial and the strategy for clinical
evaluation of protection were previously published [17]. Briefly, from the moment
infants received their second dose of vaccine/placebo, they were contacted every 2
weeks until they were 13 months old to identify cases of GE. Of the 319 children
who received two doses of vaccine/placebo, a subgroup of 119 was randomly
selected for immunological assessments (50 vaccinees and 69 placebo recipients).
Plasma samples from all these children, except seven placebo recipients (children
in whom the informed consent form did not authorize further studies), were
included in the present studies. Plasma samples were collected 14–16 days after
receiving each dose of RIX4414 or placebo.
57
All plasma samples from previous studies [17, 94, 268], which had been stored at 80°C, were thawed, diluted in 50% glycerol and preserved at -20°C for use. All
assays, except competitive binding assays, were blinded experiments.
9.1.2 ELISA for measuring plasma RV secretory immunoglobulin
96-well vinyl microtiter ELISA plates (Thermo Electron Corporation, Cat. No. 2401)
were coated with 70 l of 1/10,000 dilution (in phosphate-buffered saline, PBS
[Gibco, Cat. No. 21600-069]) of an anti-human SC monoclonal Ab (clone GA-1)
(Sigma-Aldrich, Cat. No. I 6635), and incubated overnight at 4°C. After discarding
this solution, 150 l of 5% blotto (5% non-fat powdered milk plus 0.1% Tween-20)
was added to the plate as a blocking solution and incubated at 37°C for 1h. Then,
the blotto was discarded and 70 l of serial dilutions of plasma samples in 2.5%
blotto were deposited in each well. After 2 h incubation at 37°C, samples were
discarded and the plates were washed three times with PBS-Tween 20 and 70 l
of 1/10 dilutions in PBS of a supernatant from RF (Bovine RV strain P6[1]G6, 10 7
focus forming units/ml) virus-infected MA104 cells or the supernatant of mockinfected cells (negative control) were added and incubated at 37°C for 1h. Notably,
most antibodies detected with this antigen are specific for the major capsid protein
VP6, which contains group- and subgroup-specific antigenic determinants and
exhibits a high level of sequence conservation [201, 269]. The wells were then
washed three times and 70 l of guinea pig anti-rhesus RV hyperimmune serum
diluted 1/4,000 in 2.5% blotto was added to each well and incubated for 1h at
37°C. After three washes, 70 l of biotinylated goat anti-guinea pig serum (Vector
Laboratories, Cat. No. BA-7000) diluted 1/2,000 in 2.5% blotto was added to each
well. After 1h of incubation at 37°C, the wells were washed three times and 70 l of
peroxidase-labeled streptavidin (Kirkegaard and Perry Laboratories [KPL], Cat. No.
14-30-00) diluted 1/1,000 in 2.5% blotto was added to each well and incubated for
1h at 37°C. After three washes, plates were developed using 70 l tetramethyl
benzidine substrate (Sigma-Aldrich, Cat. No. 50-76-00). The reaction was stopped
by the addition of 17.5 l 2 M sulfuric acid. Absorbance was read at a wavelength
of 450 nm on an ELISA plate reader (Multiskan EX; Thermo Labsystems). Serial
dilutions of a pool of plasmas from children with RV-SIg was used as a positive
control and a plasma from a child without evidence of previous RV infection (RVIgA-) was used as a negative control in each plate. Samples were considered
positive if the optical density in the experimental well was > 0.1 optical density units
and 2-fold greater than the optical density in the corresponding negative control
wells. To be accepted for analysis, the titer of the positive control plasma could not
differ by more than one dilution from plate to plate. A schematic representation of
this ELISA is available in Figure A1 of the appendix.
58
9.1.3 ELISA for measuring total plasma secretory IgA
To detect total SIgA, a sandwich ELISA was developed using a previously
described approach [270]. Briefly, 96-well vinyl microtiter ELISA plates were coated
with 70 l of 1/10,000 dilution of the anti-human SC monoclonal Ab or PBS
(negative control) and incubated overnight at 4°C. 5% blotto was added and
incubated at 37°C for 1h. Then, 70 l of serial dilutions of plasma samples were
applied in each well. After 2 h incubation at 37°C, the plates were washed three
times with PBS-Tween 20 and 70 l of biotin-labeled goat anti-human IgA (KPL,
Cat. No. 16-10-01) diluted 1/1,000 in 2.5% blotto was added and incubated for 1h
at 37°C. After three washes, 70 l of peroxidase-labeled streptavidin diluted
1/1,000 in 2.5% blotto was added and the plates were incubated for 1h at 37°C.
After three washes, plates were developed and analyzed as described above for
measuring plasma RV-SIg. The concentration of total SIgA in the plasma pool,
used as a positive control, was interpolated from a standard curve generated with
purified SIgA from human colostrum (AbD Serotec, Cat. No. PHP133). The
corresponding concentration for each plasma sample tested was in turn
interpolated from the plasma pool curve using a four-parameter logistic-log function
[271]. A schematic representation of this ELISA is available in Figure A2 of the
appendix.
9.1.4 ELISA for measuring plasma RV-IgM
RV-IgM ELISA was performed as previously described [43], with minor
modifications. Briefly, 96-well Immulon 2 microtiter ELISA plates (Dynex
Technologies) were coated overnight at 4°C with 70 l of Goat F(ab’)2 anti-human
IgM (Invitrogen, Cat. No. AHI1601) diluted 1/500 in PBS. The plates were then
blocked and incubated with plasma samples diluted in 5% blotto. After five washes
with PBS-Tween 20, the remaining steps of the assay continued as described
above for measuring plasma RV-SIg. Samples were considered as positive using
the same criteria previously described.
9.1.5 Recombinant human SC and competitive binding assays
The recombinant human secretory component (rhSC) was kindly provided by
Doctor Blaise Corthésy. To obtain rhSC, a expression vector pcDNA3:SC was
created by inserting into XbaI/EcoRI-cut pcDNA3 plasmid (Invitrogen) the 1.85 kb
fragment carrying the cDNA for human SC recovered from XbaI/EcoRI-treated
plasmid pBS-hSC:End. After transfection into CHO dhfr- cells (CHO DUK-; ATCC
CRL 9096) and selection in the presence of G418, clones isolated by FACS scan
were tested by ELISA [272]. The clone producing the highest amount of rhSC
59
represents the source of rhSC used in the paper to ensure the activity of the
capture antibody in the SIg ELISAS. Bovine serum albumin (Merck, 1120180100)
(used as a negative control) or rhSC was added in 1/2 serial dilutions (starting from
6.1 μg/ml onwards) after the blocking step, and incubated for 10 min at 37°C. Next,
purified SIgA was added at a concentration of 0.076 μg/ml (the concentration of
SIgA present in a 1/200 dilution of the positive control plasma pool), plates were
incubated for 2 h, and the assay continued as described above. A similar strategy
was used for RV-SIg, using a dilution of plasma samples (1/200), either with only
plasma RV-IgA or with only plasma RV-IgM, which gave a sub-saturating signal in
the ELISA. After this, plates were incubated for 2 h and the assay continued as
previously described.
9.1.6 Subjects, sample collection and processing: patients with autoimmune
diseases and healthy volunteers
Fourteen patients, twelve females and two males, nine of them diagnosed with RA
and five with SLE, according to the American College of Rheumatology
international criteria [273, 274], were included. All patients had moderate or high
disease activity, despite treatment with disease-modifying anti-rheumatic drugs or
standard immunosuppressive therapy, measured by the disease activity score of
28 joint counts (DAS28) or SLE Disease Activity Index (SLEDAI), respectively.
Given the failure to standard treatment regimens, they were selected to receive
RTX by their treating rheumatologist. Additional clinical manifestations considered
to use RTX as the treatment of choice in patients included: lupus nephritis (n = 2),
autoimmune thrombocytopenia (n = 1), overlap of RA and SLE (n = 4), and
antiphospholipid syndrome (n = 2). The treatment regimen included two infusions
of intravenous RTX (1,000 mg), 14 days apart, in combination with intravenous
methylprednisolone (100-250 mg) [275, 276].
The median age at RTX treatment was 46 years (range 26 – 69) and the median
disease duration to the time of RTX treatment was 3 years (range 1 – 30); eight
patients had been diagnosed (within three years or less). Clinical follow up could
be done in eleven patients within the following six months post B-cell depletion
therapy. Out of these, seven patients showed subjective and objective clinical
improvement. Supplementary Table S1 in the second article describes
accompanying autoimmune diagnoses, concomitant and relevant previous
pharmacologic treatment, baseline disease activity, and clinical follow up. Ten age
and sex matched healthy volunteers were used as controls.
PBMC were isolated by LymphoSep (MP Biomedicals, Solon, OH) density-gradient
centrifugation from heparinized samples from ten of the fourteen patients described
above, immediately before and four to six months after RTX infusion (in the
remaining four patients PBMC samples were only available after RTX treatment),
and from ten age and sex matched healthy controls. A complete blood count test
60
was performed for each volunteer after each blood draw, and plasma was collected
and stored at −80 °C for subsequent total and antigen-specific Igs assessment and
auto-Abs measurement.
9.1.7 Production of fluorescent virus like particles (VLPs)
Fluorescent RV VLPs were a kind gift of Annie Charpilienne and were produced
using baculovirus expression vectors, as previously described [202]. Briefly, Sf9
cells were co-infected with 2 recombinant baculoviruses at a multiplicity of infection
greater than 5 PFU/cell. One baculovirus expressed RF (bovine RV) VP6 and the
other a fusion protein consisting of green fluorescent protein (GFP) fused to the N
terminus of RF VP2 deleted in the first 92 amino acids. Infected cultures were
collected five to seven days post infection and purified by density gradient
centrifugation in CsCl. The optimal concentration of the RV VLPs for labeling of
specific mBc was determined using PBMC from healthy volunteers. Notably, RV
VP6 is an immunodominant protein, and the majority of human RV-specific B cells
bind to VP6 [102]. Additionally, the majority of RV antibodies in infected animals
and humans recognize the VP6 protein on the outer shell of the VLPs [201].
9.1.8 Biotinylated - TT antigen
Tetanus Toxoid (Statens Serum Institute, Denmark) for in vitro tests was
biotinylated using the EZ-Link Photoactivatable Biotin kit (Pierce Protein Biology
Products, Cat. No. 29987) according to manufacturer's instructions, as previously
described [43], with minor modifications. A total of 750 μg of TT protein was
incubated with biotin at a molar ratio of 20 mol of dye per mole of protein, for 20
min on ice under a UV (365 nm) lamp, and then dialyzed against PBS for 18 hours
to remove excess biotin, using a Slide-A-Lyzer dialysis cassette (Thermo Scientific
Pierce, Cat. No. 66333). The optimal concentration of the biotinylated TT for
labeling of specific mBc was determined using PBMC from healthy and recently TT
vaccinated volunteers.
9.1.9 Flow cytometry assays
Fresh PBMC, 4 to 6 x 106, were washed twice with PBS (Gibco-BRL, Cat. No.
70011-044) and incubated with the GFP labeled VLPs (0.9 μg/test) or without this
reagent (negative control) for 45 minutes in the dark, at room temperature (RT).
The cells were then washed with PBS – 1% bovine serum albumin (Merck, Cat No.
1120180100), 0.02% sodium azide (Mallinckrodt Baker, Cat No. V015-05) (staining
buffer), and surface stained with Abs against CD19-allophycocyanin (APC)-H7
(SJ25C1 clone; Becton Dickinson [BD], Cat No. 643078), IgD-HorizonTM V450
(IA6-2 clone; BD, Cat. No. 561309), CD27- phycoerythrin (PE)-Cy7 (M-T271 clone;
61
BD, Cat No. 560609), goat anti-IgA-R-PE (Jackson ImmunoResearch, Cat. No.
109-115-011), IgG-APC (G18-145 clone; BD, Cat. No. 550931) and custom-made
IgM-Alexa Fluor 700 (145-8 clone; BD). Biotinylated-TT, or no reagent, was also
added at this step and incubated for 30 minutes in the dark, at RT. Cells were then
washed with staining buffer and the biotinylated-TT was detected using
streptavidin-peridinin chlorophyll protein (PerCP) (BD, Cat. No. 340130).
Streptavidin-PerCP was also added to the PBMCs without biotinylated-TT to
assess the background generated by this reagent (negative control). After staining,
the cells were washed and resuspended in staining buffer. At least 200,000 B cells,
gated on a CD19+ window, were acquired on a FACSAria (BD) or LSRFortessa
(BD) flow cytometer. Application settings were used to obtain constant
fluorescence intensity values among experiments run on different days and
regardless of the flow cytometer used. Fluorescence minus one controls were used
to determine the cut-off between positive and negative cell populations for each
marker [277].
Notably, Abs against CD3/CD14-HorizonTM V500 (UCHT1 and M5E2 clones,
respectively; BD, Cat. No. 561416 and 561392) were used as a dump channel in
samples taken from patients after RTX treatment. However, since conclusions did
not change with or without its use, the dump channel was not considered in the
final analyses.
Results for total and antigen-specific mBc were expressed as absolute numbers of
CD19+ B cells/mL, calculated after background subtraction, based on the total
lymphocyte numbers in the patient's complete blood count test results, according to
the following equation: Absolute number of CD19 + B cells/mL per mBc
subpopulation =
(absolute number of lymphocytes in complete blood
count/mL)×(percentage of CD19+ B cells in each subpopulation).
Evaluable total B cell subpopulations were defined as those with 10 acquired
events [278]. The frequency, in terms of CD19+ B cells, of those 10 events was
calculated considering the median CD19+ events acquired after RTX treatment,
and multiplied by the median absolute B cell count after RTX treatment. This value,
3.5 CD19+ B cells/mL, corresponds to the dotted lines present in each total B cell
subpopulation of Figure 2 and represents the flow cytometry detection limit for total
B cell subpopulations. A common detection limit could not be determined for
antigen-specific mBc subsets due to the variable background. Nevertheless, the
reported values correspond to antigen-specific mBc subsets with 10 acquired
events, and at least two-fold greater than the observed background. Flow
cytometry analysis was performed using FlowJo software version 9.6.2 for Mac
(Treestar, Ashland, OR).
62
9.1.10 Measurement of total immunoglobulins (IgA, IgG and IgM) and IgM
Rheumatoid Factor
Plasma samples were thawed and simultaneously assessed by kinetic
nephelometry on an IMMAGE® immunochemistry system (Beckman Coulter,
Fullerton, CA), following the manufacturer’s instructions. Measuring ranges are as
follows: IgA: 40 – 700 mg/dL (normal reference range values: 82 – 453 mg/dL);
IgG: 200 – 3,600 mg/dL (normal reference range values: 751 – 1,560 mg/dL); IgM:
25 – 400 mg/dL (normal reference range values: 46 – 304 mg/dL); and IgM
rheumatoid factor (RF): 20 – 800 IU/mL (negative cut off value: <20 IU/mL), with a
sensitivity level reported by the manufacturer of 70-75%.
9.1.11 ELISAs for detection of RV-specific IgA, IgG, and IgG1 and TT-specific
IgG in plasma
RV and TT antibodies were assessed as previously described with minor
modifications [43]: 96-well vinyl microtiter ELISA plates (Thermo Electron
Corporation, Milford, MA) were coated with 70 l of either a supernatant from RF
virus-infected MA104 cells or the supernatant of mock-infected cells (negative
control) diluted 1/10 in PBS, for RV-specific ELISAs, or 0.5 μg/mL of TT or PBS
(negative control), for TT-specific IgG ELISA, and incubated overnight at 4°C. After
blocking with 150 l of 5% blotto during 1h at 37°C, 70 l of serial dilutions of
plasma samples were deposited in each well. After 2 h incubation at 37°C,
samples were discarded and the plates were washed three times with PBS-Tween
20. Depending on the type of ELISA performed, 70 l of a 1/1,000 dilution in 2.5%
blotto of biotin-labeled goat anti-human IgA or IgG (KPL, Cat No. 16-10-01 and
Cat. No. 16-10-06, respectively), or biotin-labeled mouse anti-human IgG1 (8c/6-39
clone, Sigma-Aldrich, Cat. No. B 6775) diluted 1,2000 in 2.5% blotto were added to
each well. After three washes, 70 l of a 1/1,000 dilution of peroxidase-labeled
streptavidin were added. Plates were developed and analyzed as previously
described for measuring plasma RV-SIg. Pools of plasmas from children with RVIgA or RV-IgG, and from adults with TT-IgG were used as positive controls.
Plasmas from a child without evidence of previous RV infection (RV-IgA-), and
without RV-IgG from placental transfer of maternal IgG antibodies, and from an
adult negative for TT-IgG were used as negative controls.
9.1.12 Measurement of anti-CCP and anti-dsDNA autoantibodies (IgG isotype)
Plasma samples were simultaneously assessed by a fluorescence enzyme
immunoassay (EliA test), following the manufacturer’s instructions, on a Phadia
ImmunoCAP 100 system (Phadia AB, Uppsala, Sweden). The anti-CCP test has a
measuring range of 0.4 to at least 340 U/mL, a negative cut off value of < 7 U/mL,
an equivocal range of 7 – 10 U/mL, and a positive cut off value of > 10 U/mL; the
63
manufacturer’s reported clinical sensitivity and specificity are 87.8% and 96.7%,
respectively. The anti-dsDNA test has a measuring range of 0.5 to at least 400
IU/mL, a negative cut off value of < 10 IU/mL, an equivocal range of 10 – 15 IU/mL,
and a positive cut off value of > 15 IU/mL; the manufacturer’s reported sensitivity
for active SLE is 70.8% and clinical specificity of 93.2%.
9.1.13 Statistical analyses
Analyses were performed with SPSS software version 20.0 (IBM Inc.) and with
GraphPad Prism version 6. Differences between groups were evaluated with
nonparametric Mann–Whitney and Wilcoxon tests, as required. Correlations were
evaluated with Spearman's test. When data followed a normal distribution, or were
normalized, a Student’s t-test was applied. Significance was established if p <
0.05, in 1 or 2 tailed tests, if the direction of hypothesis was known or unknown,
respectively.
64
9.2 Figure A1: Schematic representation of the ELISA for measuring plasma RV secretory immunoglobulin
65
9.3 Figure A2: Schematic representation of the ELISA for measuring total plasma secretory IgA
66
9.4 Relevant data not shown from the first article: “Rotavirus Specific Plasma
Secretory Immunoglobulin in Children with Acute Gastroenteritis and
Children Vaccinated with an Attenuated Human Rotavirus Vaccine”
9.4.1 Concentration of total SIgA in plasma and serum samples from the
same adult healthy volunteers
The concentration of SIgA for each sample was determined by ELISA based on a
standard curve of plasma with a known SIgA concentration (14.6 g/ml). The limit
of detection was 4.8 ng/ml. Lines and error bars denote the mean and SEM,
respectively. Differences between groups (n = 10) were evaluated with the
nonparametric Mann–Whitney test. There was no significant difference when
plasma or serum samples were evaluated.
67
9.5 Relevant data not shown from the second article: “Simultaneous
Assessment of Rotavirus-Specific Memory B Cells and Serological Memory
after B Cell Depletion Therapy with Rituximab”
9.5.1 RV-mBc enrichment in the CD27-IgG+ subset in healthy volunteers when
the analysis was done in terms of the CD27- mBc
Summary of the frequencies of three subsets of total and RV-mBc assessed by
multiparametric flow cytometry and expressed in terms of CD27- mBc in healthy
volunteers (n = 10). All p values reported are 2-tailed (p < 0.05, Wilcoxon test).
Lines and error bars denote the median and interquartile range, respectively.
9.6 Journal paper: “Circulating human rotavirus specific CD4 T cells
identified with a class II tetramer express the intestinal homing receptors
α4β7 and CCR9”
68
Elsevier Editorial System(tm) for Virology
Manuscript Draft
Manuscript Number: VIRO-13-631R1
Title: Circulating human rotavirus specific CD4 T cells identified with a class II tetramer express the
intestinal homing receptors α4β7 and CCR9
Article Type: Regular Article
Section/Category: 8 Immunity
Keywords: Rotavirus (RV); T cells; intestinal homing receptors; MHC class II tetramers.
Corresponding Author: Dr. Juana Angel,
Corresponding Author's Institution: Pontificia Universidad Javeriana
First Author: Miguel H Parra
Order of Authors: Miguel H Parra; Daniel Herrera; J. Mauricio Calvo-Calle; Lawrence J Stern; Carlos A
Parra-López; Eugene Butcher; Manuel Franco; Juana ANGEL
Abstract: Using a consensus epitope prediction approach, three rotavirus (RV) peptides that induce
cytokine secretion by CD4 T cells from healthy volunteers were identified. The peptides were shown to
bind HLA-DRB1*0101 and then used to generate MHC II tetramers. RV specific T cell lines specific for
one of the three peptides studied were restricted by MHC class II molecules and contained T cells that
bound the tetramer and secreted cytokines upon activation with the peptide. The majority of RV and
Flu tetramer+ CD4 T cells in healthy volunteers expressed markers of antigen experienced T cells, but
only RV specific CD4 T cells expressed intestinal homing receptors. CD4 T cells from children that
received a RV vaccine, but not placebo recipients, were stained with the RV-VP6 tetramer and also
expressed intestinal homing receptors. Circulating RV-specific CD4 T cells represent a unique subset
that expresses intestinal homing receptors..
*Manuscript
Click here to view linked References
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Circulating human rotavirus specific CD4 T cells identified with a class II
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tetramer express the intestinal homing receptors 47 and CCR9
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4
Miguel Parra1, Daniel Herrera1, J. Mauricio Calvo-Calle2, Lawrence J. Stern2,
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Carlos A. Parra-López3, Eugene Butcher4, Manuel Franco1, and Juana Angel1,&
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Javeriana, Bogotá, Colombia.
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Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad
Department of Pathology, University of Massachusetts Medical School,
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Worcester, MA 01655.
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de Colombia, Bogotá, Colombia.
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Stanford University School of Medicine, Stanford, California , USA.
Facultad de Medicina, Departamento de Microbiología, Universidad Nacional
Laboratory of Immunology and Vascular Biology, Department of Pathology,
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Running title: Rotavirus specific CD4 T cells express intestinal homing
17
receptors.
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Keywords: Rotavirus (RV), T cells, intestinal homing receptors, MHC class II
20
tetramers
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&
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Pontificia Universidad Javeriana, Carrera 7 No. 40-62, Bogotá, Colombia.
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Email: [email protected]. Phone: 57-1-3208320 Ext 2790. Fax: 57-1-
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2850356.
Address Correspondence to: Dr. Juana Angel. Instituto de Genética Humana,
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ABSTRACT
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Using a consensus epitope prediction approach, three rotavirus (RV) peptides
29
that induce cytokine secretion by CD4 T cells from healthy volunteers were
30
identified. The peptides were shown to bind HLA-DRB1*0101 and then used to
31
generate MHC II tetramers. RV specific T cell lines specific for one of the three
32
peptides studied were restricted by MHC class II molecules and contained T
33
cells that bound the tetramer and secreted cytokines upon activation with the
34
peptide. The majority of RV and Flu tetramer+ CD4 T cells in healthy volunteers
35
expressed markers of antigen experienced T cells, but only RV specific CD4 T
36
cells expressed intestinal homing receptors. CD4 T cells from children that
37
received a RV vaccine, but not placebo recipients, were stained with the RV-
38
VP6 tetramer and also expressed intestinal homing receptors. Circulating RV-
39
specific CD4 T cells represent a unique subset that expresses intestinal homing
40
receptors.
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43
INTRODUCTION
44
Rotavirus (RV) is the leading worldwide cause of severe gastroenteritis in
45
children under the age of 5 years (Tate et al., 2012). Two vaccines (RotarixTM
46
and RotateqTM) have been included in national immunization programs in many
47
countries (Glass et al., 2012), but their efficacy is relatively low in some African
48
and Asian countries, where they are most needed (Angel et al., 2012). Thus,
49
improvement of current vaccines or generation of new RV vaccines is desirable;
50
however, an important drawback to this end is the lack of optimal immune
51
correlates of protection after vaccination (Angel et al., 2012).
52
The protective immune response to RV in humans and animals is mediated by
53
intestinal IgA (Blutt et al., 2012; Franco et al., 2006), which is, at least partially,
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T cell dependent: on the one hand, in the murine model the CD4 T cells are
55
essential for the development of RV-specific intestinal IgA (Franco and
56
Greenberg,
57
immunodeficiencies get chronically infected with RV, suggesting that both arms
58
of the immune system are important in clearance of RV infection (Gilger et al.,
59
1992). Our group has observed that higher frequencies of RV-specific CD8 and
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CD4 T cells secreting IFN- circulate in symptomatically infected adults and RV-
61
exposed laboratory workers, compared with healthy volunteers. (Jaimes et al.,
62
2002). In contrast, children with RV diarrhea had low or below detection levels
63
of RV-specific CD8 and CD4 T cells secreting IFN- (Jaimes et al., 2002; Mesa
64
et al., 2010; Rojas et al., 2003). Consequently, monitoring of RV-specific CD4 T
65
cells responses after vaccination may require a direct and highly sensitive
66
assay.
1997).
On
the
other
hand,
children
with
T
and/or
B
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The expression of tissue-homing receptors defines subsets of memory T cells
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that preferentially home to the skin or gut (Butcher and Picker, 1996; Sallusto
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and Lanzavecchia, 2009). These receptors are imprinted by dendritic cells in
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developing T cells and promote trafficking properties based on the site-specific
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expression of their ligands (Sigmundsdottir and Butcher, 2008). Gastrointestinal
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associated lymphoid tissue dendritic cells metabolize food-vitamin A into
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retinoic acid, which induces the T cells to express high levels of the gut-homing
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receptors 47 and CCR9, predisposing the migration of the recent activated T
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cells from the blood to the effector sites in the gut mucosa (Mavigner et al.,
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2012). The natural ligand of the 47 integrin is the mucosal addressing cell
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adhesion molecule-1 (MAdCAM-1), which is expressed by endothelial cells of
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the lamina propria along the entire intestine. Whereas, CCL25, the ligand of
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CCR9, is only expressed by small intestine endothelial and epithelial cells
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(Mavigner et al., 2012). Thus, T cells that express both markers are conditioned
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to home to the small intestine.
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RV predominantly replicates in mature enterocytes of the small intestine, but
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also has a systemic dissemination (Blutt et al., 2003) and, consequently, both
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intestinally and systemically primed T cells are expected to be generated
85
(Franco et al., 2006). Using purified subsets of peripheral blood mononuclear
86
cells (PBMC) that express the intestinal homing receptor 47, we found that
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RV-specific CD4 T cells secreting IFN- from adult volunteers preferentially
88
express this receptor (Rojas et al., 2003). With similar studies other
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investigators have shown that in healthy adults circulating CD4 T cells that
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proliferate in vitro in response to RV also express 47 (Rott et al., 1997). All of
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these studies have the drawback that subsets of T cells expressing the homing
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receptor have to be purified before their identification in the functional studies,
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which may change their phenotype. Moreover, no studies have assessed the
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expression of CCR9 on human RV-specific T cells.
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Recently, the use of MHC class II tetramers for characterization of CD4 T cells
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populations (Vollers and Stern, 2008) has appeared as a new important tool to
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characterize antigen-specific T cells against different viruses (Nastke et al.,
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2012; Nepom, 2012). Also, tetramers have been used to examine the CD4 T
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cells responses after vaccination against influenza (Flu) (Danke and Kwok,
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2003) and anthrax (Laughlin et al., 2007). The tetramers permit the ex vivo
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quantification and phenotypic characterization of T cells without T cell activation.
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In the present study, we identified the first HLA-DR1-restricted human RV-
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specific CD4 T cell epitope, and used MHC class II tetramers to characterize
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the phenotype of the T cells specific to this epitope. T cells specific for the RV
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peptide tetramer, but not for a Flu virus peptide-tetramer, expressed intestinal
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homing receptors. Moreover, antigen experienced CD4 T cells from children
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that received a RV vaccine, but not from placebo recipients, were stained with
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the RV tetramer and expressed intestinal homing receptors.
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METHODS
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Epitope prediction and peptides synthesis
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To predict HLA-DR1 (DRB1*0101) binding epitopes, we used the sequences of
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the RV strain KU G1P[8] form the NCBI genome databases (BAA84966,
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BAA84967, Q82050.1, BAA84969, BAA84970, AAK15270.1, BAA84962,
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BAA84963, BAA84964, P13842, BAA84965, BAA03847). Each potential 9-mer
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binding frame was evaluated using two independent prediction algorithms: P9
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(Calvo-Calle et al., 2007; Hammer et al., 1994; Nastke et al., 2012; Sturniolo et
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al., 1999) and SYFPEITHI (Schuler et al., 2007), as previously described
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(Calvo-Calle et al., 2007; Nastke et al., 2012). Potential epitopes were selected
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using cutoff scores of ≥1.5 for P9 and ≥29 for SYFPEITHI. Overall, 1,440
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possible 9-mer minimal epitopes were evaluated and 39 potential epitopes,
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scoring highly for both algorithms, were selected (Supplementary table 1),
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extended by six residues on each side, and synthesized (Sigma-Aldrich
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PEPscreen®) with an acetylated N-terminal and amidated C-terminal.
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Subjects
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After written informed consent was signed, blood samples were obtained from
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52 healthy volunteers, 23 to 52 years old that, as expected, had serum
130
antibodies against RV. Frozen PBMC from 35 RV IgA seropositive vaccinated
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children and 24 RV seronegative placebo recipient children (samples from a
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previous study (Rojas et al., 2007)), in whom the informed consent authorized
133
further studies, were also assessed. This was a double-blind randomized
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controlled study, in which children received two doses of either placebo (n =
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160) or 10
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(precursor of the RotarixTM vaccine, n = 159). The first and second doses were
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administered at 2 and 4 months of age, respectively, and children were bled
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14–16 days after each dose. Studies were approved by the Ethics Committee of
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the San Ignacio Hospital and Pontificia Universidad Javeriana.
6.7
focus-forming units of the attenuated RIX4414 human RV vaccine
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141
Human haplotype determination
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DNA was obtained from blood samples using Illustra blood genomicPrep Mini
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Spin Kit (G/E healthcare, UK Buckinghamshire), according to manufacturer’s
144
instructions. The HLA class II haplotype was determined using All set Gold-SSP
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HLA DRDQ low resolution Kit (Invitrogen Corporation, Wisconsin USA), PCR-
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based protocols, according to manufacturer’s instructions. All samples identified
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as a DRB1*01 in low resolution were analyzed for high-resolution using the All
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set Gold-SSP HLA DRB1*01 high-resolution Kit (Invitrogen Corporation,
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Wisconsin USA), according to manufacturer’s instructions. (Supplementary
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table 2).
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Antigen stimulation of PBMC and intracellular cytokine staining (ICS)
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PBMC were purified from heparinized whole-blood samples by Ficoll-Hypaque
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gradients (Lympho Separation Medium, MP Biomedicals). The cells were
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washed twice with RPMI containing 20 mM HEPES, 100 U of penicillin/ml, and
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100 mg of streptomycin/ml plus 10% fetal bovine serum (FBS) (all from GIBCO,
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Carlsbad, CA, USA) (complete medium) and re-suspended in 1ml of AIM-V
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medium (life technologies, Carlsbad, CA, USA). PBMC (1x106 cells in a final
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volume of 1 ml) were stimulated with the supernatant of MA104 cells infected
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with RRV (Rhesus RV, MOI:7), the supernatant of mock-infected MA104 cells
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(negative control), the superantigen staphylococcal enterotoxin B (SEB; Sigma,
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St Louis, Mo, USA), peptides pools (5 peptides per pool at 1 µg/ml each) or
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individual peptides at different concentrations. Anti-CD28 (0.5 µg/ml) and anti-
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CD49d (0.5 µg/ml) (Both from BD Biosciences, San Jose, CA) monoclonal
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antibodies were added to each sample as co-stimulators (Waldrop et al., 1998;
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Waldrop et al., 1997). Antigen stimulation was done in 15 ml polystyrene tubes
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(Becton Dickinson Falcon Labware, Franklin Lakes, N.J., USA) incubated with a
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5° slant for 10 h at 37°C with 5% CO2. The last 5 h of the incubation included
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brefeldin A (10 µg/ml; Sigma) to block the secretion of cytokines. At the end of
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the incubation, the cells were washed once with PBS–0.5% bovine serum
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albumin (Merck, Darmstadt, Germany) 0.02% sodium azide (Mallinckrodt
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Chemicals, Paris, Ky.) (Staining Buffer). Then, a 2 mM final concentration of
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EDTA (GIBCO, N.Y. USA) in PBS was added for 10 min to detach plastic-
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adherent cells, and the samples were washed once more with Staining Buffer.
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PBMC were stained with Aqua viability reagent (Invitrogen Molecular Probes,
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Eugene, OR) in PBS for 10 min at room temperature (RT), and the cells were
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stained with monoclonal antibodies (Mabs) against CD14-V500 and CD19-
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V500, as a dump channel, CD3-pacific blue, CD4-PerCP-Cy5.5, and CD8-APC-
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H7 for 20 min at RT (all from BD Biosciences, San Jose, CA). After two wash
180
steps with Staining Buffer, cells were treated with Citofix/Citoperm solution (BD
181
Biosciences, San Jose, CA) for 30 min at 4°C and washed twice with 1 ml of
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Perm/Wash solution (BD Bioscience, San Jose, CA). Then, Mabs against IL-2-
183
FITC, IFN--PE-Cy7 and TNF--APC (all from BD Biosciences) were added and
184
incubated for 20 min at RT. Production of IL-2 and IFN- by RV stimulated T
185
cells was observed in our previous work; TNF- secretion was also evaluated to
186
make a more comprehensive analysis of multifunctional T cells (Gattinoni et al.,
187
2011). Finally, the cells were washed twice with Perm/Wash, resuspended in
188
250 µl of Perm/Wash, acquired in a FACSAria flow cytometer (BD Biosciences,
189
San Jose, CA) and analyzed with FlowJo software v.9.3.2.
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191
Peptide binding assays
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Peptide binding to DR1 and DR4 was analyzed using an ELISA-based
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competition assay, as previously described (Parra-López et al., 2006). Purified
194
HLA-DR1 or DR4 molecules (0.05 µM) were diluted with freshly prepared
195
binding buffer (100 mM citrate/phosphate buffer [pH 5.4], 0.15 mM NaCl, 4mM
196
EDTA, 4% NP-40, 4 mM PMSF, and 40 µg/ml for each of the following protease
197
inhibitors: soybean trypsin inhibitor, antipain, leupeptin and chymostatin)
198
containing 0.025 µM biotin-labeled hemagglutinin HA306–318, a well described
199
binding peptide from Flu hemagglutinin (PKYVKQNTLKLAT) (Roche and
200
Cresswell, 1990), and various concentrations of unlabeled competitor peptide
201
(HA306-318,
202
(YNALIYYRYNYAFDLKRWIYL) and VP6-7 (DTIRLLFQLMRPPNMTPAVNA)) in
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a total volume of 120 µl. Peptides were diluted from stock solutions in binding
204
buffer. After 48h of incubation at RT, 100 µl were transferred to 96-well ELISA
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microtiter plates (Immuno Modules MaxiSorp, Nunc, Denmark), which had been
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previously coated overnight with a 10 µg/ml anti-HLA-DR Mab LB3.1, washed,
207
and subsequently blocked with PBS containing 3% bovine serum albumin. After
208
2 hours of incubation at RT, plates were washed with PBS, 0.05% Tween-20
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and incubated for 1h with phosphatase-labeled streptavidin (KPL, Maryland,
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USA). Captured biotin-labeled peptide/DR complexes were revealed with 4-
211
nitrophenylphosphate substrate (KPL, Maryland, USA). For determining peptide
212
binding to HLA-DR molecules a Ultramark ELISA plate reader (Bio-Rad, CA,
213
USA) with a 405 nm filter was used. Results at each concentration are
214
expressed normalized with respect to the maximum observed binding, and used
215
to calculate IC50 values.
NSP2-3
(SGNVIDFNLLDQRIIWQNWYA),
VP3-4
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Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay
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Proliferation of cells was evaluated by CFSE staining as reported by Quah
219
(Quah and Parish, 2010; Quah et al., 2007), with some modifications. Briefly, 4-
220
10x106 fresh PBMC were resuspended in 1 ml of PBS 1X, placed in a 15 ml
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conical tube, and stained with CFDA-SE (CellTrace™ CFSE Cell Proliferation
222
Kit). The cells were incubated 5 min at RT, protected from light, and then
223
washed 3 times with 10 ml of PBS 1X-FBS 5% at RT. Finally, the cells were
224
diluted in 1 ml of RPMI supplemented with 10% AB+ human serum (Multicell,
225
human serum AB, Wisent INC, Canada). After CFSE staining, PBMC were
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stimulated with SEB and three RV peptides (NSP2-3, VP3-4, and VP6-7) for 5
227
days at 37°C with 5% CO2. Then, cells were harvested, washed twice with PBS
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1X, and stained with Violet viability reagent (Invitrogen Molecular Probes,
229
Eugene, OR) in PBS for 10 min; CD3-PE, CD4-PerCP-Cy5.5 and CD8-APC-H7
230
were added and incubated for 30 min at RT. Finally, the cells were
231
resuspended in 250 µl of PBS, acquired in a FACSAria flow cytometer (BD
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Biosciences, San Jose, CA) and analyzed with FlowJo software v.9.3.2.
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234
T cell lines
235
T cell lines were generated from PBMC. 1-2x106 PBMC per well were incubated
236
in 24-well plates in 1 ml of RPMI 1640 supplemented with AB+ human serum
237
10% (Multicell), 100 U/ml penicillin, 100 g/ml streptomycin, 1 mM sodium
238
pyruvate, 2 mM L-glutamine, and 1 mM nonessential amino acids. Antigen-
239
specific populations were expanded by culture in the presence of NSP2-3, VP3-
240
4 or VP6-7 peptides (10 µg/ml) or RRV infected MA104 cell lysate (MOI:7).
241
After 48h of incubation at 37°C with 5% CO2, fresh T cell medium
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supplemented with 100 U/ml IL-2r (Proleukin; Chiron Corporation, Emeryville,
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CA) was added. This last step was repeated every 2 days until the culture
244
completed 12 days. Then, autologous non-irradiated PBMC (ratio 2:1), pulsed
245
with or without peptide (10 µg/ml) for 1h, were used as source of antigen-
246
presenting cells to stimulate the T cell lines. Finally, T cell lines were evaluated
247
by ICS, as described above. In some cases, the cells were stained as described
248
below with the class II tetramer.
249
250
Class II tetramer staining of PBMC from adults and children
251
Biotinylated HLA-DR1-peptide complexes were prepared using proteins
252
produced in insect cells, as previously described (Cameron et al., 2002). Class
253
II tetramers with different peptides (HA306-318, a transferrin [TRF] control peptide
254
[RVEYHFLSPYVSRKESP (Chicz et al., 1992)], NSP2-3, VP3-4 or VP6-7) were
255
prepared with streptavidin-PE (Invitrogen, MD, USA) from a stock solution at a
256
final concentration of 1 µg diluted in 50 µl of PBS 1X. Fresh PBMC obtained
257
from 8 DR1 healthy adults were washed twice in PBS 1X and distributed in 5 ml
258
polystyrene tubes (5x106 cells/50µl per tube). Frozen PBMC of 3 vaccinated or
259
3 placebo recipient HLA-DR1 children were thawed at 37°C and washed twice
260
with a 5 ml Benzonase® (Novagen, San Diego, CA) solution pre-heated at
261
37°C. Then, the cells were distributed in 5 ml polystyrene tubes. 50 µl of
262
Dasatinib® (Bristol-Myers Squibb Company Princeton, NJ 08543 USA) solution
263
(100 µM) was added to each tube and incubated for 30 min at 37°C (Lissina et
264
al., 2009). Later, 10 µl of AB+ human serum was mixed with the cells just before
265
tetramer solution (1 µg) was added and incubated for 120 min at RT. Unlabeled
266
mouse anti-CCR9 Mab was added and incubated for 20 min at RT, then the
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cells were washed once with PBS 1X, followed by addition of goat anti-mouse
268
antibodies (AF-488 or Pacific Blue [Invitrogen Corporation, Wisconsin USA])
269
and incubated for 20 min at RT. The cells were washed once with PBS 1X, then
270
Aqua reagent was added and incubated for 10 min at RT; subsequently, Mabs
271
against CD14-V500 and CD19-V500, as a dump channel, CD3 (Pacific Blue or
272
AF-700), CD4-PerCP-Cy5.5, CD8-APC-H7, CD45RA (FITC or PE-Cy7),
273
CD62L-V450 or CCR7-PE-Cy7, and 47-APC (ACT-1) were added and
274
incubated for 20 min at RT. Cells were washed and resuspended in 400 µl of
275
PBS-BSA-sodium azide, acquired in a FACSAria flow cytometer (BD
276
Biosciences, San Jose, CA) and analyzed with FlowJo software v.9.3.2.
277
278
ELISA for RV-specific IgA in plasma
279
For detection of RV-specific IgA, 96-well vinyl micro titer plates were coated
280
with 70 µl of a 1:10 dilution (in PBS, pH 7.4) of supernatant from RF (bovine
281
RV) virus-infected MA104 cells or the supernatant of mock-infected MA104 cells
282
(negative control) and incubated overnight at 4°C. The wells were then blocked
283
with 150 µl of 5% nonfat powdered milk plus 0.1% Tween-20 in PBS (5%
284
BLOTTO) and the plates were incubated at 37°C for 1 h. Then, the BLOTTO
285
was discarded, and 70 µl of serial plasma dilutions in 2.5% BLOTTO were
286
deposited in each well. After 2h of incubation at 37°C, the plates were washed
287
three times with PBS-Tween-20, and 70 µl of biotin-labeled goat anti-human IgA
288
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted (1:1000) in 2.5%
289
BLOTTO was added to the plates. The plates were then incubated for 1h at
290
37°C. After three washes with PBS-Tween-20, 70 µl of streptavidin-peroxidase
291
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted (1:1000) in 2.5%
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BLOTTO was added, and the plates were incubated for 1h at 37°C. After three
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washes with PBS-Tween-20, the plates were developed using 70 µl of
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tetramethyl benzidine substrate (TMB; Sigma, St. Louis, Mo.). The reaction was
295
stopped by the addition of 17.5 µl of sulfuric acid (2 M). Absorbance was read at
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450 nm wavelength on an ELISA plate reader (Multiskan EK, Thermolab
297
Systems). Samples were considered positive if optical density (OD) in the well
298
was >0.1 OD units and were two times higher than the OD of the negative
299
control (Rojas et al., 2007).
300
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Statistical analyses
302
Analysis was performed with GraphPad Prism version 6. Differences between
303
groups were evaluated with the nonparametric Wilcoxon test.
304
305
RESULTS
306
307
Prediction of RV epitopes
308
We used a consensus approach that combined P9 binding and SYFPEITHI
309
presentation algorithms to predict HLA-DR1-restricted T cell epitopes (Calvo-
310
Calle et al., 2007) from the KU strain of RV (Table S1). Thirty-nine 9-mer
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sequences with high scores were selected: 11 were derived from non-structural
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proteins and the remaining 28 were derived from structural proteins (Table S1).
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Peptides were synthesized as 21-mers, containing the 9-mer sequence of
314
interest flanked by 6 aa residues on each side.
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Screening of peptide pools and identification of individual peptides
317
recognized by CD4 T cells from healthy adults
318
The 39 peptides were organized in 8 pools of 5 peptides each (pool 8 only had
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4 peptides) according to the algorithm score; the 5 peptides with the lowest
320
scores were arranged in pool 1, and the 4 peptides with highest scores were
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joined in the pool 8. We expected that HLA-DR1-peptides selected would be
322
recognized by CD4 T cells from individuals of other MHC haplotypes, because
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of the broadly specific “promiscuous” peptide binding motifs characteristic of
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most human MHC class II protein, that give rise to the concept of “MHC class II
325
supertypes” (Greenbaum et al., 2011). For this reason, all 18 volunteers
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selected for our screening experiments expressed MHC haplotypes belonging
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to the DR-1 supertype (Table S2) (Greenbaum et al., 2011).
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PBMC of 18 healthy volunteers were obtained and CD4 T cell responses to
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pools of peptides were evaluated by ex-vivo ICS of IFN- and IL-2. CD4 T cells
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from 4 volunteers (HA-02; HA-04; HA-05 and HA-06) responded against the
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peptide pools: cells from volunteer HA-06 recognized pools 7 and 8 (Fig. 1A),
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cells from volunteer HA-05 recognized pool 5 (CD4 IL-2+: 0,0232%), and cells
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from volunteers HA-02 (CD4 IL-2+: 0,0208%) and HA-04 (CD4 IL-2+: 1,284%)
334
recognized pool 7 (data not shown). Positive pools were deconvoluted to
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individual peptides using the same assay. Fig. 1B shows the frequency of cells
336
from volunteer HA-06 producing IL-2 in response to peptides from pools 7 and
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8. Three peptides (VP6-7, NSP2-3, and VP3-4) were found to induce IL-2
338
production by CD4 T cells from the HLA-DR1-volunteers: VP6-7 induced IL-2
339
production in cells from volunteers HA-06 (Fig. 1B), HA-02, and HA-04; and
340
also IFN- production in cells from HA-06 (data not shown). In addition, VP3-4
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341
induced IL-2 production in cells from volunteer HA-06 (Fig. 1B), and NSP2-3 in
342
cells from HA-04 (data not shown). No response was seen to individual
343
peptides of pool 5 from volunteer HA-05 (data not shown).
344
The percentage of cells producing IL-2 in response to VP6-7 peptide used to
345
stimulate PBMC from the volunteers HA-06 (Fig. 1C), HA-02, and HA-04 (data
346
not shown) was dose dependent. A similar result was obtained stimulating cells
347
from volunteer HA-06 with VP3-4 peptide (Fig. 1C), but no dose effect was
348
observed with cells from volunteer HA-04 stimulated with NSP2-3 (data not
349
shown).
350
351
RV peptides bind HLA-DR1 molecules
352
To evaluate if the initial 9-mer sequences defined using prediction algorithms
353
bind to recombinant DR1 and DR4 MHC molecules, the 9 aa core of the
354
peptides NSP2-3, VP3-4 and VP6-7, with 1aa addition at both ends, were
355
synthetized and tested for HLA-DR1 and HLA-DR4 binding in a competition
356
binding assay with a biotinylated high affinity DR1- and DR4-binding peptide
357
from Flu haemagglutinin HA306–318 (Fig. 2). Compared to HA peptide (IC50 96
358
nM), VP6-7 (IC50 2 nM) and NSP2-3 (IC50 15 nM) peptides bound to the HLA-
359
DR1 molecules with relatively higher affinity and VP3-4 peptide bound with
360
similar affinity (IC50 73 nM). In contrast, all RV peptides show low affinity binding
361
to the HLA-DR4 molecules (IC50 from 0.8 M to 55 M) (Fig. 2). Based on these
362
results, DR1 tetramers were synthetized with the RV peptides NSP2-3, VP3-4,
363
and VP6-7.
364
365
RV peptide VP6-7 is recognized by RRV specific T cell lines
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366
To evaluate if the selected peptides were processed and presented after viral
367
infection, RRV-T cell lines were derived from two HLA-DR1 healthy volunteers
368
(HA-02 and HA-41) and stained with the three RV DR1-tetramers. RRV-T cell
369
lines derived from both volunteers only stained with VP6-7-tetramers (Fig. 3A
370
and data not shown). In addition, after restimulation of RRV-T cell lines from
371
volunteers HA-02 and HA-52 with RRV and VP6-7 peptide, CD4 T cells
372
producing TNF- and/or IFN- were identified in both cases (data not shown).
373
Only one of these cell lines produced cytokines after restimulation with NSP2-3
374
and neither of them after restimulation with the VP3-4 peptide (data not shown).
375
376
RV peptides are presented to CD4 T cells in the context of HLA-DR
377
molecules
378
To determine if the three RV peptides were presented in the context of HLA-DR
379
molecules, peptide specific T cell lines from two individuals (HA-02
380
[DRB1*01:01]
381
corresponding or control peptides in the absence or presence of LB3.1 or SPV-
382
L3 antibodies (directed against HLA-DR or HLA-DQ molecules, respectively)
383
and IFN- production was evaluated by intracellular cytokine staining. The
384
frequency of CD4 T cells specific for VP6-7, NSP2-3 and VP3-4 producing IFN-
385
 decreased in the presence of LB3.1, but not in the presence of SPV-L3 (Fig.
386
3B and data not shown): IFN- production of VP3-4-T cell line from HA-02
387
volunteer was reduced approximately 55% in the presence of LB3.1 and only
388
6% in the presence of SPV-L3 (Fig. 3B). From volunteer HA-06, only a VP6-7
389
specific T cell line was obtained and the response of these cells was also
390
specifically inhibited by LB3.1 (data not shown). These results provide
and
HA-06
[DRB1*01:02])
were
restimulated
with
the
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391
additional evidence that RV peptides are presented in the context of HLA-DR
392
molecules, as indicated by tetramer staining.
393
394
VP6-7-MHC class II tetramers recognize functional peptide-specific CD4 T
395
cells in T cell lines
396
To provide additional evidence that VP6-7-tetramers recognize functional VP6-
397
7-specific CD4 T cells, T cell lines from two volunteers (HA-02 and HA-19) were
398
stained with tetramers (TRF or VP6-7) and restimulated with VP6-7 peptide or
399
DMSO (as negative control), and the production of IFN- and TNF- was
400
evaluated by intracellular cytokine staining. When the VP6-7-T cell line from
401
HA-02 was restimulated with VP6-7 peptide almost 70% of the VP6-7-tetramer
402
positive CD4 T cells produced cytokines (TNF- and/or IFN-), whereas in the
403
control stimulated T cell line only a low number (10%) of tetramer positive cells
404
produced cytokines (Fig. 3C). Similar results were obtained with a VP6-7- T cell
405
line derived from HA-19 volunteer (data not shown).
406
407
CD4 T cells from DR1 healthy adults proliferate and produce cytokines
408
after stimulation with RV peptides.
409
Fresh PBMC from eight DRB1*0101 (HA-02, HA-15, HA-19, HA-23, HA-25, HA-
410
41, HA-51 and HA-52) and two DRB1*0102 healthy volunteers (HA-04 and HA-
411
06) were stimulated with the three RV peptides and evaluated 10h later by
412
intracellular cytokine staining (IL-2, TNF- and IFN-), and 5 days later for
413
proliferation using CFSE staining. Five DRB1*0101 (HA-15, HA-19, HA-29, HA-
414
51 and HA-52) and one DRB1*0102 (HA-04) healthy adults had detectable
415
levels of cytokine producing CD4 T cells (Table 1). CD4 T cells from HA-15 and
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5
6
7
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416
HA-19 produced IL-2 after stimulation with VP6-7. CD4 T cells from HA-51 and
417
HA-52 produced TNF- after stimulation with VP6-7. Cells from HA-29
418
responded to VP3-4 stimulation producing TNF- and cells from HA-04
419
responded to VP3-4 producing TNF- and to VP6-7 producing IFN- (Table 1).
420
CD4 T cells of HA-02 proliferated in response to the three RV peptides (NSP2-
421
3, VP3-4, and VP6-7), whereas CD4 T cells obtained from HA-19 and HA-23
422
only proliferated to NSP2-3 stimulus. Thus, although approximately 60% of DR1
423
individuals have peptide-specific T cells detectable by intracellular cytokine
424
staining or proliferation, these responses do not seem to occur simultaneously.
425
426
Antigen experienced CD4 T cells of healthy volunteers stained with VP6-7-
427
tetramer are enriched in the populations expressing the intestinal homing
428
receptors
429
Fresh PBMC from the same eight DRB1*0101 healthy donors described above
430
were stimulated with the three RV peptides in order to generate specific T cell
431
lines. Specific T cell lines for VP6-7 were obtained in all eight individuals and
432
specific T cell lines for NSP2-3 and VP3-4 each in four individuals (Table 2). T
433
cell lines from three volunteers (HA-02, HA-51 and HA-52) were expanded with
434
three peptides; T cell lines from two volunteers (HA-15 and HA-19) were
435
expanded with two peptides and T cell lines from three volunteers only
436
recognized VP6-7 (Table 2).
437
To compare the phenotype of RV- and Flu-specific T cells of the volunteers in
438
which T cell lines were expanded, PBMC were stained with the TRF-, Flu-, and
439
the corresponding RV-peptide tetramers (Figs 4A and 4B). The relative
440
numbers of the CD4 T cells that stained with the tetramers VP6-7 and Flu
19
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38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
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65
441
(medians of 27/6.0x105 and 45/6.0x105 CD4 T cells, respectively) were higher
442
than those stained with the TRF-tetramer (median of 9/6.0x105 CD4 T cells)
443
(Wilcoxon test p=0.011 and p=0.003, respectively). Most of the CD4 T cells
444
stained with VP6-7- and Flu-tetramers (medians of 63.5% and 67.05%,
445
respectively) showed a phenotype of antigen experienced cells (CD62L
446
CD45RA
447
in the frequencies of these cells (Table 2, Fig. 4C). Antigen experienced CD4 T
448
cells stained with VP6-7-tetramer were enriched in the populations expressing
449
the intestinal homing receptors: 47 CCR9 and 47 CCR9 (Table 2, Figs
450
4B and 4D). In contrast, antigen experienced CD4 T cells stained with Flu-
451
tetramer were enriched in the 47 CCR9 population (Table 2, Figs 4B and
452
4D).
453
RV-tetramer
454
populations expressing the intestinal homing receptors
455
To evaluate if RV vaccination induced the expansion of RV-CD4 T cells of
456
children, PBMC from three HLA-DR1-vaccinated and three HLA-DR1-placebo
457
recipient children, obtained two weeks after the second dose of the RIX4414
458
human attenuated RV vaccine or placebo (Rojas et al., 2007), were stained with
459
the VP6-7- or control TRF-tetramers, and the same panel of markers previously
460
used in adults. Because of the limited amount of sample available from
461
vaccinated children only direct PBMC staining and not T cell line experiments
462
were performed. The vaccinated, but not placebo recipient children, had
463
detectable serum levels of RV-specific IgA after two doses of RV vaccine (data
464
not shown). In vaccinated children and placebo recipient children from 40-71%
465
and 0-8%, respectively, of VP6-7-tetramer+ cells had the phenotype of antigen-
+/-
+
-
-
and CD62L CD45RA ) and no statistical differences were observed
+
-
+
+
+
-
-
CD4 T cells in vaccinated children are enriched in the
20
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6
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65
466
experienced cells (data not shown). In both groups of children the CD4 T cells
467
stained with the TRF-tetramer only 0-30% of cells had this phenotype (data not
468
shown). In vaccinated children, VP6-7 antigen experienced CD4 T cells were
469
detected at low frequencies (0.001-0.1%). In all 3 cases, most of the antigen
470
experienced CD4 tetramer T cells expressed 47, and in two children (Fig.
471
5A, two top panels), most cells expressed both, 47 and CCR9, homing
472
receptors. In vaccinated children the TRF-tetramer (Fig. 5A, left panels) stained
473
from 4 to 10 times less antigen experienced cells than the VP6-7-tetramer (from
474
<0.0001-0.01%). In placebo recipients (Fig. 5B), VP6-7- and TRF-tetramers
475
detected antigen experienced CD4 T cells at similar low levels (<0.0001-
476
0.003%).
+
477
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478
DISCUSSION
479
We have identified a RV CD4 T cell epitope (VP6-7) and shown that HLA-DR1
480
tetramers loaded with this peptide, but not a Flu peptide, expressed intestinal
481
homing receptors (Fig. 4). Moreover, studies with a low number of vaccinated
482
children showed that this type of reagent might be useful to monitor the RV CD4
483
T cell response in vaccine trials (Fig. 5).
484
Of 39 predicted epitopes, three peptides were identified by screening of PBMC
485
from healthy adults by intracellular cytokine staining; all of them also bound to
486
HLA-DRB1*0101 molecules (Figs. 1 and 2). The VP6-7 peptide is probably
487
processed and presented after RV infection, since it was recognized by a
488
rotavirus-T cell line (Fig. 3A). Moreover, peptide specific T cell lines were DR-
489
MHC restricted (Fig. 3B) and VP6-7-tetramers were recognized by cells of
490
vaccinated but not placebo recipient children (Fig. 5), characterizing this peptide
491
as a RV epitope. This epitope overlaps totally with one previously found in mice
492
(Baños et al., 1997) and partially with a VP6 epitope found in Rhesus
493
macaques (Zhao et al., 2008), which suggests that this region is particularly
494
prone to be recognized by CD4 T cells. Although a RV specific class II restricted
495
human T cell epitope had been previously described (Honeyman et al., 2010),
496
our studies are the first to characterize epitope specific CD4 T cells with
497
tetramers.
498
HLA supertypes are defined as a set of HLA (class I or II) associated with
499
largely overlapping peptide/binding repertoires. Recently, three different class II
500
DR supertypes were classified (main DR, DR4 and DRB3) (Greenbaum et al.,
501
2011), and all of the healthy adults volunteers for our screening experiments
502
were selected for having the main DR supertype, which includes HLA-DR1 and
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7
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65
503
many other class II molecules. Contrary to our expectations, only HLA-DR1
504
healthy adults recognized three peptides. However, in one DRB1*0102
505
individual VP6-7 T cell line were generated and their stimulation was inhibited
506
by an anti-DR monoclonal antibody, suggesting that at this level promiscuity for
507
HLA binding exists. The levels of promiscuity of viral epitopes varies between
508
studies from low (Kwok et al., 2008; Nepom, 2012) to moderate (Roti et al.,
509
2008). Further studies are necessary to clarify the reasons for these
510
differences.
511
Ex vivo tetramer staining of PBMC from healthy adults showed that CD4 T cells
512
specific for the VP6-7 peptide expressed intestinal homing receptors. This result
513
is in agreement with our previous report, in which we observed that RV-specific
514
IFN- secreting CD4 T cells from adult volunteers preferentially express the
515
intestinal homing receptor α4β7 (Rojas et al., 2003). The present findings
516
extend these results by showing that RV-tetramer
517
(CD62L CD45RA
518
CCR9 and, thus, are prone to home to the small intestine. Compared to total
519
non-antigen specific T cells, VP6-7 specific T cells are enriched approximately
520
10 times in CCR9 expressing cells (Figs 4A and 4B), indicating that this is
521
indeed a unique subset. Further studies are necessary to determine if these
522
circulating dual 47 and CCR9 expressing RV-specific T cells have a unique
523
TCR repertoire.
524
The generation of RRV- or RV peptide-specific T cell lines support the results
525
obtained with ex vivo tetramer staining. Characterization of T cell lines
526
expanded with RRV showed that VP6-7 and NSP2-3 are processed and
527
presented by infected cells (Fig. 3A and data not shown). However, peptide-
+
-
+/-
+
antigen experienced
-
and CD62L CD45RA ) CD4 T cells express both 47 and
23
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4
5
6
7
8
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49
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53
54
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57
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65
528
specific T cell lines were obtained after stimulation of PBMC with all three
529
peptides (Table 1). Moreover, responses of T cell lines specific for the three
530
peptides generated from PBMC obtained from an HLA-DRB1*0101 healthy
531
adult were significantly blocked with an antibody against DR and poorly against
532
an antibody to DQ molecules (Fig. 3B and data not shown), suggesting that the
533
VP6-7 epitope and the two candidate RV-epitopes (NSP2-3 and VP3-4) were
534
presented in the context of HLA-DR molecules. T cell lines expanded with virus
535
lysate preparations might select clonotypes associated with immunodominant
536
peptides over low frequency and/or slowly growing clonotypes (Nastke et al.,
537
2012). Thus, in some of the experiments with the RRV-T cell lines the
538
responses to the NSP2-3 and VP3-4 candidate epitopes might have been
539
masked.
540
The simultaneous staining of the majority of cytokine secreting cells from a
541
VP6-7 T cell line with the VP6-7 tetramer (Fig. 3C) supports the specificity of the
542
tetramer staining. Moreover, they show that most cells stained with the tetramer
543
are functional. A correlation between cells staining with the tetramer and those
544
producing cytokines ex vivo is difficult to establish, because of the low or
545
inexistent level of cytokine secreting cells specific for the RV peptides (Table 1).
546
However, the capacity of the tetramers to identify specific T cells ex vivo seems
547
more sensitive than the intracellular cytokine staining and proliferation assays
548
(Table 1) and, thus, more suited to vaccine studies in children. Nonetheless, it is
549
possible that some of the cells stained with the tetramer might be secreting
550
cytokines not evaluated.
551
Staining of cells from vaccinated and placebo recipient children with the VP6-7
552
tetramer showed that VP6-7-specific CD4 T cells could be expanded after RV
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65
553
vaccination. Although the numbers of cells observed in the vaccinated children
554
were low (Fig. 5A), they were all, as expected, 47 and in two cases they also
555
expressed CCR9. The cells used in the present study had been frozen over
556
seven years (Rojas et al., 2007), and it is expected that studies with fresh cells
557
may permit to obtain more cells to study an increase in the sensitivity of the
558
assay. Further studies are necessary to confirm that CD4 T cells from HLA-DR1
559
vaccinated children recognize the RV epitope we have identified.
560
In conclusion, we have shown that CD4 T cells specific for a RV epitope
561
express intestinal homing receptors, which supports the hypothesis that cells
562
primed in peripheral compartments may constitute a separate lineage (Sallusto
563
and Lanzavecchia, 2009). We also describe MHC tetramers that could be used
564
to analyze RV-specific CD4 T cell responses in vaccine studies. Similar studies
565
in the context of other MHC haplotypes are needed to expand the number of
566
tetramers available to monitor the CD4 T cell response to RV vaccines and,
567
hopefully, develop better correlates of protection.
568
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569
ACKNOWLEDGMENTS
570
This work was financed by grants from Colciencias 1203-408-20481 and the
571
Pontificia Universidad Javeriana (ID5195). LJS and JMCC received support
572
from NIH grant U19-AI057319 and EB funding from R37-AI047822 and RC1-
573
AI087257 also from the NIH, which funded Ab production. Miguel Parra was
574
funded by a scholarship from Colciencias.
575
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690
Sturniolo, T., Bono, E., Ding, J., Raddrizzani, L., Tuereci, O., Sahin, U.,
691
Braxenthaler, M., Gallazzi, F., Protti, M.P., Sinigaglia, F., Hammer, J., 1999.
692
Generation of tissue-specific and promiscuous HLA ligand databases using
693
DNA microarrays and virtual HLA class II matrices. Nat Biotechnol 17, 555-561.
694
Tate, J.E., Burton, A.H., Boschi-Pinto, C., Steele, A.D., Duque, J., Parashar,
695
U.D., 2012. 2008 estimate of worldwide rotavirus-associated mortality in
696
children younger than 5 years before the introduction of universal rotavirus
697
vaccination programmes: a systematic review and meta-analysis. Lancet Infect
698
Dis 12, 136-141.
699
Vollers, S.S., Stern, L.J., 2008. Class II major histocompatibility complex
700
tetramer staining: progress, problems, and prospects. Immunology 123, 305-
701
313.
702
Waldrop, S.L., Davis, K.A., Maino, V.C., Picker, L.J., 1998. Normal human
703
CD4+ memory T cells display broad heterogeneity in their activation threshold
704
for cytokine synthesis. J Immunol 161, 5284-5295.
705
Waldrop, S.L., Pitcher, C.J., Peterson, D.M., Maino, V.C., Picker, L.J., 1997.
706
Determination of antigen-specific memory/effector CD4+ T cell frequencies by
707
flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism
708
in HIV-associated immunodeficiency. J Clin Invest 99, 1739-1750.
30
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57
58
59
60
61
62
63
64
65
709
Zhao, W., Pahar, B., Sestak, K., 2008. Identification of Rotavirus VP6-Specific
710
CD4+ T Cell Epitopes in a G1P[8] Human Rotavirus-Infected Rhesus Macaque.
711
Virology (Auckl) 1, 9-15.
712
713
714
31
1
2
3
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5
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7
8
9
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57
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59
60
61
62
63
64
65
715
FIG. LEGENDS
716
Fig. 1. CD4 T cells from one healthy adult recognize peptide pools and
717
individual RV peptides. PBMC from HA-06 were stimulated with peptide pools
718
(A), or individual peptides (B), at a concentration of 1 µg/ml or with different
719
doses (C) during 10h at 37oC and for the last 5h, 1 µg/ml of brefeldin A was
720
added. The frequencies of T cells producing IL-2 and IFN- were evaluated by
721
intracellular cytokine staining. SEB was used as a positive control. Responses
722
were considered positive if the number of IL-2 or IFN--producing CD4 T cells
723
was at least twice that of the DMSO control and above 0.02% (dashed lines). In
724
the experiments shown T cells did not produce IFN-
725
726
Fig. 2. RV peptides bind HLA-DR1 but not HLA-DR4. Competition binding
727
assays for RV (NSP2-3, VP3-4, and VP6-7) and Flu (HA306-318) peptides were
728
performed. The graphics show inhibition of binding of the biotinylated HA 306-318
729
peptide to HLA-DR1 (A) and HLA-DR4 (B) by increasing amounts of RV and flu
730
peptides. Results were analyzed using GraphPad Prism Software version 6.
731
732
Fig. 3. Response of RRV and peptide specific T cell lines. (A) A RRV-T cell
733
line was obtained from a healthy adult (HA-02), then restimulated with RRV,
734
NSP2-3, VP3-4 or VP6-7, and finally stained with the TFR-tetramer or the
735
corresponding RV-tetramers. Left panel: RRV-T cell line restimulated with RRV
736
and stained with the TRF-tetramer. Middle panel: RRV-T cell line restimulated
737
with NSP2-3 peptide and stained with the NSP2-3-tetramer. Right panel: RRV-T
738
cell line restimulated with VP6-7 peptide and stained with the VP6-7-tetramer.
739
This T cell line only recognizes the VP6-7 tetramer. (B). A VP3-4 specific T cell
32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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31
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37
38
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40
41
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49
50
51
52
53
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56
57
58
59
60
61
62
63
64
65
740
line from HA-02 was restimulated with DMSO (upper left panel), VP1-4 as a
741
control peptide (upper right panel), or VP3-4 in the absence (upper center
742
panel) or in the presence of an isotype control antibody (lower left panel), LB3.1
743
(lower center panel), or SPV-L3 (lower right panel) antibodies, which are
744
directed against HLA-DR or HLA-DQ molecules, respectively, and the
745
frequencies of cells producing IFN- were evaluated by intracellular cytokine
746
staining. (C) A VP6-7 specific T cell line from HA-02 was stimulated with DMSO
747
(left panels) or VP6-7 (middle and right panels) for 6h at 37oC and stained with
748
the VP6-7-tetramer (upper right and left panels) or TRF-tetramer (upper center
749
panels). Tetramer cells were evaluated by ICS for TNF- and IFN- production
750
(lower panels). Numbers inside all graphics represent percentages of
751
populations. Responses were considered positive if the frecuency of TNF-
752
and/or IFN--producing CD4 T cells was at least twice that of the DMSO control
753
stimulated cells.
+
754
+
755
Fig. 4. Phenotype and expression of homing receptors of CD4 tetramer T
756
cells of healthy adults. Fresh PBMC were obtained from HLA-DRB1*0101
757
healthy adults and stained with the Flu-tetramer (left panels), the TRF-tetramer
758
(center panels) or the VP6-7-tetramer (right panels) and antibodies against
759
differentiation markers and intestinal homing receptors. After gating on live CD3
760
T cells, total (A) or tetramer (B) CD4 T cells (top panels) were analyzed to
761
identify antigen experienced T cells CD62L CD45RA
762
(middle panels). The expression of 47 and CCR9 intestinal homing receptors
763
was evaluated in antigen experienced cells (lower panels). Numbers in
+
-
+/-
+
-
and CD62L CD45RA
33
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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24
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
764
parenthesis in the lower row dot plots indicate number of events. Results from
765
the eight volunteers for antigen experienced T cells VP6-7-tetramer and Flu-
766
tetramer cells (C) and the expression of intestinal homing receptors (D) are
767
summarized. Statistically significant differences determined with the Wilcoxon
768
test are shown.
+
+
769
-
770
Fig. 5. Expression of homing receptors on antigen experienced (CD62L
771
CD45RA
772
placebo recipient children. Frozen PBMC from HLA-DRB1*0101 RV
773
vaccinated (A) or placebo recipient children (B) were stained as in Fig. 4 with
774
the TRF-tetramer (left panels) and VP6-7-tetramer (right panels). Dot plots
775
show the expression of 47 and CCR9 intestinal homing receptors of CD4-
776
tetramer
777
parenthesis in the dot plots indicate number of events.
+/-
+
+
-
and CD62L CD45RA ) CD4 T cells from RV vaccinated and
antigen experienced cells from individual children. Numbers in
778
34
Figure
Click here to download Figure: figuras 221213.pdf
Figure 1
A.
16
14
12
% CD4-IL2+
10
8
1.5
1.0
SEB DMSO
1
2
3
4
5
6
7
8
NSP1-4
0.0
VP4-5
0.5
Peptide pool number
B.
20
18
14
12
10
0.05
0.04
0.03
0.02
NSP2-3
NSP1-5
VP6-7
NSP6-1
VP3-3
VP2-3
VP3-4
DMSO
0.00
SEB
0.01
C.
100
DMSO
SEB
VP1-1
VP3-4
VP6-7
10
1
% CD4-IL2+
% CD4-IL2+
16
0.1
0.01
0.0
0.5
Peptide Concentration ug/ml
1.0
Figure 2
% HA-Bio Peptide
A.
B.
120
120
110
110
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
100
101
102
103
Peptide Concentration (nM)
104
105
0
100
HA peptide
VP3-4a
VP6-7a
NSP2-3a
101
102
103
Peptide Concentration (nM)
104
105
Figure 3
!
C.
D.
CD4 Tet VP6-7+
CD4 Tet Flu+
% CD4 Tetramer+ cells
!
Figure 4
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
CD4 Tet+Antigen Exp.
0
p=0.0078
p=0.0195
p=0.0039
!4"7+CCR9+
!4"7+ CCR9-
!4"7-CCR9-
!4"7-CCR9+
Figure 5
B.
A.
TRF Tetramer
VP6-7 Tetramer
TRF Tetramer
CHP-1
CHV-2
CHP-2
CHV-3
CHP-3
α4β7
α4β7
CHV-1
CCR9
CCR9
VP6-7 Tetramer
Tables
Table 1. Cytokine production and proliferation of CD4 T cells
after stimulation with RV peptides.
Healthy Adult
Peptide
Stimulus
HA-02
NSP2-3
-
+
VP3-4
-
+
VP6-7
-
+
NSP2-3
HA-15
HA-19
HA-23
HA-29
HA-41
HA-51
HA-52
HA-04*
HA-06*
CD4 T cytokine
production
CD4 T
proliferation
-
-
VP3-4
-
-
VP6-7
IL-2(+)
-
NSP2-3
-
+
VP3-4
-
-
VP6-7
IL-2(+)
-
NSP2-3
-
+
VP3-4
-
-
VP6-7
-
-
NSP2-3
-
-
VP3-4
TNF-α(+)
-
VP6-7
-
-
NSP2-3
-
-
VP3-4
-
-
VP6-7
-
-
NSP2-3
-
N.D.
VP3-4
-
N.D.
VP6-7
TNF-α(+)
N.D.
NSP2-3
-
N.D.
VP3-4
-
N.D.
VP6-7
TNF-α(+)
N.D.
NSP2-3
-
N.D.
VP3-4
TNF-α(+)
N.D.
VP6-7
IFN-γ(+)
N.D.
NSP2-3
-
-
VP3-4
-
-
VP6-7
-
-
*HLA DRB1 0102 Healthy volunteer. N.D. Not Done.
+, at least two-times background values.
+
Table 2. Ex vivo phenotyping of Tetramer CD4 T cells of DRB1*0101 healthy adults with RV
peptide specific TCLs.
+
Healthy Adult
Peptide
Tetramer
#Tet /6.0x105
+
CD4 T cells
HA-02
TRF
8
54.5
HA-15
HA-19
HA-23
HA-29
HA-41
HA-51
HA-52
% Antigen % α4β7(+)
experienced CCR9(-) #
T cells*
% α4β7(+)
CCR9(+)#
% α4β7(-)
CCR9(-)#
% α4β7(-)
CCR9(+)#
33.3
33.3
33.3
0
FLU
36
68.4
42.3
7.69
50
0
NSP2-3
10
53.8
85.7
14.3
0
0
VP3-4
16
61.1
54.5
9.09
27.3
9.09
VP6-7
28
85.7
63.3
26.7
3.33
6.67
TRF
4
0
¥
¥
¥
¥
FLU
30
83.3
25
12.5
62.5
0
VP3-4
16
23.1
50
0
50
0
VP6-7
22
63.2
63.6
27.3
9.09
0
TRF
10
85.7
8.33
33.3
33.3
25
FLU
24
65.7
21.7
0
78.3
0
NSP2-3
18
48.1
30.8
7.69
61.5
0
VP6-7
26
64.1
28
4
64
4
TRF
8
57.1
25
25
50
0
FLU
60
91.2
32.7
3.85
61.5
1.92
VP6-7
24
87
75
15
10
0
TRF
14
50
0
50
50
0
FLU
48
59.3
6.25
12.5
81.2
0
VP6-7
32
38.9
57.1
14.3
28.6
0
TRF
3
100
33.3
0
66.7
0
FLU
42
97.1
3.03
0
90.9
6.06
VP6-7
25
67.3
50
0
50
0
TRF
98
36.7
44.8
20.7
27.6
6.9
FLU
155
40.6
50.7
15.5
31
2.82
NSP2-3
63
31.7
34.6
30.8
26.9
7.69
VP3-4
54
37.5
45.8
12.5
29.2
8.33
VP6-7
50
41.5
58.8
23.5
5.88
11.8
TRF
72
27.3
66.7
0
33.3
0
FLU
284
25.4
40.3
8.06
50
1.61
NSP2-3
140
17
55.6
11.1
27.8
5.56
VP3-4
149
22.6
35.7
0
57.1
7.14
VP6-7
134
16.5
31.2
25
43.7
0
+/-
+
-
-
* Antigen experienced cells are CD62L ,CD45RA and CD62L CD45RA (Figure 4B). # Percentages
correspond to antigen experienced cells. ¥, No antigen experienced cells were identified.
Supplementary Material (To be Published)
Click here to download Supplementary Material (To be Published): Suplementary tables 201213.docx
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