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The Innate and Adaptive Immune Response
to Measles Virus
By:
Nicole Putnam
A thesis submitted to Johns Hopkins University in conformity
with the requirements for the degree of Master of Science.
Baltimore, Maryland
April 2014
© Nicole Putnam
All Rights Reserved
i
Abstract
Measles is one of the most important causes of childhood morbidity
and mortality worldwide. Although a vaccine is available, the high
transmission rate of measles virus requires population of 95% to interrupt its
transmission. The World Health Organization and the United Nations
Children’s Fund recommend that children that develop measles receive
vitamin A supplementation, as a safe, cheap, and efficacious way to reduce
the burden of disease. Due to differences between strains and confounding
data of measles stocks contaminated with defective interfering RNA
particles, the immune response to measles virus infection has not been well
defined. Furthermore, the mechanism by which vitamin A protects against
severe measles-induced disease is unknown.
In this thesis, I investigate the innate and adaptive immune response
to measles virus infection. Measles virus strains were purified of defective
interfering RNA particles and used for in vitro infections of monocyte derived
dendritic cells. Gene expression changes of interferon-stimulated genes and
viral stress-induced genes, IFIT1 and Mx1, were upregulated in response to
infection with the Edmonston measles virus vaccine strain, as well as the
wild-type strains of Bilthoven, IC-B, and C- and V-protein knockout strains,
as compared to mock infected cells. Unexpectedly, there were no differences
between transcript levels of these genes between C and V protein knockout
strains and the respective wild-type infection. Additionally, the absence of
ii
type I interferon production supports the theory that measles virus induces
the transcription of these genes through the viral stress-induced pathway,
and not the interferon-stimulated pathway.
While a previous study had detected measles virus-specific IL-17producing T cells in measles virus-infected rhesus macaques, the Th17
response to measles virus has not been characterized. Th17 cell
differentiation was inhibited early after measles virus infection in vitro.
There was a significant decrease in IL-23A transcript ts and a significant
increase in IL-27 transcripts, both of which affect Th17 cell differentiation
negatively. However, in a rhesus macaque model of infection, a biphasic Th17
response was observed with peaks at days 18 and 56.
The effects of vitamin A supplementation following measles virus
infection on the immune response was explored in a rhesus macaque model
using supplemented and non-supplemented groups. While some data has yet
to be explored, major differences were not observed between the two groups
up to three months following infection, in regards to clearance of infectious
virus, immune cell composition, or immune cell function. Archived data will
elucidate the role of vitamin A in measles virus RNA persistence, and Th1
and T follicular helper cell responses. Data will continue to be analyzed out to
six months post infection. A larger cohort will be necessary to elucidate the
role of vitamin A in protection against severe disease and death due to
measles.
iii
Acknowledgements
First and foremost, I would like thank my advisor, Dr. Diane E.
Griffin, for allowing me to do my master’s research in her laboratory. Her
guidance and support was invaluable throughout my time here. I would like
to thank her for the opportunity to get involved in the dynamic, challenging,
and rewarding research that she had entrusted with me. Furthermore, a
huge thank you to Dr. Rupak Shivakoti for passing down his knowledge of
the basics of how to work with measles virus and acquainting me with Dr.
Griffin’s lab in general. Additional thanks go out to Rupak to teaching me
many, many techniques. Although he was available to ask questions while he
was here, it was helpful that he encouraged me to ―jump right in‖ and
conduct my experiments independently early on. I would like to also thank
Rupak for being responsive to questions much after he had graduated from
the laboratory, which was especially helpful.
I would like to thank Dr. Wendy Lin, for providing me with her
knowledge of the logistics of working with measles virus in rhesus macaques.
Her ability to pass down her understandings and techniques was invaluable.
Furthermore, I would like to thank Wendy for taking time from her career at
Columbia University to come down to Baltimore to meet with us, as well as
making herself available to talk about techniques or data analysis.
Importantly, this project would not have run as smoothly as it did without
the help of Ashley Nelson, the PhD student with whom I shared
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responsibility in this project. With Ashley’s flexibility to work around my
schedule, we were able to make sure the assays for the monkey study could
be completed and analyzed in a timely manner so I could complete my thesis
work. I would also like to thank Ashley for her support and friendship
throughout my time here!
The vitamin A/monkey study was largely a success due to the time and
effort of Dr. Bob Adams and Dr. Tori Baxter. I would like to thank them
immensely for their time and expertise in handling the monkeys, obtaining
samples, and for being flexible with their schedules around the holidays,
while also granting this project many of their early mornings.
I would like to give a huge thanks to my roommate, Dr. Cailin Deal,
who was able to provide me her knowledge and skills in so many areas of
virology and immunology as a whole. Her expertise in writing in science was
crucial to the process of editing my thesis, as well as her general knowledge
of techniques and data analysis. Furthermore, I would like to thank Debbie
Hauer for her assistance in teaching me techniques and processing samples
that were essential for my projects and this thesis.
I would like to extend my warmest thanks to the rest of Dr. Griffin’s
laboratory for being so welcoming to me as a master’s student, for passing
down their expertise and insight when I needed assistance, and for their
general support and friendship. Dr. Kim Shulz, Dr. Tori Baxter, Dr. Kirsten
Kulscar, Stephen Goldstein and Siva Manivannan, your help was
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instrumental towards my experience as a student in this laboratory.
Additionally, I would like to acknowledge Gui Nilaratanakul, Rachy
Abraham, and Nina Martin for their presence and livelihood in the lab.
Finally, I would like to thank my mother, father, and brother Ryan, as
well as my extended family and friends for providing their unwavering
support. As a little girl, my parents told me I could do whatever I put my
mind to, and when I decided to pursue a science and research their
enthusiasm was there to match my own. This thesis is the product of the
hard work and support of many people, and I would again like to extend a
tremendous thanks to all of the people by my side!
vi
Table of Contents
Abstract………………………………………………………………………………. ii
Acknowledgements……………………………………………………………….. iv
List of Tables……………………………………………………………………...... ix
List of Figures………………………………………………………………………. x
Chapter 1: Introduction to measles virus…………………………………… 1
Public health implications…………………………………………………... 2
Measles virus pathogenesis…………………………………………………. 2
Prevention of measles virus infection……………………………………... 3
Measles virus virology……………………………………………………….. 6
Measles virus infection………………………………………………………. 7
Defective replication of measles virus genome…………………………… 8
Innate immune response to viral infection……………………………….. 9
TLRs…………………………………………………………………….. 9
Cytoplasmic PRRs…………………………………………………… 10
Type I interferon…………………………………………………….. 11
Interferon-inducible antiviral proteins…………………………... 12
Innate immune response to measles virus infection…………………… 12
Role of dendritic cells……………………………………………….. 14
Adaptive immune response to measles virus infection………………... 14
Antibody response…………………………………………………… 15
T lymphocyte response……………………………………………… 16
Effector CD4+ T lymphocytes……………………………………… 16
Immunosuppression following measles virus infection……………….. 18
Figures………………………………………………………………………… 20
Chapter 2: Comparision of in vitro immune responses to wild-type
measles virus with C and V protein-knock out strains; wild-type and
vaccine strains of measles virus……………………………………………… 24
Introduction…………………….……………………………………………. 25
Measles virus immune evasion……………………………………………. 25
Block of type I interferon production…………………………………….. 25
Block of type I interferon signaling………………………………………. 27
Interferon-stimulated genes (ISGs) and virus stress-induced
genes (VSIGs)……………………………………………………..…………. 28
Role of dendritic cells in measles virus infection……………………….. 29
Defective interfering (DI) particles……………………………………….. 29
Th17 response to viral infection…………………………………………... 30
Th17 response to measles virus infection……………………………….. 30
Materials and methods……………………………………………………... 31
Results………………………………………………………………………… 38
Discussion…………………………………………………………………….. 46
Tables…………………………………………………………………………. 53
Figures………………………………………………………………………... 54
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Chapter 3: Effects of vitamin A supplementation on the immune
response and the Th17 response to measles virus infection in rhesus
macaques……………………………………………………………………………. 63
Introduction………………………………………………………………….. 64
Vitamin A and measles infection…………………………………………. 64
Role of vitamin A in CD4+ T cell differentiation……………………….. 66
Vitamin A supplementation……………………………………………….. 67
Vitamin A: Potential roles in improving measles outcome………..….. 69
Repair of lung epithelium………………………………………….. 69
Effect on lymphopenia and T cell-mediated viral clearance….. 69
Inhibition of viral replication……………………………………… 70
Enhanced antibody response……………………….……………… 71
Th17 response to measles infection………………………………………. 71
Materials and methods……………………………………………………... 72
Results………………………………………………………………………… 81
Discussion…………………………………………………………………….. 91
Tables…………………………………………………………………………. 98
Figures………………………………………………………………………. 100
Chapter 4: Discussion of the immune responses to measles virus
infection in vitro and in vivo………………………………………………… 120
The innate immune response to measles virus infection……………. 121
Type I interferon…………………………………………………… 121
The role of measles virus and its C and V proteins on interferonstimulated genes (ISGs) and virus stress-induced genes
(VSIGs)………………………………………………………………. 122
The early adaptive immune response to measles virus infection…... 122
Th17 regulatory cytokine expression to measles virus infection…… 123
The Th17 response to measles virus infection………………………… 124
The measles virus antibody response…………………………………… 124
The role of vitamin A on the immune response to measles virus
infection……………………………………………………………………… 125
References……………………………………………………………………….... 127
Curriculum vitae……………………………………………………………....... 144
viii
List of Tables
Chapter 2:
Table 1: PCR primers, targets, and cycling conditions for P gene sequencing
and detection of measles virus standard and defective genomes……………. 53
Chapter 3:
Table 1: PCR primers, targets, and cycling conditions for detection of the
measles virus N gene.…………….…………….…………….…………….………. 98
Table 2: Measles virus shedding in respiratory secretions…….…………….. 98
Table 3: IL-17A ELISAs…….……….…….……….…….……….…….………… 99
ix
List of Figures
Chapter 1:
Figure 1: Measles virus infection and pathogenesis…………..……………… 20
Figure 2: 2012 immunization coverage rates with measles-containing
vaccines in infants……….………………………………………………………….. 21
Figure 3: Measles virus structure, RNA genome, and replication………..… 21
Figure 4: Defective interfering particle formation from the measles virus
genome……………………………………………………………………………...… 22
Figure 5: Biopsies of the measles virus rash show CD4+ and CD8+
lymphocyte infiltration……………………………………………………………... 22
Figure 6: Measles virus RNA over the course of infection………………..…. 23
Figure 7: Potential mechanisms leading to measles virus-induced immune
suppression…………………………………………………………………………... 23
Chapter 2:
Figure 1: Signaling pathways leading to virus stress-inducible gene (VSIG)
induction…………………………………………………………………………….... 54
Figure 2: Generation of wild-type measles virus defective for the C or V
protein……………………………………………………………………………….... 55
Figure 3: Primer binding sites for sequencing……………………………….... 55
Figure 4: Sequencing confirms correct viral sequences. …………………..… 56
Figure 5: Gel of measles virus stocks. ………………………………………..… 57
Figure 6: Measles virus standard and DI genome PCR products of measles
virus stocks used in in vitro experiments. ……………………………………… 58
Figure 7: mRNAs for interferon-stimulated genes, IFIT1 and Mx1, are
upregulated in moDCs by the Edmonston measles virus vaccine strain and
Bilt Wt strain at MOIs of 0.4 and 4.0…………………………………………..… 59
Figure 8: mRNAs for interferon-stimulated genes, IFIT1 and Mx1, are
comparably upregulated moDCs in response to Wt measles virus, and its
respective C- and V-protein KO strains at MOIs of 0.01 and 0.1………….… 59
Figure 9: Positive regulators of Th17 cell differentiation, IL-1β, IL-23A and
IL-6, mRNA expression levels from moDCs in response to Edmonston and
Bilthoven measles virus infection at MOIs of 0.4 and 4.0………………….…. 60
Figure 10: Positive regulators of Th17 cell differentiation, IL-1β and IL-6,
mRNA expression levels from moDCs in response to wild-type measles virus
and its respective C- and V-protein knock out strains at MOIs of 0.01 and
0.1…………………………………………………………………………………….... 61
Figure 11: Negative regulators of Th17 cell differentiation, IL-27 and IL-10,
mRNA expression levels from moDCs in response to Edmonston and
Bilthoven measles virus infection at MOIs of 0.4 and 4.0..…………………… 61
x
Figure 12: Negative regulators of Th17 cell differentiation, IL-27 and IL-10,
mRNA expression levels from moDCs in response to wild-type measles virus
and its respective C- and V-protein knock out strains at MOIs of 0.01 and
0.1…………………………………………………………………………………….... 62
Chapter 3:
Figure 1: Vitamin A (retinol) status and usage is impaired during
infection……………………………………………………………………………… 100
Figure 2: Plasma retinol levels averaged between two rhesus macaques after
measles infection.………………………………………………………………..… 100
Figure 3: Time course of measles virus clearance…………………………… 101
Figure 4: Viremia is present by day 7, and is cleared in all animals by day
18. ………………………………………………………………………………….... 101
Figure 5: Change in total body weight over course of measles virus
infection.……………………………………………………………………….……. 102
Figure 6: Maculopapular rash was very robust on monkey 50Y on day 10
post-infection……………………………………………………………………….. 102
Figure 7: Rash histology of skin biopsies……………………………………... 103
Figure 8: Histology of lymph node biopsies…………………………………… 104
Figure 9: Vitamin A levels begin to drop at day 21 in the non-supplemented
group of monkeys (17Y, 31Y, 46Y) but remain stable in vitamin Asupplemented monkeys (14Y, 24Y, 50Y). ……………………………………… 105
Figure 10: Comprehensive blood counts and differential leukocyte counts
following rash.…………………………………………………………………….... 106
Figure 11: Frequency of CD4+ and CD8+ cells within the CD14-CD20- live
cell population, and CD4:CD8 cell ratio……………………………………...… 107
Figure 12: Measles virus H, N, and F protein-specific IFN-γ secreting T cells
peak at 21 days post-infection………………………………………………….... 108
Figure 13: Intracellular staining for IL-17A……..…………………………... 109
Figure 14: Intracellular staining for IL-21…………………………………… 110
Figure 15: Frequency of IL-17+ cells as a percentage of total CD4+ cells
peaked at day 18…………………………………………………………………... 111
Figure 16: Frequency of IL-21+ cells as a percentage of total CD4+ T cells
showed peaks at day 18 and day 39 post-infection…………………………… 112
Figure 17: RORγt expression was upregulated in CD4+ T cells by day 18
post-infection, and is higher in IL-17+ cells than IL-17- cells………………. 113
Figure 18: IL-21 production begins to increase by day 18, and is much
greater by day 56 post-infection in IL-17+ cells than IL-17- cells…..……… 114
Figure 19: IL-17A-secreting T cells are present in a biphasic response, with
an early peak at day 14 and a late peak at day 52.…………………….…..… 115
Figure 20: Measles virus-specific IgG as detected by ELISA………….…... 116
Figure 21: Total antibody- and measles virus-specific antibody-secreting
cells in PBMCs as detected by ASC assays………………………..…….….…. 117
xi
Figure 22: Total antibody- and measles virus-specific antibody-secreting
cells in BM as detected by ASC assays…………………………………………. 118
Figure 23: Neutralizing antibody response as detected by PRNTs……….. 119
xii
Chapter One:
Introduction to Measles Virus
1
Public health implications
Measles virus is one of the most important causes of childhood
morbidity and mortality throughout the world (1, 2, 3, 4). Mortality can result
from complications due to young age (9), viral dose as a result of
overcrowding (10) and immunosuppression (11). Additionally, mortality due
to measles can be further increased by malnutrition (12) and hyporetinolemia
(13). Death due to acute measles virus infection is most common in young
children, and is most often attributed to viral and bacterial secondary
infections that are acquired during a stage of measles-induced immune
suppression (1).
Measles virus pathogenesis
Measles virus infects cells in the respiratory mucosa, such as epithelial
cells, dendritic cells and macrophages (61, 62, 63). Infected immune cells then
traffic to local lymph nodes, where measles virus can establish a viremia and
disseminate into the blood stream through measles virus-infected cells (62).
From the blood stream, measles virus is spread systemically to various
tissues that are subsequently infected (Figure 1a).
The incubation period of measles lasts for approximately ten days.
This is followed by a prodromal phase characterized by generalized fever,
cough, coryza, conjunctivitis (7). Subsequent development of a maculopapular
rash on the trunk and limbs of the body can be used to clinically diagnose
measles (7). Clinical symptoms begin to develop after the systemic spread of
2
measles virus, and correspond to the development of adaptive immune
responses to the virus (Figure 1b, 1c).
During the prodrome and rash, measles virus can spread from the
infected host to susceptible individuals through direct transmission (7). This
allows measles virus to be spread in a susceptible population before measures
are taken to prevent transmission. Measles virus is a human virus that is not
found in any reservoir populations and is maintained in the population by an
unbroken chain of acute infections, as latent or persistent measles virus
infections do not have the ability to be spread to new hosts (6).
Before the development of a measles vaccine, measles was estimated to
claim between 5-8 millions deaths annually (6,7). Recently, measles deaths
globally have decreased to 158,000 in 2011, down 71% from 2000 (8). A safe,
efficacious measles virus vaccine is available, but it has proven difficult to
reach a high proportion of children in developing countries. In 2004 alone,
almost half of the estimated measles deaths were in sub-Saharan Africa (7).
Measles virus is one of the most highly contagious infectious agents, with
outbreaks occurring even in populations where only 10% of individuals are
susceptible (6).
Prevention of measles virus infection
Prevention of measles virus infection is important, because of the
significant case fatality rates. In different locations in Africa, case fatality
rates due to measles can range between 5-10% (3, 14). Vaccination against
3
measles virus infection as introduced in the 1960s, in the forms of attenuated
and killed vaccines (6). The killed virus vaccine was associated with
complications and withdrawn.
Currently, safe and efficacious live attenuated vaccines are available
either as a measles-only vaccine or coupled with other vaccine viruses, such
as mumps and rubella (MMR) (1, 7). Most measles vaccines currently in use
have been derived from the Edmonston strain of measles virus that was
isolated in 1954 by Enders and Peebles (7,19). Despite variations in
attenuation of vaccines and sequence differences among wild-type measles
virus strains, measles virus is an antigenically stable virus with only one
serotype (60).
Though vaccination is an important method of prevention, interruption
of measles transmission in a population requires that approximately 95% of
the population is immune (7,15). Notably, that doesn’t guarantee protection.
Once measles virus transmission has been halted in a geographical area,
introduction of measles virus and outbreaks can still be imported via an
infected individual (7). In 2007 in Quebec, Canada, population immunity was
estimated to be at 95% when an outbreak leading to 94 measles cases
occurred, when several groups of unvaccinated people became exposed to
measles virus (16). Clusters of unvaccinated individuals can create pockets of
susceptible individuals, allowing for sustained transmission of measles virus.
Recently, measles vaccination rates have dropped in some developed nations
4
due to complacency, public concerns of safety, and philosophical objections
(17,18).
Unfortunately, immunization rates upwards of 90% are difficult to
establish in many nations (Figure 2). Current vaccination strategies rely on
subcutaneous inoculation and are difficult to sustain in developing countries
for financial and logistical reasons (1). Attenuated measles vaccines are
inactivated by heat and light, which means that immunization requires a
cold chain (7). Furthermore, once the vaccines do reach these areas, trained
health care workers, sterile needles and syringes are needed for proper and
safe vaccination.
In developed nations, where there are few measles virus infections, the
measles vaccine is usually given in the form of the MMR vaccine at 12-15
months of age. However, in countries where measles is endemic, measles
vaccines are typically administered at 9 months of age (7). It is thought that
this is a reasonable time to vaccinate infants, because maternal antibodies to
measles virus begin to wane around 6 months, and the development of
protective antibody responses to measles vaccination is inhibited by maternal
antibody (7,20). Not all vaccinated individuals will develop protective
immunity, so a single dose of measles vaccine will not achieve levels of
population immunity necessary for the elimination of measles. To reach
levels of population immunity greater than 90%, it is necessary to provide a
second measles virus vaccination (7).
5
In regions of the world where measles remains endemic, the burden of
disease can be lessened through secondary prevention mechanisms. The
World Health Organization (WHO) and United Nations Children’s Fund
(UNICEF) have recommended two large doses of vitamin A to be given at the
time of measles diagnosis in children under 5 years of age (21, 22). Vitamin A
decreases morbidity and mortality due to measles infection, but the
mechanism is unclear (23-29).
Measles virus virology
Measles virus is a member of the family Paramyxoviridae, and is in the
morbillivirus genus. Measles virus has a 16 kb negative-sense RNA genome
that is non-segmented, encapsidated, and found within a lipid envelope (1).
The genome encodes six structural and two non-structural proteins (Figure
3b). The structural proteins are associated with the viral RNA and the
envelope (Figure 3a). The hemagluttinin (H) and fusion (F) glycoproteins, are
embedded in the lipid envelope and interact with host cells for attachment,
fusion and entry (7). The interior of the lipid bilayer is lined with the matrix
(M) protein. Inside the enveloped virus, the genomic RNA is maintained in a
helical nucleocapsid by interaction with the nucleocapsid (N) protein, which
also associates with the phosphoprotein (P) and large polymerase (L)
proteins. The gene encoding the P protein also encodes the two nonstructural proteins. The C protein is translated from a separate start codon
downstream in the gene, whereas the V protein is a product of RNA editing.
6
The C and V proteins regulate the cellular response to measles virus infection
(7).
Measles virus infection
Measles virus H protein is responsible for virus attachment to cellular
receptors, which determines the cell type specificity for infection through
receptor protein interactions. Three host cell receptors have been identified.
CD46 is ubiquitously expressed on all nucleated cells and can be utilized by
vaccine strains of measles virus for attachment (30). Signaling lymphocyte
activation molecule (SLAM) or CD150 is found on activated immune cells and
can interact with the H protein from wild-type measles virus and vaccine
strains; it is normally used as a co-stimulatory molecule (31). The association
between the H protein and nectin-4 has recently been discovered (32, 33).
Nectin-4 is an adherens junction protein expressed on epithelial cells. It is
possible that other host receptors play a role in measles virus infection, but
these are not yet identified. In persistent infections, glial cells and neurons in
the central nervous system are targeted, supporting the notion that other
receptors may be important in the infection process (1).
Fusion with the cellular membrane and entry into the cell requires
interaction between the F and H proteins, along with the cellular receptor (1).
Following fusion with the viral envelope, host cells express viral glycoproteins
at the cellular membrane (Figure 3c). This allows for subsequent fusion with
surrounding, uninfected host cells, forming giant cells. Giant cells are one
7
hallmark of measles virus infection, and although they do not occur in all cell
types, these multinucleated cells can be found in the lung, skin, and
lymphatic tissue (1).
Release of the measles virus genome into the cytoplasm occurs
following fusion of the viral and cellular membranes. In the cytoplasm,
transcription of the measles virus genome occurs first, producing mRNAs for
viral proteins. Once translation of the mRNA transcripts forms sufficient
nucleocapsid proteins to encapsidate other genomes, the viral RNA
polymerase begins to read through the intergenic regions to replicate the
genome. The negative strand RNA genome that enters the cell is used to form
a positive, antigenome strand template for replication of negative strand
RNA genomes. The new genomes are encapsidated by the N protein,
packaged with other structural proteins and released by budding from the
cellular membrane (Figure 3c).
Defective replication of measles virus genome
Replication of the measles virus genome can result in RNA forms other
than the full length antigenome and genomes. Defective replication results in
an incomplete form known as defective interfering (DI) particles or virus (55).
These forms may be produced as a method of attenuation, to allow
persistence in host cells (55). One theory of DI generation of negative-strand
viruses such as measles virus, proposes a 5’ copy-back model (55) (Figure 4).
In this model, the polymerase copying the template RNA begins to form the
8
negative sense measles virus genome, but the polymerase detaches from the
template strand and resumes replication on the nascent chain. This partially
synthesized chain is then copied back and a stem-loop structure is formed as
the final RNA product (55). DI particle formation in vitro is partially
dependent on the cell type used to grow measles virus (56). The frequent
presence of DI RNA in measles virus stocks has confounded data from several
in vitro studies looking at the innate immune response (1).
Innate immune response to viral infections
Measles virus infection introduces foreign RNA and proteins into the
cell. Typically, during viral infections the host immune system will respond to
these structures, pathogen-associated molecular patterns (PAMPs), that are
recognized by host pathogen recognition receptors (PRRs) (34). The activation
of PRRs results in cell signaling to produce various cytokines, which affect
the immune environment in the host (35). In response to many viral
infections, these cytokines are often pro-inflammatory and include
interferons (34). Cytokines that are released early in response to a viral
infection are important for inducing an antiviral state to protect uninfected
cells and to modulate the induction of the adaptive immune response (34).
TLRs
Virus antigens and RNA at the cell surface or in the endosome, may be
detected by Toll-like receptors (TLRs). In response to measles virus, several
TLRs may be activated. TLR2 is found on the cell surface and interacts with
9
viral glycoproteins (39). TLR3 can sense double-stranded RNAs in the
endosome, though its role in the antiviral response remains unclear (39, 157).
TLR7, which is also expressed on the endosome, has been implicated in the
innate antiviral response by detecting single-stranded RNA (158).
TLRs dimerize after binding their ligands, causing conformational
changes and allowing for the recruitment of adaptor molecules. Differential
recruitment of adaptor molecules to these TLRs leads to the activation of
distinct signaling pathways (39). MyD88 is the adaptor protein responsible
for the production of proinflammatory cytokines, whereas TIR-domaincontaining adaptor protein-inducing IFN-β (TRIF) leads to production of type
I interferons (39). TLR2 stimulation results in an inflammatory response, but
not an antiviral response, because TLR2 interacts with MyD88 but not TRIF.
Conversely, MyD88 does not interact with TLR3, but TRIF does. In fact, most
virus-infected cells use a TLR3-triggered mechanism to produce type I
interferon through its TRIF-dependent pathway (39). TLR7 and TLR8 can
also interact with MyD88 to generate proinflammatory cytokines, and TLR7
can also interact with TRIF to induce production of type I interferon as well
(39).
Cytoplasmic PRRs
Once the virus has reached the cytoplasm, it will no longer be detected
by the TLRs (39). Instead, RNA helicases are the predominant PRRs involved
in virus recognition. These helicases are retinoic acid inducible gene I protein
10
(RIG-I) and melanoma differentiation antigen 5 (MDA-5) (35). Inside the
virus-infected cell, RIG-I can recognize single-stranded RNAs with 5’triphosphate or short double-stranded RNAs and MDA-5 can detect long
dsRNAs (36, 37, 38). Studies using RIG-I-deficient cells have revealed that
RIG-I is essential for induction of type I IFN responses following many RNA
virus infections (40).
Following activation, these RNA helicases associate with the adaptor
protein IPS-1 via their CARD domains (39). IPS-1 then functions to activate
specific kinases that phosphorylate Interferon-regulatory factors, IRF-3 and
IRF-7 (6). IRF-3 is constitutively expressed, whereas IRF-7 must be
transcriptionally activated along with other interferon-induced genes. The
phosphorylated IRFs then translocate to the nucleus and function as
transcription factors leading to the expression of type I interferons (39, 42).
Type I interferon
Type I interferons are a family of cytokines that include 12 subtypes of
IFNα, IFNβ, IFNε, IFNκ, and IFNω (43). Primarily referring to IFNα and
IFNβ, type I interferons are produced in direct response to many viral
infections by the mechanisms outlined above (34). In response to TLR
stimulation, type I interferons are produced by macrophages, conventional
dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and epithelial
cells (43). However, once the virus is cytoplasmic, multiple cell types may
11
produce interferon through interaction with RNA helicases and other
intracellular PRRs (43).
After production and release, type I IFNs bind to the two-chain IFNα/β
receptor IFNAR1/IFNAR2. Receptor binding induces dimerization and
phosphorylation, followed by the phosphorylation of receptor-associated
Janus kinases (Jaks), Jak1 and Tyk2 (34). Jaks then phosphorylate signal
transducer and activator of transcription proteins (STATs), STAT1 and
STAT2 (34). Phosphorylated STAT1 and STAT2 heterodimerize and interact
with IRF-9 to form the transcription factor interferon-stimulated gene factor
3 (ISGF3). ISGF3 binds the interferon stimulated response element (ISRE) to
activate the transcription of interferon stimulated genes (ISGs) (34).
Interferon-inducible antiviral proteins
ISGs encode proteins that have varying abilities to inhibit viral growth
and spread through distinct mechanisms (41). Antiviral proteins that have
well-established roles in measles virus infection are protein kinase R (PKR),
2’,5’-oligoadenylate synthetase, and Mx1 (41). PKR becomes activated by
binding dsRNA and plays a role in inhibiting virus translation (44), OAS is
involved in cleaving viral RNA, and Mx1 is involved in sequestering viral N
proteins to prevent encapsidation of the viral genome (42, 45).
Innate immune response to measles virus infection
Many innate immune responses have been implicated in response to
viral infection. Measles virus is unique in the immune responses that are
12
activated upon infection. The innate immune response to measles virus
infection has not been well defined, due to differences observed in vitro and in
vivo to different strains of measles virus (1). Measles virus replication is
sensitive to the inhibitory effects of interferon in vitro (51), and some studies
have reported the production of type I interferon in vitro in response to
measles virus infection (46, 47, 48, 53). These studies could be confounded by
the presence of DI RNA in measles virus stocks, which are potent inducers of
interferon (1, 57).
However, generally little or no type I interferon is produced in vivo
during the acute response to measles virus infection (49, 50). Measles virus
efficiently inhibits the induction of interferon and interferon signaling in
infected cells (1). Nonstructural proteins V and C encoded by measles virus
interact with cellular signaling pathways to interfere with the host interferon
response.
PRRs involved in the recognition of measles virus infection include
RIG-I and MDA-5 (35). However, the V protein encoded by paramyxoviruses
can bind MDA-5, interfering with downstream activation of the interferon β
promoter (39). RIG-I plays an important role in the recognition of measles
virus, most likely through detection of leader RNA sequences (35). It remains
unclear as to why little or no interferon production is observed after
activation of RIG-I and interference with MDA-5.
13
TLR2 detects the wild-type measles virus hemagglutinin protein (39,
52). Activation of TLR2 by the wild-type H protein, but not the measles
vaccine H protein, leads to the production of TLR-responsive genes IL-1α/β,
IL-6, and IL-12 p40 in monocytes, and also increases expression of measles
virus receptor SLAM (CD150) (52). These signaling events contribute to
enhanced immune activation and measles virus spread. Cytokines IL-1 and
IL-8 have been detected in plasma of patients with measles (1).
Role of dendritic cells
Dendritic cells play a multifaceted role in the immune response to
measles virus. They are able to contribute to both the innate and adaptive
immune responses and bridge these processes through the production of proinflammatory cytokines as well as their ability to function as efficient
antigen-presenting cells (APCs) (77). Dendritic cells contribute to the innate
immune response by producing pro-inflammatory cytokines. Dendritic cells
are present at mucosal surfaces (77), such as the lung, and can become
infected with pathogens such as measles virus through direct infection or by
phagocytizing infected cells in the airways. Infection of dendritic cells
promotes their maturation, leading to increased expression of MHC class II
molecules (78).
Adaptive immune response to measles virus infection
Mature dendritic cells play a role in activating the adaptive immune
response by migrating to local lymph nodes, where they function as
14
professional APCs. Peptides from measles virus are presented to naïve T cells
to induce differentiation and expansion into effector T cell subsets (58, 59).
The inflammatory environment established by innate immune cells allows for
the expansion and differentiation of antigen-specific effector T cells that
encompasses a large portion of the adaptive immune response.
Antibody response
Measles virus infection elicits several adaptive immune mechanisms to
resolve the infection. Measles virus-specific antibody and T cell responses
appear at the same time as the characteristic rash (Figure 1). B cells are a
major component of the adaptive immune response, and are activated
through the interaction of their surface antibody B cell receptor and helper T
cells (74). Measles virus-specific IgM antibody is the sole isotype produced for
approximately a week, between 10-17 days, and then persists for about 2
months (70). The majority of these early antibodies are specific to the
nucleocapsid protein (71). Measles virus-specific B cells then undergo classswitch recombination in germinal centers to produce measles virus-specific
IgG and IgA antibodies.
Somatic hypermutation in germinal centers allows for selection of B
cells that produce antibodies with high affinity for measles virus antigens
(73). N-specific antibodies are the predominant specificity. The IgG and IgA
antibodies specific to measles virus H and F glycoproteins can be neutralizing
15
and protective (70). M protein-specific antibodies are detected at low levels
(71), and are not neutralizing (70).
T lymphocyte response
Biopsies of the measles virus rash show CD4+ and CD8+ lymphocyte
infiltration into areas of skin epithelium that are infected with measles virus
(Figure 5). The CD8+ cellular response is also present in the blood during
this time, and these cells are thought to be most important for clearance of
infectious measles virus (64). This is supported by studies in rhesus
macaques where CD8+ T cell depletion (65), but not to B cell depletion (66),
leads to prolonged viremia after measles virus infection. CD8+ T cells have
two types of effector mechanisms that contribute to clearance of measles
virus, including cytotoxicity and IFN-γ production (1). Clearance of infectious
measles virus occurs shortly after the rash fades (Figure 6).
Though infectious measles virus has been cleared by approximately
two weeks post-infection, measles virus RNA persists in peripheral blood
mononuclear cells (PBMCs), urine and/or nasopharyngeal aspirates of
hospitalized children (67, 68) and rhesus macaques (69) for months following
measles virus infection. It is thought that measles virus RNA remains
present after recovery due to slow clearance by the immune response (1).
Effector CD4+ T lymphocytes
T-helper subsets are effector cells with varying immune functions.
They are activated by the interaction with antigen presented in MHC class II
16
molecules by APCs (73). These CD4+ T helper lymphocytes can differentiate
into Th1, Th2, Th17, T regulatory cells (Tregs), or T follicular helper cells
(Tfh). Differentiation to a specific T helper cell lineage is a result of the
surrounding immune environment. Induction of Th1 cells are promoted by
IL-12, Th2 by IL-4, Th17 by IL-23, TGF-β for Tregs, and IL-6 and IL-21 for
Tfh cells (77, 83).
In response to measles virus infection, the roles of Th17 and Tfh cell
subsets have not yet been elucidated. Before and during the rash due to
measles virus infection, a Th1 response dominates the CD4+ T lymphocyte
population. Th1 cells function by producing high levels of IFN-γ and IL-2, and
are observed during the rash (75, 79). Th1 cells are considered an important
host defense mechanism to protect against and clear viral infection (73). After
the rash subsides, Th1 cytokines return to normal levels and plasma IL-4
levels increase along with the Th2 subset (75).
Th2 cells are characterized by their ability to produce cytokines IL-4,
IL-5, and IL-13 (73). IL-4 can remain elevated in some patient plasma
samples for seven weeks after measles virus infection (75). A mixed Th1/Th2
response has also been reported with significant IL-10 production. IL-10 may
be a product of monocytes, macrophages or CD4+ CD25+ Tregs (50). A
significant increase in the numbers of T regulatory cells was observed in
these patients (50).
17
Immunosuppression following measles virus infection
A state of immune suppression is another clinical feature that follows
measles virus infection. There are several factors that contribute to this
period of immunosuppression that are not completely understood. Following
measles virus infection, little, if any type I interferon is produced (84, 85).
There are abnormalities in the number and function of lymphocytes, due to
apoptosis and impaired proliferation (85). Furthermore, impairment of
maturation and antigen presentation by dendritic cells may lead to decreased
T cell activation (84, 85).
The interaction of measles virus H protein with its receptor, CD150,
results in the inhibition of IL-12 production by dendritic cells (86).
Additionally, cross-linking the CD46 receptor decreases the production of IL12 by monocytes (85). The suppression of the IL-12 response could lead to a
decrease in induction of Th1 cells (85, 86). Furthermore, the effector cytokine
IFN-γ produced by Th1 cells inhibits the proliferation of the Th2 subset, and
conversely IL-4 and IL-10 produced by Th2 cells inhibit Th1 cytokine
production (80, 81). The shift from a Th1 to a Th2 response early after
infection will suppress activation of macrophages and proliferation of T cells,
and may prevent the host from mounting an effective Th1 response upon
subsequent exposure to new pathogens, leaving the host more vulnerable to
these exposures (85, 76).
18
Furthermore, a prolonged presence of IL-10 and regulatory T cells
could play a role in immune suppression as well (50). Tregs contribute to a
favorable environment for opportunistic infections, and also can suppress T
cell responses that clear viral infections (82). There are probably multiple
mechanisms that play a role in suppression of the immune response following
measles virus infection (Figure 7). The decreased ability of the immune
response to clear virus may also be contributing to the persistence of measles
virus RNA in cells for months after infection.
19
Figure 1. Measles virus infection and pathogenesis. (a) Diagram
outlines spread of measles virus and associated viral titer, as measles virus
spreads from the initial site of infection, the respiratory epithelium, to the
local lymph nodes and blood, from which measles virus is spread
systemically. (b) Clinical symptoms are outlined in this panel as they appear
following measles virus infection. (c) This diagram summarizes the immune
response to measles virus infection over time [7].
20
Figure 2. 2012 immunization coverage rates with measles-containing
vaccines in infants. The figure was compiled using data provided by the
WHO (2012).
Figure 3. Measles virus structure, RNA genome, and replication. (a)
Picture detailing the organization of measles virus and the association of the
structural proteins as labeled in the table. (b) Measles virus single stranded,
negative sense RNA genome, with genes in different colors and letters
indicating proteins made by the labeled gene. (c) Diagram depicting measles
virus entry, transcription and translation, and replication and budding [7].
21
Figure 4. Defective interfering particle formation from the measles
virus genome. 5’ copy-back mechanism is outlined during replication of the
negative strand RNA genome, the polymerase becomes detached from the
antigenome template strand and resumes replication on the nascent chain.
This results in a final DI RNA form with a stem-loop structure [156].
Figure 5. Biopsies of the measles virus rash show CD4+ and CD8+
lymphocyte infiltration. Rash (a) due to measles virus infection of the skin
epithelium. (b) Hematoxylin and eosin staining shows immune cell
infiltration in regions infected epithelial cells, with an arrow pointing to
mononuclear cells. Immunoperoxidase staining of biopsy samples show CD4+
(c) and CD8+ (d) T cells in brown [1].
22
Figure 6. Measles virus RNA levels over the course of infection. Early
after measles virus infection, viremia is established with relatively high
levels of infectious measles virus in the blood. Following the rash phase, this
is cleared, but non-infectious measles virus RNA remains persistent for
several weeks and is slowly cleared [1].
Figure 7. Potential mechanisms leading to measles virus-induced
immune suppression. These include apoptosis of lymphocytes, impaired
lymphoproliferation, increase in production of immunomodulatory cytokines
IL-4 and IL-10 by monocytes, downregulation of IL-12 production in
monocytes, and impaired differentiation and antigen presentation by
dendritic cells (85).
23
Chapter Two:
Comparision of in vitro immune responses to wild-type measles virus
with C and V protein-knock out strains; wild-type and vaccine
strains of measles virus
24
Introduction
Infection with measles virus leads to a well-established sequence of
events following the incubation period, progressing through the prodromal
phase and transforming into the characteristic rash, followed by a period of
immune suppression. Though these generalized phases are well established,
the immune responses during the early innate period have been elusive. This
is due in part to difficulty in study of phases prior to rash in vivo because
infection is not recognized at this stage and the confounding of in vitro
studies by DI in virus stocks. Many viruses encode proteins involved in
evading the host immune response, and measles virus has this ability as well.
Measles virus immune evasion
Like other morbilliviruses, measles virus encodes nonstructural
proteins within the P gene, the V and C proteins. These proteins counter host
innate defenses to measles virus infection (88, 89, 90). Most notably, they
prevent the production and signaling of type I interferons, the main innate
cytokines produced during most viral infections (87).
Block of type I interferon production
IFNα/β transcription is regulated primarily by IRF3, IRF7 and NFkB
transcription factors. Measles virus interferes with the activities of these
transcription factors to suppress target gene expression and subsequent
production of type I interferon. The nuclear factor κB (NF-κB) transcription
factors play a role in the regulation and efficiency of IFN-β transcription, as
25
well as several other innate immune system responses (91, 92). Measles virus
proteins P, V, and C interfere with gene expression dependent on NF-κB, in
response to activation of several immune receptors, including the tumor
necrosis factor (TNF) receptor, RIG-I-like receptors or TLR receptors (93).
The measles virus V protein has the most robust ability to suppress NF-κB
activity, whereas the presence of P and C proteins lead to a moderate NF-κB
inhibition (93). Some studies have suggested that the measles virus C protein
can inhibit the interferon response (41, 94), whereas others have not found
this result (15).
The V protein specifically interacts with the NF-κB subunit p65. This
interaction prevents the translocation of the transcription factor complex to
the nucleus. The NF-κB complex is maintained in the cytoplasm by the V
protein, which also binds STAT2, IRF7 and MDA-5 via its cysteine-rich Cterminal domain. The V protein C-terminal domain interactions with these
host molecules are sufficient for the inhibition of gene transcription by NF-κB
(93). Furthermore, the measles virus V protein can interfere with the
signaling of TLR7 and TLR9 by binding IκB kinase alpha and transcription
factor IRF7, which is necessary for type I interferon production (35).
MDA-5 and RIG-I are cytoplasmic PRRs used by the host in the
recognition of viral nucleic acids (35). The V protein encoded by measles virus
can bind MDA-5 and interfere with recognition of viral RNA and downstream
activation of the interferon β promoter (35, 39). RIG-I is important for
26
production of type I interferon in response to RNA virus infection (40), and
RIG-I recognizes measles virus RNA (35). However, the lack of a type I
interferon response due to measles infection suggests interference with
signaling after RIG-I activation. Downstream in this pathway, IRF-7 is
normally activated through phosphorylation, but it is bound by the measles
virus V protein, which prevents translocation of this transcription factor to
the nucleus important for type I interferon transcription (6, 35).
Block of type I interferon signaling
Measles virus V protein also targets interferon signaling pathways (87,
106). Recently, the V proteins from several morbilliviruses were explored for
their ability to block the responses to type I and type II interferons. Measles
virus V protein blocks the activity and phosphorylation of Tyk2 necessary for
signaling of type I interferon (106). Co-immunoprecipitation studies have
shown that both Tyk2 and Jak1 interact with the measles virus V protein to
prevent the subsequent activation of the STAT transcription factors
necessary for type I interferon signaling and induction of an antiviral state
(106).
Strains of measles virus have slight differences in their P, V and C
protein sequences, and therefore have varying abilities to interfere with the
signaling of type I interferons (106). The IC-B wild-type strain of measles
virus inhibits interferon α signaling by means of the common N-terminal
domain of the P and V proteins (87). The IC-B wild-type measles virus C
27
protein does not interfere with Jak-Stat pathway of type I interferon
signaling (41, 87, 89, 90), although the C protein of the Edmonston measles
virus vaccine strain does inhibit this signaling pathway (94).
The measles virus C protein is thought to regulate the synthesis of
viral RNA (89). It is possible that the C protein of measles virus complexes
with the ribonucleoprotein (RNP) complex, comprised of the N, P and L
proteins, however data on this are inconsistent (95, 96). Either way, the role
of the C protein in regulating synthesis of measles virus RNA could be an
indirect mechanism to suppress induction of interferon (89). It is likely that
measles virus requires the varying mechanisms of the V and C proteins to
sustain viremia, and fully evade the host type I interferon response (89, 97).
Interferon-stimulated genes (ISGs) and virus stress-induced genes
(VSIGs)
ISGs include hundreds of genes whose expression is induced by the
action of interferon (98). However, some genes referred to as ISGs also fall
into a category of virus stress-inducible genes (VSIGs), that are stimulated
using different mechanisms (98). Viral infection and dsRNA can induce
certain VSIGs by an interferon-independent mechanism (153) (Figure 1).
One important VSIG is IFIT1, also known as ISG56. IFIT1
transcription is induced by viral stress quickly and transiently (99). IFIT1
binds and sequesters single stranded viral PPP-RNA, decreasing viral
replication (154, 155). Another VSIG is Mx1, an interferon-induced GTPase
28
localized in the cytoplasm that restricts replication of negative stranded RNA
viruses (100). Mx1 has been implicated in antiviral protection against
paramyxoviruses, and measles virus replication is sensitive to inhibition by
Mx1 in a cell type-specific manner (101).
Role of dendritic cells in measles virus infection
Dendritic cells are an important determinant of the host immune
response to measles virus infection. CD150+ dendritic cells are implicated
early in infection and can become infected directly by measles virus or
through phagocytosis of infected cells in the airway (61, 63, 77). They
contribute to the innate immune response by producing pro-inflammatory
cytokines, and establish a specific cytokine environment for antigen-specific
effector T cells to develop during the adaptive immune response. Mature
dendritic cells function as professional APCs in lymph nodes, where they
present measles virus peptides in MHC class II molecules to naïve CD4 T
cells to induce differentiation and expansion into effector T cells (58, 59). To
study the immune response to measles virus infection, in vitro studies have
used monocyte-derived dendritic cells (61).
Defective interfering (DI) particles
DI particles are formed through a defective replication, where the
polymerase detaches from the antigenome template while forming the
negative strand measles virus genome and then copies back on the nascent
chain it is forming (55). DI particles can efficiently induce the production of
29
type I interferon (57). The presence of DI RNA in measles virus stocks used
for in vitro studies have complicated reports of the immune response to
measles virus infection (1).
Th17 response to viral infection
IL-17-producing cells are produced in response to HIV infection in
humans (146), and to herpes simplex virus and respiratory syncytial virus
infections in mice (147, 148). IL-17 plays a role in regulating the
inflammatory response to these viral infections. Th17 cells promote viral
persistence in chronic virus infections, through an IL-17-mediated
upregulation of anti-apoptotic molecules, which promote cell survival of virusinfected cells and confer resistance to cytotoxic T cells (152).
Th17 response to measles virus infection
It is currently unknown whether the Th17 effector T lymphocyte
population is involved in the immune response to measles virus. Bi-phasic
development of measles virus-specific IL-17-producing cells has recently been
described in infected vaccinated and unvaccinated rhesus macaques (102).
Th17 cells differentiate in the presence of TGF-β in an inflammatory
environment, consisting of IL-6 or IL-1β, and IL-21 that promotes Th17 cell
differentiation through a feedback mechanism (77, 103). The development of
Th17 cells is inhibited by the Th1 effector cytokine IFN-γ (150). The
production and signaling of type I interferons has a similar antagonistic
effect on Th17 cell development (151). Additionally, IL-27 produced by
30
dendritic cells, monocytes or endothelial cells can suppress the development
of Th17 cells (105, 106).
Differentiation of Th17 cells leads to the upregulation of the receptor
for IL-23 (IL-23R), allowing for the signaling of the cytokine IL-23, which is
necessary for their survival (77). The production of the anti-inflammatory
cytokine IL-10 inhibits IL-23 production and therefore the establishment of
permanent Th17 cells (104). RORγt is the transcription factor associated with
the Th17 cell lineage, and is necessary for the expression of IL-23R and the
production of the Th17 cytokines, IL-17 and IL-22 (77). Th17 cells that
produce IL-22 have been permanently differentiated through signaling by IL23 (77). The cytokine IL-17 promotes the inflammatory response, by inducing
mediators of inflammation and leading to recruitment of neutrophils (149).
Materials and Methods
Viruses
The wild-type measles virus strain IC-B and Edmonston measles
vaccine strain were used to create the corresponding C and V knockout
strains using site-directed mutagenesis of the infectious cDNA by Dr. Roberto
Cattaneo’s lab (97). The C protein knockout strains were created using sitedirected mutagenesis to eliminate the AUG start codon by mutating this
sequence to ACG, as well as inserting a UAG stop codon downstream of the
start site (Figure 2). The V protein knockout strains were created by
mutating the RNA editing site from AAAAAGGG to AAAGAGGG, causing
31
this site to become nonfunctional, and inserting a stop codon downstream of
that site (Figure 2). These six measles virus strains were provided to our
laboratory by Dr. Cattaneo for further experimentation.
Sequencing
To sequence each virus, viral RNA was isolated using the QIAamp®
Viral RNA Mini Kit (Qiagen). The SuperScript® III One-Step RT-PCR with
Platinum® Taq (Invitrogen) was then used to amplify a 1,681 base pair
sequence, using the MVP 1745 forward primer and the MVP 3426 reverse
primer to flank the P gene (Table 1). The PCR products from each reaction
were purified using the QIAquick® PCR Purification Kit (Qiagen), following
the manufacturer’s protocols. The NanoDrop® ND-1000 spectrophotometer
was used to measure DNA concentrations and 150 ng of PCR product was
used as the template for sequencing reactions. The primer MVP1745 was
used to hybridize just upstream of the C protein start codon, and MVP2373
hybridizes just upstream of the RNA editing site for V protein synthesis
(Figure 3). Primer sequences can be found in Table 1. Two separate
sequencing reactions were done on the wild-type measles virus IC-B and
Edmonston virus strains, to sequence both of these areas. The C protein
knockout strains were amplified only with the MVP1745, whereas the V
protein knockout strains were amplified only with MVP2373. The products of
these reactions were sequenced by the JHMI Synthesis and Sequencing
Facility.
32
Cells
Vero, Vero/hSLAM, and WI-38 cells, human lung fibroblast cell line
(ATCC), were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco®)
and B95a cells (145) were grown in Roswell Park Memorial Institute 1640
(RPMI-1640, Gibco®). Cells were grown in incubators at 37°C, 5% CO2, and
all media were supplemented with 10% heat inactivated fetal bovine serum
(FBS), 1% penicillin/streptomycin and 1% L-glutamine.
Detection of DI RNA
Viral RNA was isolated from each virus stock, using the QIAamp®
Viral RNA Mini Kit (Qiagen). The SuperScript® III One-Step RT-PCR with
Platinum Taq (Invitrogen) was used according to the manufacturer’s
directions, using primers (JM396, JM402) specific for the measles virus
standard genome and for (JM396, JM403) the measles virus 5’ copy-back DI
RNA genome (Table 1). A 1% UltraPure™ Agarose (Invitrogen) gel was used
to run PCR products stained with 5X dye, in relation to the I KB Plus ladder
(Invitrogen).
Purification of DI-free viruses
All six original virus stocks were tested and found to be positive for
defective interfering (DI) particles using reverse-transcriptase PCR (Figure
5). Plaque purification was used to purify DI-free virus from stocks that
contained the DI genome. Six-well plates were infected in triplicate with each
virus using serial dilutions from 10-1 to 10-7 for one hour and overlayed with a
33
mixture of one part 1.2% bacterioagar and one part 2X MEM supplemented
with 2% FBS, 1% penicillin/streptomycin and 1% L-glutamine, before
incubating for 6 days at 37°C, 5% CO2. Plaques were detected by eye, and
individual plaques were isolated and added to a T25 flask with a confluent
monolayer of Vero/hSLAM cells. The amount of plaqued virus added to each
flask varied, ranging from 3 plaques per flask to 1/16th of a plaque. Fractions
of a plaque were added to flasks by first dissociating a plaque in 1 ml of
media and adding only a portion of that media to the flask to be infected.
Virus was propagated for five to ten days, until 70-80% of the flask exhibited
cytopathic effects, as observed by syncytia formation, cell clearings or dead
floating cells. All cells and media were used for the new virus aliquots. Cells
were separated from the media, and then subjected to three cycles of 15minute exposure to dry ice, each followed by thawing. After the last thaw,
media was added to resuspend any cell-associated virus that was freed in this
process and pooled with the original media supernatant from the flask, then
aliquoted as a new virus stock.
Each new virus stock was tested for DI particles. Virus strains that
continued to show the presence of DIs after several rounds of plaque
purification, were then grown in different cell lines thought to be less
permissive to DI formation. Rather than using Vero/hSLAM for in vitro
growth of the wild-type viruses, a semi-adherent subline of the B95a
marmoset B-lymphoblastoid cell line, B95-8 susceptible to infection with WT
34
measles virus, was used (2). The vaccine strain does not rely on the SLAM
(CD150) receptor for entry and was grown in Vero cells, which contain the
receptor CD46 necessary for the binding of measles vaccine strains (30). In an
attempt to obtain DI-free stocks, virus was also grown in the WI-38 cell line,
a human fibroblast cell line derived from embryonic lung tissue (56).
PBMC isolation
Leukopaks were obtained from healthy human adult donors at the
Johns Hopkins Hospital Blood Bank. Blood was diluted 1:3 with 1X PBS and
layered on top of Ficoll-Paque PLUS (GE Healthcare) for gradient
centrifugation. PBMCs were isolated from this gradient and any remaining
red blood cells were lysed during a 5-minute incubation with ACK Lysing
Buffer (Quality Biological, Inc). PBMCs were resuspended in 1X RPMI-1640
4% heat inactivated human AB serum (Lonza), 1% penicillin/streptomycin,
1% 200 mM L-glutamine, 1% 100 mM sodium pyruvate.
Collection of monocytes and differentiation to dendritic cells
PBMCs were fractionated using the AutoMACS pro cell sorter
(Miltenyi Biotec) and human anti-CD14 microbeads (Miltenyi Biotec) to
positively select for monocytes. Monocytes were differentiated by culturing 1
million cells/ml with 500 U/ml of recombinant human GM-CSF (R&D
Systems) and 1,000 U/ml of recombinant human IL-4 (R&D Systems) for 6
days at 37°C, 5% CO2 to create monocyte-derived dendritic cells (moDCs).
35
Infection of monocyte-derived dendritic cells
Wild-type measles virus and its C- and V-protein knockout strains
Infections of moDCs were done in Costar 96-well round bottom plates
(Corning) with 2x105 cells per well. DI-free wild-type measles virus with its
respective C- and V-protein knockout viruses obtained from plaque
purification were used for these infections. Variations of this experiment
were conducted four times. Cells were infected with each virus strain at
multiplicities of infection (MOIs) of 0.1 or 0.01, in duplicate or triplicate.
Samples were collected 12- and 24-hours following infection, with one
experiment including samples collected at 2- and 48-hours post-infection as
well. All samples were frozen at -80°C. Cells were collected as pellets, with
replicate wells pooled. Supernatant fluids were also gathered, with one
experiment pooling duplicate samples and the remaining three experiments
collecting each well’s supernatant individually. Each experiment included
non-infected controls, where samples were collected in the same manner.
Edmonston vaccine and Bilthoven wild-type measles virus strains
PBMC isolation, positive selection of monocytes, and differentiation of
monocytes to dendritic cells was done by Dr. Rupak Shivakoti using the same
methods described above. He followed the same infection protocols, but
infected with the Edmonston vaccine strain or the Bilthoven wild-type strain
of measles virus, each at MOIs of 0.4 and 4.0. Post-infection time points were
collected at 2, 12, 24 and 48 hours, also using uninfected moDCs as controls.
36
Cells were collected as pellets, with replicate wells pooled into one sample.
These samples were archived by Rupak and were used for the rest of the
experiments as described.
Measurement of mRNAs
Cell pellets from all moDC infections were used for RNA isolation
using the RNeasy® Plus MicroKit (Qiagen). The Taqman® RNA-to-CT™ 1Step Kit (Applied Biosystems) was used according to directions of the
manufacturer. Taqman® Gene Expression Assays and PrimeTime® Std
qPCR Assays were used to detect GAPDH, IL-28 (IFNλ2), IL-29 (IFNλ1),
IFN-β, and ISG56 (IFIT1) and Mx1 mRNAs. The RNAs from Rupak’s measles
virus infected moDCs, and RNA from one of the replicate experiments, were
analyzed using the Taqman® Gene Expression Assays and PrimeTime® Std
qPCR Assays to detect GAPDH, IL-28 (IFNλ2), IL-29 (IFNλ1), ISG56 (IFIT1),
Mx1, IL-23A, IL-6, IL-1β, IL-27, and IL-10.
Plates were read using the 7500 Real Time PCR System and relative
quantification was done using the 7500 System Software with GAPDH as the
endogenous control for amplification. The presence of mRNA for these genes
was calculated using Ct values and reported as fold-change relative to noninfected conditions.
Interferon bioassays
Supernatant fluids were analyzed for interferon production by
bioassays. Vero cells were grown overnight in Costar 96-well flat-bottom
37
plates (Corning). Vero cells were then incubated with either supernatant
samples, recombinant human interferon alpha A (rhIFN-α 2A, PBL
Interferon Source) or recombinant human interferon beta (IFN-β 1A, PBL
Interferon Source) for 24 hours at 37°C, 5% CO2. The following day, cells
were challenged with vesicular stomatitis virus expressing green fluorescent
protein (VSV-GFP, a gift from Sean Whelan at Harvard Medical School,
Boston, Massachusetts) at an MOI of 1.0 for 24 hours at 37°C, 5% CO2. The
negative control wells included cells that were incubated in media only and
were not challenged with VSV-GFP, and the positive control wells were
incubated in media only and were challenged with VSV-GFP.
Following infection, cells were trypsinized and washed with PBS 1%
FBS before being fixed in PBS 1% FBS 1% formaldehyde. Results were
analyzed using the BD FACS Canto II™ flow cytometer by reading signals of
VSV-GFP-infected cells on the FITC channel using BD FACSDiva Software.
Interferon standards were run as controls to show protection from infection
by inducing an antiviral state in the cells. Four different dilutions were used
for each standard; IFN-α 2A was used at 500, 50, 5, and 0.5 units/well, and
IFN-β 1A was used at concentrations of 500, 5, 0.5, and 0.05 units/well.
Results
Sequencing and DI-status of measles virus stocks
Virus stocks obtained from the lab of Dr. Roberto Cattaneo (Mayo,
Rochester, MN) were sequenced to confirm the presence of the desired
38
mutations in knock out (KO) strains and the absence of mutations in the
wild-type (Wt) and vaccine measles virus strains (Figure 4). These viruses
were created by site-directed mutagenesis (97). The start codon of the
measles C protein was mutated (AUG  ACG) to prevent translation.
Additionally, a stop codon was introduced slightly downstream of this site
(UGG  UAG), leading to the C protein KO (C KO) measles virus strain. The
measles virus V protein knockout (V KO) strains were developed by mutating
the RNA editing site (AAAAAGGG  AAAGAGGG) to prevent editing of the
P gene to produce the V protein, while retaining the ability to translate the
full length P protein. A stop codon was also introduced slightly downstream
in V protein frame (AGA  UGA) (97).
All measles virus strains had the correct sequence (Figure 4). Both the
Wt measles virus strain and Edmonston measles vaccine strain (Edm)
preserved the C protein start codon and V protein RNA editing site, with no
insertions of stop codons downstream of these sites. Wt C KO and Edm C KO
strains contained mutations in the start codon sequence and a stop codon in
the C protein reading frame. Sequencing of Wt V KO and Edm V KO strains
confirmed the mutations to the RNA editing sites and the premature stop
codon in the V protein reading frame.
Each measles virus strain was tested for the presence of DI particles
by PCR, and 5 of 6 virus strains were contaminated, with only the Edm
strain being DI-free (Figure 5). All six strains of measles virus were plaque
39
purified in order to obtain measles virus stocks free of DI particles and
subsequently grown to increase the stock viral titer, by infecting at a low
MOI (0.003-0.0001).
Four measles virus strains: Wt MV, Wt C KO, Wt V KO, and Edm,
were successfully isolated without DI particle contamination. The DI-free Wt
MV stock used for experiments was isolated after two rounds of plaque
purification and the DI-free Wt C KO virus stock used was isolated after one
round of plaque purification. These viruses grew to titers between 104 and 107
pfu/ml. The Wt V KO strain was more difficult to isolate free of DI particles,
at high enough titers. For these reasons, three different Wt V KO stocks were
used for in vitro experiments. An initial plaque purification of Wt V KO
strains yielded a low titer, DI-free virus that was subsequently grown at
MOIs of 0.0003 and 0.0001 on Vero/hSLAM cells, which yielded DI-containing
virus stocks. After one more round of plaque purification, two DI-free Wt V
KO stocks were isolated. The last DI-free Wt V KO stock was isolated after
two more rounds of plaque purification. Figure 6 shows gels with PCR
products for the measles standard genome and the DI genome from each
virus used for in vitro assays.
Infection of monocyte-derived dendritic cells
Interferon assays
To determine the effects of measles virus infection with and without C
and V on interferon production and interferon-stimulated gene (ISG)
40
induction, moDCs were infected with Wt, Wt V KO and Wt V KO strains.
Data on mRNA expression of type III interferons λ1 and λ2, also known as IL28 and IL-29, were not reported, because the results were highly variable and
no trends were observed (data not shown). Primers used for IFN-β did not
differentiate between genomic RNA and mRNA. Although a control sample
without reverse transcriptase was used, the signal from IFN-β began to
amplify at a very similar Ct value compared to the normal RT-PCR
conditions, making it difficult to determine if the reaction was amplifying
mRNA (data not shown).
Interferon bioassays were done to detect the presence of type I
interferon in supernatants from the four study replicates. No significant
protection was observed (data not shown). Although this suggests that no
interferon was present in the supernatants, the control recombinant
interferon samples prevented infection with VSV-GFP only at the higher
concentrations so sensitivity of the assay was an issue. Recombinant IFN-β
1A provided complete protection from infection at 500 units/well and partial
protection at 5 units/well, whereas IFN-α 2A conferred only partial protection
at the highest dilution of 500 units/well. It is possible that interferon α/β is
present in supernatant samples, but below the limit of detection with this
assay.
mRNA gene expression changes in monocyte-derived dendritic cells
Induction of cytokine and ISG mRNAs was explored by qPCR on RNA
41
isolated from infected monocyte-derived dendritic cells, specifically
investigating ISGs/VSIGs IFIT1 and Mx1, as well as positive (IL-1β, IL-6, IL23) and negative regulators (IL-27, IL-10) of Th17 differentiation. Time
points at 12- and 24-hours post-infection were collected for four replicates of
infection using Wt, Wt CKO, and Wt V KO at MOIs of 0.1 and 0.01. For
infections using Edm and Bilt at MOIs of 4.0 and 0.4, samples were collected
following infection at 2, 12, 24, and 48 hours and were done in duplicate.
Interferon-stimulated genes (ISGs) and viral stress-induced genes (VSIGs)
Edmonston vaccine and Bilthoven wild-type measles virus strains
For both Edm and Bilt at both MOIs, IFIT1 mRNA was upregulated
approximately 30-fold at 2 hours post-infection (Figure 7). For Edm and Bilt
at high MOIs (4.0), IFIT1 was further upregulated at 12, 24, and 48 hours
post-infection. At 24 hours, IFIT1 mRNA expression peaked, and was higher
for an MOI of 4 than 0.4. Edm had a more robust response than Bilt, with
upregulation at an MOI of 0.4 as well as 4.0 (3000X), whereas Bilt increased
FIT1 primarily at an MOI of 4 (1000X) (Figure 7a).
Mx1 mRNA was not increased until 12 hours and was maximal at 24
hours. A virus dose-response was exhibited for each strain of measles virus,
though none achieved statistical significance. Overall, infection with Edm
exhibited higher expression, with Mx1 mRNA upregulated approximately
400-fold at an MOI of 4, while Bilt infection led to a 100-fold Mx1 mRNA
42
upregulation (Figure 7b). Overall, the Mx1 pattern was similar to that of
IFIT1.
Wild-type measles virus and its C- and V-protein-knockout strains
For wild-type measles virus and the corresponding C- and V-protein
KO strains, only lower MOIS of 0.1 and 0.01 could be used for infection. The
patterns of IFIT1 and Mx1 mRNA increases at both MOIs were similar
(Figure 8). IFIT1 mRNA levels were not increased from baseline at 12 hours
but by 24 hours post-infection, all viruses upregulated IFIT1 mRNA except
Wt V KO at the low (0.01) MOI.
Mx1 mRNA expression was upregulated approximately 5-fold at the
24-hour time point for each Wt virus at the higher MOI (0.1), although only
the Wt V KO strain showed a statistically significant difference from baseline
(Figure 8b). An increase in Mx1 mRNA expression was not observed at low
MOIs (0.01) for Wt or KO infection. Statistically significant increases in
IFIT1 or Mx1 gene expression were observed only in the absence of C and V
proteins at the high MOI (0.1) (Figure 8).
Positive regulators of Th17 differentiation
Edmonston vaccine and Bilthoven wild-type measles virus strains
Expression of positive and negative regulators of Th17 differentiation
was investigated in the RNA samples from Edm- and Bilt-infected moDCs
(Figure 9, 11) and Wt-, Wt C KO-, and Wt V KO-infected moDC mRNAs
(Figures 10, 12).
43
mRNA expression of IL-1β, IL-23A, and IL-6, positive regulators of
Th17 differentiation, were explored following measles virus infection with
Edm and Bilt (Figure 9). IL-1β mRNA was upregulated only in response to
Edm at a high MOI (4.0) where a 6-fold increase in IL-1β mRNA expression
was observed at 48 hours, though this was not statistically significant (Figure
9a). The primers used to detect IL-23A detected genomic DNA and mRNA.
For Edm and Bilt infections, large enough differences in the Ct values were
observed between the control lacking reverse transcriptase, and the normal
RT-PCR conditions to be confident that induction could have been detected
(Figure 9b). At 12 hours post-infection, with the high MOI (4.0) of Bilt, IL23A transcripts were significantly downregulated, otherwise there was little
evidence that either Edm or Bilt altered IL-23A mRNA expression (Figure
9b).
A robust increase in expression occurred of IL-6 mRNA after infection
with Edm and Bilt at both MOIs, but none reached statistical significance
(Figure 9c). At 2 hours post-infection, all viruses showed approximately 30fold upregulation of IL-6 mRNA. By 48 hours post-infection, Edm infection
led to a substantial increase in IL-6 mRNA expression with the high MOI
inducing IL-6 mRNA approximately 106-fold and the lower MOI
approximately 104-fold compared to levels in mock-infected moDCs. There
was variability in the IL-6 mRNA expression following infection with Bilt and
overall little evidence of induction.
44
Wild-type measles virus and its C and V protein-knockout strains
The effects of the C and V protein deletion on induction of positive and
negative regulators for Th17 expression were also investigated a tMOIs of 0.1
and 0.01 (Figure 10, 12). IL-1β and IL-6 mRNAs were downregulated at 2
hours but were generally back to baseline by 12 hours (Figure 10).
Upregulation of IL-1β mRNAs were observed at 12 or 24 hours post-infection,
for infection at the high MOI (0.1) (Figure 10a). The Wt measles virus had an
IL-1β mRNA fold-induction around 30 at 24 hours post-infection. The C and V
protein-KO measles virus strains showed lower levels, ranging between 6and 12-fold, respectively. IL-6 mRNA expression demonstrated the same
trends, with similar fold changes as for IL-1β (Figure 10b). The numbers of
replicates did not allow for any statistical analyses.
Negative regulators of Th17 differentiation
Edmonston vaccine and Bilthoven wild-type measles virus strains
RNA expression of IL-27 and IL-10, negative regulatory cytokines for
the development of Th17 cells, were explored (Figure 11, 12). IL-27 mRNA
expression was increased compared to mock-infected moDCs (Figure 11a),
while IL-10 mRNA was not (Figure 11B). For both Edm and Bilt measles
virus strains, the high MOI (4.0) infection increased IL-27 transcripts 2 hours
post-infection to approximately 30-fold, peaked at 24 hours, and subsequently
declined at 48 hours post-infection. At 24 hours post-infection, IL-27 mRNA
45
was significantly upregulated 1000-fold in response to Edm infection (p<
0.01), and 100-fold in response to Bilt infection (not statistically significant).
Wild-type measles virus and its C and V protein-knockout strains
IL-27 transcript levels were variable for all Wt and KO measles virus
strains (Figure 12a) with no clear dose-response and high variation between
samples making it difficult to conclude if and how IL-27 expression was
regulated by measles virus infection in moDCs. There was a downregulation
of IL-10 transcripts at 2 hours post-infection, followed by a 5- to 20-fold
upregulation 12- and 24-hours post infection at high MOI infections (Figure
12b). The Wt C KO showed similar IL-10 regulation at both MOIs, whereas
Wt and Wt V KO viruses showed a dose-dependent response. Trends for Th17
negative regulator mRNAs were not statistically significant when compared
to mock-infected moDCs (Figure 12).
Discussion
In these studies we were unable to detect type I interferon production
by measles virus-infected moDCs, but demonstrated MOI-dependent
induction of ISGs/VSIGs, IFIT1 and Mx1. IFIT1 and Mx1 mRNAs were
significantly increased by Wt V KO, and IFIT1 mRNAs were also
significantly increased by Wt C KO and Edm. For all strains of measles virus
tested, IFIT1 was upregulated to a greater extent than Mx1. Over the first 48
hours following infection, cytokines induced would inhibit a Th17 response,
as IL-23A mRNA is downregulated and IL-27 mRNA is upregulated. It
46
appears that a proinflammatory (IL-1β, IL-6) response may be occurring as
well, but this did not reach significance.
Type I interferons were not detected
Little or no biologically active type I interferon as detected from the
supernatants of measles virus-infected moDCs, using the Wt, Wt C KO and
Wt V KO strains. It was expected that some may be present in the Wt C KO
and Wt V KO conditions, due to the absence of immunomodulatory C and V
proteins that have the ability to inhibit type I interferon production. The
sensitivity of this assay was low, and there may have been small amounts of
interferon α/β present, below detectable levels. Similarly, previous studies
done by Dr. Rupak Shivakoti showed that in the absence of DI RNA, Bilt
infection of moDCs produced no functional type I interferon. Although Edm
produced low levels of interferons α/β, this effect returns to a similar level
when levels of infection were adjusted between Edm and Bilt (data not
shown).
ISGs/VSIGs were upregulated in response to measles virus infection
IFIT1 and Mx1 both function to decrease viral replication (100, 154,
155). IFIT1 mRNA increased to a greater degree to measles virus infection
than Mx1 mRNA and was more highly upregulated by Edm than Bilt.
Upregulation continually increases up to 24 hours, and then begins to
decrease. This result agrees with previous obervations with dsRNA that
IFIT1 transcription is induced quickly and is transient (99). Although
47
upregulation of Mx1 transcripts did not reach statistical significance, because
of the low number of replicates (2) and increase in Mx1 is likely to be
biologically significant, because measles virus glycoprotein synthesis and
replications are sensitive to the inhibitory effects of Mx1(159).
Absence of measles virus C and V proteins may enhance induction of
ISGs/VSIGs
The regulation of transcription of ISGs/ VSIGs in response to measles
virus infection and the role of the nonstructural proteins, C and V, have not
been explored, although these proteins contribute to evasion of the host
interferon α/β response (34, 41, 97, 161). Our data suggest that the C and V
proteins may play a role in regulating the induction of ISGs/VSIGs.
Upregulation of IFIT1 and Mx1 mRNA transcripts were observed 24 hours
after infection at an MOI of 0.1 for the Wt V KO infection, and Wt C KOinduced Mx1 transcripts. at high MOI conditions (0.1) at 24 hours postinfection for the the Wt V KO-induced upregulation of IFIT1 and Mx1
transcripts, and the Wt C KO-induced upregulation of Mx1 transcripts.
Although statistically significant upregulation of IFIT1 and Mx1
occurred with C KO and V KO infections compared to mock infected baseline
expression, the increase was not significantly different than the upregulation
after Wt infection. Coupled with the lack of detection of interferon,
upregulation of IFIT1 and Mx1 are likely due to activation of the viral stressinduced, interferon-independent pathway by infection. ISG/VSIG
48
upregulation by Wt C KO and Wt V KO infections, supports the idea that
IFIT1 and Mx1 can be induced by more than one mechanism. However, the
ability of our bioassay to detect interferon suggests the need to increase the
sensitivity of this assay to detect low levels of type I interferons. This is
supported by studies done with the same Wt C KO and Wt V KO viruses in
vivo, where interferon α/β as detected at day 7 and day 14 following infection
with these viruses in rhesus macaques (97).
The C and V proteins are important for inhibition of interferon
production, and the V protein plays an additional role in inhibition of
interferon cell signaling (87, 93, 106). This additional ability of the V protein
to interfere with interferon cell signaling, may have increased induction of
ISG/VSIG mRNAs for IFIT1 and Mx1.
Inhibition of Th17 cell differentiation early after measles virus infection
The Th17 effector cell subset is regulated by several cytokines
produced by APCs and innate immune cells (103). Differentiation occurs in
an inflammatory environment, but the survival and expansion of Th17 cells
requires IL-23A signaling (77). Our results suggest that measles virus
infection tends to upregulate pro-inflammatory cytokines IL-1β and IL-6. The
upregulation of IL-1β, although it did not reach significance in our results, is
supported by the detection of IL-1β in plasma of children infected with
measles (144). However, there was a significant downregulation of IL-23A
mRNA which is needed to establish long-lasting Th17 cells (77). Though
49
there is an early downregulation, IL-23A could play an important role in
development of a Th17 response at later time points following measles virus
infection.
IL-10 transcripts did not vary significantly from baseline following
measles virus infection of moDCs. This is not surprising early following viral
infection. Although IL-10 plays a role in preventing the formation of Th17
cells through inhibition of IL-23 production (104), it also inhibits T cell
proliferation and suppresses the production of pro-inflammatory cytokines
necessary for the effector responses to measles virus infection (160).
Furthermore, Th17 cell differentiation is inhibited by IL-27 (105, 106),
and a statistically significant increase in IL-27 transcripts at 24 hours
following infection with Edm was observed. A similar trend was observed in
response to Bilt infection at a lower magnitude. IL-27 upregulation early in
response to measles virus infection may support Th1 cell differentiation,
which assists in the clearance of infectious measles virus (138). Overall, the
downregulation of IL-23A and upregulation of IL-27 support inhibition of the
Th17 cell differentiation early after infection.
Conditions, limitations, and future directions
DI-free viruses were difficult to grow at high titers. Virus stocks with
high titers were often contaminated with DI particles and could not be used
for these experiments. Due to the low titer of virus stocks, infections were
only done at low MOIs and results might be different with infections at
50
higher MOIs. Nevertheless, significant upregulation of IFIT1 and Mx1
mRNAs were observed. Importantly, we can confirm that ISG/VSIG induction
was not a result of aberrant activation of innate immune responses leading to
the production of type I interferon.
In future studies, it would be beneficial to use higher MOIs to try to
elucidate clear trends in the regulation of ISGs/VSIGs and cytokines. To do
this, viruses must be isolated at higher titers that are DI-free. This is
especially important for comparison of the Wt, Wt C KO, and Wt V KO
viruses and the Edm, Edm C KO, and Edm V KO measles viruses to more
definitiviely determine the roles of V and C in modulating the responses of
moDCs to infection with Wt and vaccine strains of measles virus.
Preparation of DI-free KO viruses were important because the
knockout system of a measles virus IC-B represents a complete system for
determining accurate physiological effects of measles virus infection, with no
confounding by DI particles. Other studies using whole measles virus KO
strains have been used, and support our findings. In rhesus macaques, Wt C
KO and Wt V KO viruses induced interferons α and β and spread less
efficiently, but still induced strong adaptive immune responses (97). Tober et
al. also detected a decrease in titers in cotton rats with a measles virus V KO
virus (161), and Yanagi et al. used measles virus C KO in A549/hSLAM cells
and measles virus V KO in H358 cells to confirm the roles of the C and V in
inhibition of interferon induction (35, 41). With more time, the limited
51
analysis of mRNAs for Th17 can be further expanded with more replicates of
Wt, Wt C KO, and Wt V KO infections.
Conclusions
Although little or no biologically active type I IFNs were detected in
response to MV infection of moDCs, there may be trace amounts produced by
infection with Wt C KO and Wt V KO viruses that were below the limit of
detection. In the future, it would be beneficial to increase the sensitivity of
interferon assays to detect low levels of interferons α/β. IFIT1 and Mx1
transcript levels are likely induced by the viral stress-induced pathway in
response to Wt measles virus infection, whereas the Wt C KO and Wt V KO
viruses may also induce the transcription of IFIT1 and Mx1 through the
interferon-stimulated pathway. The absence of C and V proteins during virus
replication likely lessens the ability to interfere with production and
signaling of interferon, though interference with these pathways may not be
complete with the KO of only one immunomodulatory protein. It would be
interesting to study ISG/VSIG mRNA regulation in response to infection with
a measles virus C- and V-protein double knockout strain. IFIT1 appears to be
more vigorously induced in response to measles virus infection than Mx1.
Furthermore, early after measles virus infection, moDCs do not support the
formation of long-lasting Th17 cells. Current and future studies will help us
delineate the role of C and V protein and the immune response to measles
virus infection, as well as Th17 cytokine regulation.
52
Table 1. PCR primers, target and cycling conditions for P gene
sequencing, and detection of measles virus standard and defective
genomes.
Primers for Sequencing P gene (Biosource International)
Sequencing
Primer and Sequence
Target
Measles
virus V
protein RNA
editing site
MVP 2373F
5’-GATCCACGAGCTCCTGAGAC-3’
MVP 3426R
5’-GGAGGCAATCACTTTGCTCCTAAG-3’
Measles
virus C
protein start
codon
MVP 1745F
5’-CTTAGGAACCAGGTCCACACAGCC-3’
MVP 3426R
5’-GGAGGCAATCACTTTGCTCCTAAG-3’
Cycling
Conditions
45°C, 30 min
94°C, 2 min
[94°C, 30 s; 55°C,
30 s; 68°C, 2 min
for 40 cycles]
68°C, 5 min; 4°C,
hold
45°C, 30 min
94°C, 2 min
[94°C, 30 s; 55°C,
30 s; 68°C, 2 min
for 40 cycles]
68°C, 5 min; 4°C,
hold
Primers for Detection of Measles Virus Standard and Defective
Genomes (IDT)
Target
Primer and Sequence
Measles
virus
standard
genome
JM 396
5’-TATAAGCTTACCAGACAAAGCTGG
GAATAGAAACTTCG-3’
JM 402
5’-TTTATCCAGAATCTCAAGTCCGG-3’
Measles
virus 5’ copyback DI
genome
JM 396
5’-TATAAGCTTACCAGACAAAGCTGG
GAATAGAAACTTCG-3’
JM 403
5’-CGAAGATATTCTGGTGTAAGTCTA
GTA-3’
53
Cycling
Conditions
45°C, 30 min
94°C, 2 min
[94°C, 30 s; 53°C,
30 s; 68°C, 2 min
for 40 cycles]
68°C, 5 min; 4°C,
hold
45°C, 30 min
94°C, 2 min
[94°C, 30 s; 53°C,
30 s; 68°C, 2 min
for 40 cycles]
68°C, 5 min; 4°C,
hold
Figure 1. Signaling pathways leading to virus stress-inducible gene
(VSIG) induction. Type I interferon interacts with the IFN receptor
(IFNAR) and activates the Jak-STAT signaling pathway, leading to the
activation of the interferon-sensitive response element (ISRE), which is
present in interferon stimulated gene (ISG) promoters. TLR3 is activated by
dsRNA, which activates transcription factors NFκB and IRF3 to induce
VSIGs. Additionally, a TLR3- and IFN-independent pathway exists that
activates transcription factors ATF2, IRF3, and NFκB [98].
54
Figure 2. Generation of wild-type measles virus defective for the C or
V protein. Two mutations were used to silence C protein expression, or V
protein expression, creating two new measles virus strains. For the wild-type
measles virus C knockout (KO) strain, the nucleotide sequence was altered to
render the start codon nonfunctional and also introduce a stop codon slightly
downstream of the start site. The wild-type measles virus V KO strain, was
altered to inactivate the RNA editing sequence necessary for V protein
expression and add a stop codon downstream of that site [97].
Figure 3. Primer binding sites for sequencing. Blue arrows correspond
to locations on or near the P gene where primers hybridize to amplify RNA
for sequencing of viruses. The arrows facing right, from left to right, are MVP
1745F and MVP 2373F, and were used to amplify the start sequence of the C
gene and the latter half of the V gene, respectively. The left-facing arrow,
primer MVP 3426R was used in both PCR conditions. Adapted from [93].
55
Figure 4. Sequencing confirms correct viral sequences. Wild-type (Wt)
measles virus RNA genome sequences are boxed in blue. (a) Measles virus (Cprotein knockout (C KO) mutation [97]. In red, the triplet sequence targeted
for mutations is boxed. Below, sequencing of Wt and Edm strains confirmed
the wild-type sequence, and Wt C KO and Edm C KO strains confirmed the
existence of mutations. (b) Wt V-protein knockout (V KO) mutations as done
by the laboratory of Dr. Roberto Cattaneo [97]. In red, the triplet sequence
targeted for mutations is boxed. Below, sequencing of Wt and MV Edm
strains confirmed the wild-type sequence, and Wt V KO and Edm V KO
strains confirmed the existence of mutations.
56
Figure 5. Gel of measles virus stocks. (a) PCR products of the measles
virus N gene (primers JM396, JM402), indicative of the presence of the
measles virus standard genome. (b) PCR products of the measles virus 5’
copy-back DI RNA genome (primers JM396, JM403). All molecular weight
standards and measles virus PCR products were run on the same gel. Lane 1
has 1 KB Plus molecular weight standards (Invitrogen). Lane 2 contains PCR
products with no template measles virus. Lanes 3-8 corresponded to a specific
virus strain: lane 3 – Wt measles virus IC-B, lane 4 – Wt C KO, lane 5 – Wt V
KO, lane 6 – Edmonston (Edm) measles virus vaccine, lane 7 – Edm C KO,
lane 8 – Edm V KO.
57
Figure 6. Measles virus standard and DI genome PCR products of
measles virus stocks used in in vitro experiments. Lane 1 of each figure
is 1 KB Plus molecular weight standards (Invitrogen). (a,b) Lanes 2 and 5
have no measles virus template in the PCR product, Lanes 3 and 4 detected
the presence of the measles virus standard genome, whereas lanes 6 and 7
did not detect the presence of the measles virus DI genome. (a) contains Wt
and Wt C KO viruses and (b) contains two Wt V KO stocks. (c) Lanes 2 and 4
have no measles virus template, lane 3 detects the measles virus standard
genome, and lane 5 detects the measles virus DI genome. The small band in
(b) lane 6 is indicative of extra primers.
58
Figure 7. mRNAs for interferon-stimulated genes, IFIT1 and Mx1, are
upregulated in moDCs by the Edmonston measles virus vaccine
strain and Bilt Wt strain. IFIT (a) and Mx1 (b) mRNA fold changes were
log transformed and compared to the baseline expression of mock-infected
moDCs using a two-way ANOVA, with a Bonferroni multiple comparisons
correction. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.
Figure 8. Interferon-stimulated genes, IFIT1 and Mx1, are
comparably upregulated in monocyte derived dendritic cells
(moDCs) in response to Wt measles virus, and its respective C- and Vprotein KO strains at MOIs of 0.01 and 0.1. IFIT (a) and Mx1 (b) mRNA
fold changes were log transformed and compared to the baseline expression of
mock-infected moDCs using a two-way ANOVA, with a Bonferroni multiple
comparisons correction. (*) p < 0.05.
59
Figure 9. Positive regulators of Th17 cell differentiation, IL-1β, IL23A and IL-6, expression levels from moDCs in response to
Edmonston and Bilthoven measles virus infection at MOIs of 0.4 and
4.0. IL-1β (a), IL-23A (b), and IL-6 (c) mRNA fold changes were log
transformed and compared to the baseline expression of mock-infected
moDCs using a two-way ANOVA, with a Bonferroni multiple comparisons
correction. (**) p < 0.01.
60
Figure 10. Positive regulators of Th17 cell differentiation, IL-1β and
IL-6, expression levels from moDCs in response to wild-type measles
virus and its respective C- and V-protein knock out strains at MOIs
of 0.01 and 0.1. IL-1β (a) and IL-6 (b) mRNA fold changes were log
transformed and compared to the baseline expression of mock-infected
moDCs using a two-way ANOVA, with a Bonferroni multiple comparisons
correction. None of the changes in transcript levels were significant.
Figure 11. Negative regulators of Th17 cell differentiation, IL-27 and
IL-10, mRNA expression levels from moDCs in response to
Edmonston and Bilthoven measles virus infection at MOIs of 0.4 and
4.0. IL-27 (a) and IL-10 (b) mRNA fold changes were log transformed and
compared to the baseline expression of mock-infected moDCs using a two-way
ANOVA, with a Bonferroni multiple comparisons correction. (**) p < 0.01.
61
Figure 12. Negative regulators of Th17 cell differentiation, IL-27 and
IL-10, mRNA expression levels from moDCs in response to wild-type
measles virus and its respective C- and V-protein knock out strains
at MOIs of 0.01 and 0.1. IL-27 (a) and IL-10 (b) mRNA fold changes were
log transformed and compared to the baseline expression of mock-infected
moDCs using a two-way ANOVA, with a Bonferroni multiple comparisons
correction. None of the changes in transcript levels were significant.
62
Chapter Three:
Effects of vitamin A supplementation on the immune response and
the Th17 response to measles virus infection in rhesus macaques
63
Introduction
Immunization is the most effective way to reduce the morbidity and
mortality due to measles virus infection. However, many regions of the world
do not have the resources to efficiently deliver two doses of vaccine to their
populations. To reduce the morbidity and mortality in children that develop
measles, vitamin A supplementation is recommended by the World Health
Organization and the United Nations Children’s Fund (119, 120). Vitamin A
therapy is inexpensive to administer at just $0.35 in US dollars per patient
(122). Additionally, this supplementation decreases the amount of money
spent on healthcare by shortening the time spent in the hospital due to
measles by 2.9 days on average (122). However, the mechanism by which
vitamin A protects against severe measles virus-induced disease is currently
unknown.
Vitamin A and measles virus infection
Vitamin A is obtained from the diet in several forms and can be
acquired from fruits, vegetables and leafy greens as β-carotene, or from
animal-based food products, such as meat and dairy, that offer preformed
vitamin A. (141, 142) Vitamin A is an essential vitamin that is involved in a
wide range of biological processes, including vision, immunity, growth and
development, as well as cellular differentiation (143). For the immune system
vitamin A is necessary for B and T cell proliferation, T cell activation, and
enhances the antigen-presenting ability and maturation of dendritic cells
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(139). In its bioavailable form, vitamin A is found in the body as serum
retinol bound to retinol binding protein (RBP) (114).
Serum retinol is transferred to peripheral tissues from the liver, where
it is stored. Serum retinol levels can be depleted during the acute phase of
infection with many pathogens and epidemiologic data indicate that infection
with measles virus often precedes vitamin A deficiency (27, 108, 115, 122,
123, 124). Importantly, hyporetinolemia due to measles occurs regardless of
vitamin A status of the individuals before infection, and this has been
observed in both vitamin A sufficient and deficient populations (116, 117).
Plasma vitamin A depletion may be due to increased metabolic utilization of
vitamin A in peripheral tissues (26, 108, 119), less hepatic RBP production
(118), or increased losses of RBP and retinol through urinary excretion (119121) (Figure 1). Increased urinary excretion is associated with decreased
reabsorption of fat-soluble vitamins and low-molecular weight proteins such
as RBP after these molecules are filtered through the glomerulus of the
kidney, due to damaged proximal tubular epithelium (119). Renal tubular
damage can be mediated by inflammatory cytokines, such as IL-1, IL-6, and
TNF, released during acute phase responses to infection (128).
It is important to note that, serum retinol levels may not correlate with
body stores of vitamin A (109). In healthy human adults fed a vitamin Adeficient diet, vitamin A levels can remain stable through usage of hepatic
stores for months before an effect is noticed (129). However, after several
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days of continued retinol loss, liver vitamin A stores may be significantly
diminished (119). A diet high in vitamin A is not necessary for healthy
adults, due to the storage capacity of the liver. However, the continued loss of
vitamin A that may accompany measles virus infection could deplete liver
stores, leading to low serum retinol levels.
While a sharp drop in vitamin A levels has been observed (Figure 2),
during the acute phase of measles virus infection in rhesus macaques, this is
reversible (108, 115-117). As children with measles recover and enter the
convalescent phase, vitamin A levels in their plasma begin to return to
normal levels (113). Peripheral tissues may then have access to normal
amounts of circulating vitamin A. However, total body stores may still be
depleted for months post-infection, as has been observed in cases of influenza
in children (132). This is problematic, because vitamin A deficiency increases
the severity of disease, resulting from bacterial, viral or parasitic infections
(131), and children in developing countries show increased levels of morbidity
and mortality due to respiratory infections and diarrhea for a year after
apparent recovery from measles virus infection (133, 134, 135, 136).
Role of vitamin A in CD4+ T cell differentiation
Vitamin A supplementation has been associated with improved clinical
outcomes in patients with measles when given during the acute rash phase of
disease (27-29, 122, 123). However, there is no evidence for a direct effect of
this supplementation on cytokine production or lymphocyte activation (162).
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Though, it does appear that in vaccination models, signaling of retinoic acid
occurs in the presence of an inflammatory environment (163).
The metabolites of vitamin A play critical roles in the differentiation of
T helper cell subsets. Retinoic acid plays a direct role in suppression of the
Th1 response, while simultaneously enhancing Th2 development (165-167).
The interaction of vitamin A with differentiating regulatory T cells and Th17
cells is slightly more complex. Activated CD4+ T cells in the context of TGF-β
differentiate into T regulatory cells, unless IL-6, IL-1β, and IL-23 or IL-21 are
also present (168). Low retinoic acid concentrations are necessary for Th17
cell differentiation (169). However, all-trans retinoic acid has been implicated
in inhibition of the Th17 response, and promotion of transcription factor
Foxp3 expression in Treg cells in vitro (163, 164). After a certain threshold in
vitamin A concentration is reached, CD4+ T cells will differentiate down the
T regulatory cell pathway in the context of TGF-β, regardless of the presence
of an inflammatory environment (169). It has been hypothesized that
immune tolerance in mucosal tissues, could provide beneficial immune effects
for the outcome of measles (170).
Vitamin A supplementation
Vitamin A supplementation to mitigate infection-induced deficiency is
thought to be one of the safest, most efficacious and affordable therapeutic
approaches to decreasing measles mortality (140). In the early 1930’s,
vitamin A supplementation was first found to positively affect the outcome of
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measles virus infection in children less than five years of age, by leading to a
decrease in deaths and less severe pulmonary complications (107). The
beneficial effects of vitamin A in children with measles are numerous (27,
121,122). Vitamin A supplementation during the acute phase of measles
virus infection leads to better health outcomes (27-29, 122, 123) and does not
increase retinol or retinyl acetate excretion (119), suggesting that vitamin A
supplementation is an effective way to increase retinol levels.
A study of children in New York City with measles in 1992
demonstrated that nearly 25% were hyporetinolemic, although vitamin A
deficiency is rarely seen in this location. In this study, low plasma vitamin A
levels were associated with more severe measles, as indicated by higher and
more prolonged fever and a longer hospital stay (113). A study of children in
Milwaukee, Wisconsin also associated depressed vitamin A levels with
increased severity of measles (159). In Zaire, low serum vitamin A levels were
associated with increased measles mortality in children less than 2 years old
(13). Because many studies demonstrate that vitamin A supplementation
decreases the risk of mortality in children less than 2 years of age, risk from
low vitamin A levels may be age-dependent. However, the specific function
and mechanism by which vitamin A alters the course of infection with
measles virus is unknown.
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Vitamin A: Potential roles in improving measles outcome
Repair of lung epithelium
It is possible that vitamin A functions in a multi-faceted way to protect
against measles virus-associated morbidity and mortality. Vitamin A may
mitigate measles virus-induced damage to respiratory epithelial surfaces
(108). During measles virus infection of the respiratory tract, the lung shows
epithelial changes similar to those that occur during vitamin A-deficiency
(107). This may lead to a need for additional vitamin A for repair and healing
of the lung epithelium following infection with measles virus (108).
Effect on lymphopenia and T cell-mediated viral clearance
The effects of borderline vitamin A deficiency on immune function was
explored by Clive West, using Newcastle disease virus, a paramyxovirus that
infects poultry with many characteristics in common with measles virus. In
this study, vitamin A deficiency was associated with lymphopenia, which was
further exacerbated by infection (137). Lymphopenia describes a condition
where the number of circulating lymphocytes are decreased compared to
normal and is a characteristic of the febrile stage of measles virus infection
(138). Measles-induced lymphopenia may be due to an elevated cortisol
response due to stress and the acute phase reaction, to altered trafficking and
sequestration of lymphocytes in peripheral lymphoid tissues, or to
destruction of measles virus-infected T and B cells (130). Thus, vitamin A
69
deficiency could further exacerbate measles-related lymphopenia, which
suggests that vitamin A supplementation may lessen lymphocyte loss.
It is possible that measles- or vitamin A-related lymphopenia could
weaken the T cell response to infection through a decrease in circulating T
lymphocytes. Clearance of infectious measles virus and RNA is accomplished
by the adaptive cellular immune responses (1). Viremia in measles virus
infections lasts until approximately two weeks post-infection (68). When
CD8+ T cells are depleted, rhesus monkey studies show that peak viral titers
are elevated and the period of viremia is extended (65, 66).
Inhibition of viral replication
Vitamin A supplementation could also have effects on viral replication
and clearance. In vitro, retinoids induce type I interferon and directly inhibit
replication of measles virus (110, 111). However, in vivo there is little
evidence that type I interferons (α/β) are produced during the early stage of
disease (144) and vitamin A supplementation had no effect on the viral load
of a related morbillivirus, canine distemper virus in ferrets (112).
Once infectious virus has been cleared, measles virus RNA can persist
for several months in the respiratory tract, urine, PBMCs and lymphoid
tissues (1, 68, 125) (Figure 3). Sequencing suggests that the prolonged
presence of measles virus RNA is due to slow clearance rather than mutation
to evade the host immune response (68). Clearance of measles virus is
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delayed in cases of malnutrition, supporting the idea that vitamin A
supplementation may facilitate viral clearance (126, 127).
Enhanced antibody response
Vitamin A could also play a role in improving the host antibody
response, as it is required for the development of both B cells and T helper
cells (137). In children with measles, low plasma retinol levels are correlated
with a low measles virus-specific antibody response (113) and decreased
levels of measles virus antibodies have been associated with increased
measles mortality (130). This suggests that vitamin A deficiency may
exacerbate measles, by preventing a robust antibody response to infection.
Measles virus-specific IgG antibody concentrations correlate closely with
disease outcome, with higher antibody responses associated with less severe
morbidity and mortality (29). A randomized trial of vitamin A
supplementation in African children two years or younger showed higher
measles virus-specific IgG antibody concentrations compared to the nontreated group at day 8 after onset of rash, as well as a significant reduction in
mortality (29).
Th17 response to measles virus infection
It has been noted that measles induces an early CD8+ T cell response
along with a CD4+ Th1 polarized response, which later shifts towards a
CD4+ Th2 response (138). However, it has not been determined how CD4+
Th17 cells may contribute to the measles immune response. Th17 cells are a
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unique lineage of CD4+ T helper cells, defined by their ability to secrete IL17A, IL-17F, and IL-17AF (103). Recently, measles virus-specific IL-17producing cells were shown to display a bi-phasic pattern with peaks at day
18 and day 35 in response to measles virus challenge in a naïve monkey and
at day 10 and day 35 in monkeys vaccinated with a poorly protective vaccine
(102). It is currently unknown whether or not Th17 cells play a role in the
immune response to natural measles virus infection in humans, or if vitamin
A plays a role in this process.
Materials and methods
Animals
Six male 3-year-old juvenile rhesus macaques (Macaca mulatta) that
were measles naïve (14Y, 17Y, 24Y, 31Y, 46Y, and 50Y) were obtained from
the Johns Hopkins Primate Breeding Facility. All studies were performed in
accordance with experimental protocols approved by the Animal Care and
Use Committee for Johns Hopkins University and animals were maintained
within these guidelines. Before infection, animals were shaved and baseline
measurements were taken for all PBMC and plasma parameters to be
assessed after infection.
Measles virus infection and vitamin A supplementation
The DI-free stock of wild-type Bilthoven measles virus (a gift from
Albert Osterhaus at Erasmus University, Rotterdam, Netherlands) used to
infect the monkeys was grown in human cord blood cells by Wendy Lin,
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assayed by plaque formation on Vero/hSLAM cells, and stored at -80°C. All
six monkeys were challenged intratracheally with 104 plaque-forming units
(pfu) in 1 ml PBS.
After development of the characteristic measles rash, three monkeys
(14Y, 24Y, and 50Y) were given vitamin A in the form of retinol palmitate
(Vitamin Angels, Parsippany, NJ). The oral liquid contents from each 100,000
IU capsule were mixed with strawberry jam and two doses were given to
monkeys at the time of the rash, days 10 and 11 post-infection. Monkeys 17Y,
31Y and 46Y received the strawberry jam without vitamin A. In collaboration
with the laboratory of Dr. Richard Semba, HPLC analysis was done on
plasma samples from all monkeys to monitor retinol levels before and after
infection.
Sample collection
For all procedures, monkeys were anesthetized with 10-15 mg/kg body
weight of ketamine. Approximately 5 ml of heparinized blood was collected
from the femoral vein of each animal before infection and on days 7, 10, 18,
21, 28, 35, 52, 56, 71/72, and 84 after infection. Comprehensive complete
blood counts (CBCs) were done on each animal from day 10, onwards.
Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from
blood by whole blood gradient centrifugation on Lympholyte®-Mammal
(Cedarlane Labs). Plasma samples were stored at -20°C and 2 million PBMCs
were flash frozen for RNA isolation at -80°C. Bone marrow biopsies were
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collected at days 14, 28, 39, 56, and 84. Mononuclear cells were isolated from
bone marrow using a Lympholyte®-Mammal (Cedarlane Labs) gradient
centrifugation. Any mononuclear cells from blood or bone marrow that were
not used fresh in assays were transferred to 1 ml of cold freezing media (90%
FBS, 10% DMSO) and stored in a Nalgene Mr. Frosty™ Freezing Container
for 1 hour at 4°C and -80°C overnight before moving to permanent storage in
liquid nitrogen.
Nasal secretions were collected using one sterile cotton swab per
nostril on days 0, 7, 10, 14, 17, 21 and 39 to monitor measles virus shedding
from the respiratory tract. Swabs were immersed in PBS, which was
centrifuged to collect the cell and supernatant portions and stored at -80°C.
Skin biopsies of the rash were obtained using a skin punch, collected into
PBS, transferred to 4% paraformaldehyde and stored at 4°C for several days
before transferring back to PBS for long-term storage at 4°C. Lymph node
biopsies were collected from 3 monkeys (14Y, 17Y, 24Y) on day 71 and the
remaining 3 monkeys (31Y, 46Y, 50Y) on day 78.
Virus infection assays
The amount of infectious virus present in the blood was measured by
co-cultivating serially diluted fresh PBMCs (105 to 100) with Vero/hSLAM
cells. After incubation for 5-6 days at 37°C 5% CO2, the tissue culture
infectious dose 50 (TCID50) was determined by assessing the cytopathic
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effects, observed as cell clearing, syncytia, or dead floating cells. Viremia was
then expressed as the number of infected cells per million PBMCs.
Nasal swab RNA analyses
Debra Hauer did the following RNA analyses, using the RNeasy® Plus
Micro Kit (Qiagen) to isolate RNA from nasal swabs. RNA was eluted in 30 ul
of RNase-free water (Qiagen), and 10 ng or 10 ul were used for RT-PCR.
Primers MV41 and MV42 were used to amplify a 350 base pair sequence
within the measles virus N gene (Table 1), and human β-actin RT-PCR
primers (Agilent) were used as a control.
Skin punch and lymph node biopsies
Skin punch biopsies were taken of an area of rash on the belly of each
monkey on day 10. Biopsies were fixed in 4% PFA at 4°C for 48 hours and
stored in PBS. Skin samples were sent to the Johns Hopkins Hospital
Reference Histology Lab for paraffin embedding, sectioning, and hematoxylin
and eosin staining. Pathology was read by Victoria Baxter, DVM.
Portions of lymph nodes from each monkey were fixed in 4% PFA at
4°C overnight, and stored longer term in PBS at 4°C prior to sending lymph
nodes to the Johns Hopkins Hospital Reference Histology Lab for paraffin
embedding, sectioning, and hematoxylin and eosin staining.
Antibody and cytokine assays
Plaque reduction neutralization tests (PRNTs) were done to measure
the amount of antibody in plasma samples needed to reduce plaque formation
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by a DI-free stock of the Edmonston measles virus vaccine strain on Vero
cells by 50%. Plasma was serially diluted from 1:3 to 1:30,000 in DMEM
supplemented with 10% FBS, 1% L-glutamine, and 1%
penicillin/streptomycin. Each dilution was incubated with 250 pfu of
Edmonston measles vaccine strain virus in 200 ul, plated in a 6-well plate,
and incubated in triplicate at 37°C, 5% CO2 for one hour, shaking plates
every 20 minutes. Each well was then overlayed with a mixture of one part
1.2% bacterioagar and one part 2X MEM supplemented with 2% FBS, 1%
penicillin/streptomycin and 1% L-glutamine, before incubating for 5 days at
37°C, 5% CO2. Data are expressed as the geometric mean of the titer.
Enzyme immunoassays (EIAs) were conducted to determine the levels
of measles virus-specific IgG and IL-17 in plasma. For the measles virusspecific IgG EIA, Nunc 96-well Maxisorp plates were coated with 50 ul of
virus antigen (Rubeola/Measles Edmonston Strain Inactivated Vero Cell
Extract; ABI) at a 1:25 dilution and incubated overnight at 4°C. The next
day, the plate was blocked with 1% bovine serum albumin (BSA) in PBS for 2
hours. Plasma was added to wells neat, diluted 1:25 in PBS 10% FBS and
then serially diluted two-fold to 1:25,600 for another overnight incubation at
4°C. On day three, anti-monkey horseradish peroxidase (HRP)-conjugated
IgG antibody (Southern Biotech) was diluted 1:5000 in PBS 10% FBS and 50
ul was added to each well and incubated at 37°C for one hour. The plates
were then developed in the dark with 3,39,5,59-tetramethylbenzidine (TMB)
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substrate solution. After 5-7 minutes, sulfuric acid (2M) stop solution was
added and the optical density was read at 450 nm. Data are presented as the
reciprocal of the highest dilution with an OD absorption value greater than
two times the average background.
IL-17A EIAs were done using the ELISA for Monkey IL-17A kit
(Mabtech). Corning Costar ELISA plates were coated with monoclonal
capture antibodies and incubated overnight at 4°C. The next day, the plate
was blocked with PBS 0.05% Tween 20 0.1% BSA for one hour at room
temperature. Standard curves were established by using 2-fold dilutions of
recombinant human IL-17A (Mabtech) from 1000 pg/ml to 7.5 pg/ml,
accounting for background levels at 0 pg/ml. Plasma samples were diluted 2fold from 1:30 to 1:3840, added to the plate, and incubated at room
temperature for 2 hours. Biotinylated monoclonal detection antibody
(Mabtech) was then added to each well and the plate was incubated at room
temperature for one hour. Streptavidin-HRP (Mabtech) was diluted 1:1000
and added to the plate, then incubated at room temperature for 1 hour. After
washing, the plate was developed at room temperature by adding TMB
substrate solution in the dark for 15-30 minutes. Sulfuric acid (2M) stop
solution was added and the plate was read immediately at 450 nm. Because
concentrations were too low to extrapolate from the human recombinant IL17 standard curve results were reported as a positive or negative for plasma
neat or for the 1:30 dilution.
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Antibody-secreting cell assays
Mononuclear cells from the blood and bone marrow were used to
measure antibody-secreting cells (ASC). A Multiscreen® HTS HA Opaque 96well filtration plate (Millipore) was coated overnight with either
Rubeola/Measles (Edmonston Strain) Inactivated Vero Cell Extract (ABI) at a
1:25 dilution, or anti-monkey IgG, IgA, IgM (Sigma) at a 1:50 dilution and
then stored overnight at 4°C. The plate was blocked the next day with RPMI
media/10% FBS for 1 hour at 37°C. This was then replaced with fresh
RPMI/10% FBS and 5x105 fresh PBMCs, and serial two-fold PBMC dilutions.
If bone marrow mononuclear cells were used, the same dilutions were used
for the total Ig. To detect the measles virus-specific ASC response; 5x105 cells
were added to replicate wells. Plates were then incubated for 5-6 hours at
37°C 5% CO2. Goat anti-monkey IgG-HRP conjugated antibody (Nordic
MUbio) was added to each well and incubated overnight at 4°C. The next day,
the plate was developed with stable diaminobenzidine (DAB) solution
(Invitrogen) for 5-7 minutes in the dark. The plate was thoroughly rinsed
with DI water and left to dry before being read with the ImmunoSpot® plate
reader. ImmunoSpot® 5.0 software was used for counting spots and quality
control analysis to provide final counts, which are reported as measles virusspecific ASCs or spot-forming cells (SFCs)/106 PBMCS or ASCs/106 bone
marrow mononuclear cells.
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T cell assays
Enzyme-linked Immunosorbent Spot (ELIspot) Assays were used to
monitor T cells producing IFN-γ and IL-17. Multiscreen® HTS HA Opaque
96-well filtration plates (Millipore) were coated with purified mouse antihuman IFN-γ antibody (BD Biosciences) at 2ug/ml or anti-human IL-17A
(eBioscience) at 5 ug/ml overnight at 4°C. The plates were blocked for an hour
at 37°C with RPMI 10% FBS blocking media. Peptides or cell stimulants
were then added to the plate. IFN-γ assays were in duplicate and were either
non-stimulated, or stimulated with 1 ug/ml of pooled H, N, or F measles virus
overlapping peptides or 5 ug/ml of concanavalin A (ConA). IL-17 ELIspot
assays were done in duplicate and were either non-stimulated or stimulated
with 5.8 ug/ml ABI measles virus-infected Vero cell lysate, or 5 ug/ml ConA.
PBMCs were then added, with 105 PBMCs for IFN-γ and ConA and 5x105
PBMCs for IL-17 non-stimulated and measles virus lysate wells. Plates were
then incubated at 37°C, 5% CO2 for 40-42 hours. Biotinylated anti-human
IFN-γ antibody (Mabtech AB) was diluted in PBS 2% FBS 0.05% Tween, to 1
ug/ml and biotinylated anti-human IL-17A antibody (eBioscience) was diluted
to 2 ug/ml and added to appropriate wells before incubating at 37°C for two
hours. Avidin-HRP (BD Biosciences) diluted 1:2000 was added to all wells
and incubated at 37°C for one hour. Stable DAB substrate (Invitrogen) was
then added and incubated in the dark at room temperature for 5-7 minutes.
After rinsing the plate thoroughly with DI water and allowing it to dry
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completely, the plates were read and analyzed using the ImmunoSpot plate
reader and ImmunoSpot 5.0 software. Data are presented as SFCs/106
PBMCs for spontaneous production and for measles virus-specific SFCs after
subtracting the no cell/no in vitro stimulation SFCs.
Flow Cytometry
Flow cytometry was used to evaluate the Th17 response in PBMCs
from day 0, 10, 18, 28, 39, 56, and 84. PBMCs were stimulated under four
separate conditions with peptide mixes for measles virus-specific H protein
and N protein, dimethyl sulfoxide (DMSO) as a negative control or
staphylococcal enterotoxin B (SEB) as a positive control. Anti-CD28 and antiCD49 were included with the H and N peptides and DMSO. All stimulation
mixes included GolgiStop (BD Biosciences) and GolgiPlug (BD Biosciences).
The Th17 panel surface markers assessed varied slightly, but
stimulation conditions and intracellular staining targets remained the same.
Live/Dead® Fixable Violet Dead cell Stain Kit (Invitrogen) was used to stain
and gate out dead cells. Prior to surface staining, cells were incubated with a
human FcR binding inhibitor (eBiosciences). Surface markers that remained
constant in all Th17 panels were anti-CD4 and anti-CD3. For earlier time
points a ―dump gate‖ was used to gate out CD14- and CD20-positive cells and
anti-CD8 was present in the panel.
Intracellular staining was done following fixation and permeabilization
of cells. For early time points the Cytofix/Cytoperm™ Fixation and
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Permeabilization Kit (BD Biosciences) was used according to directions. At
later time points, the Foxp3 Staining Buffer Set (eBioscience) was used.
Intracellular staining was done to detect the transcription factor RORγt as
well as IL-17A and IL-21 cytokines. Samples were run on the BD FACS
Canto II™ flow cytometer and were analyzed using BD FACSDiva and
FlowJo software.
Results
Measles virus infection of monkeys
Six naïve rhesus macaques were successfully infected with measles
virus after intratracheal challenge with 104 pfu of DI-free wild-type Bilthoven
measles virus. Monkeys 14Y, 24Y and 50Y received vitamin A
supplementation at days 10 and 11 post-infection, while monkeys 17Y, 31Y
and 46Y did not. The presence of infectious virus in the blood was monitored
using PBMC co-cultivation. All six monkeys established viremia by day 7
post-infection, and cleared infectious virus from PBMCs by day 18 (Figure 4).
Nasal swabs were used to collect RNA from respiratory secretions, and
RT-PCR confirmed that measles virus N gene RNA was present in the nasal
passages of all monkeys (Table 2). All monkeys were shedding measles virus
in respiratory secretions early in infection, though the duration of shedding
varied. Monkeys 17Y and 50Y were positive for measles virus in respiratory
secretions from day 7 to day 21, whereas, for 24Y, measles virus was only
detected on day 14 post-infection.
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Over the course of infection, the weights of monkeys were monitored as
a measure of morbidity, and were reported as percent change from day 0
(Figure 5). Weights fluctuated over time between individual monkeys, but
most exhibited weight gain throughout day 70. By day 84, two of the six
monkeys returned to their original weight at time of infection. Two of the
three monkeys supplemented with vitamin A gained approximately 10% of
their body weight, while the remaining two monkeys (one treated, and one
non-treated with vitamin A) lost about 4% of weight by day 84.
Measles virus maculopapular rash
The characteristic maculopapular rash also developed around day 10
in all monkeys, but to varying degrees (Figure 6). Rash biopsies confirmed a
mild to moderate infiltration of inflammatory cells into the skin epithelium at
this time (Figure 7). Monkeys that shed measles virus for longer periods of
time (17Y, 50Y) exhibited a more extensive rash and more severe
inflammatory infiltration in the skin biopsy sections. Monkeys 17Y, 14Y, and
50Y showed mild-to-moderate infiltration of inflammatory cells into the
dermis, consisting primarily of mononuclear cells (lymphocytes and
macrophages), with some eosinophils and neutrophils (Figure 7a, 7b, 7c).
Severe inflammation was evident in 17Y by cells forming large aggregates or
found individually across the dermis. Perivascular cuffing by macrophages
was also seen in the biopsy section of monkey 14Y. All three of the
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aforementioned monkeys had a notable amount of debris, suggesting necrosis
of the superficial dermis, which correlates with the severity of their rash.
Monkeys 46Y, 31Y, and 24Y showed mild infiltration of inflammatory
cells into the dermis, listed in decreasing order of severity. Of the
inflammatory cells present, most were lymphocytes and macrophages, with
occasional eosinophils. Inflammatory aggregates were observed in rash
biopsies of 46Y and 31Y. Some perivascular cuffing, without major
aggregation was observed in the biopsy of 24Y (Figure 7d). While none of the
rash biospies showed inflammatory infiltration into the epidermis, a majority
of the inflammatory cells were present in the superficial dermis in 31Y.
Lymph node biopsies are depicted for vitamin A-supplemented
monkeys 14Y (day 71) and 50Y (day 78) (Figure 8). Lymph nodes were
reactive, with increased cellularity that included plasma cells and
macrophages in addition to lymphocytes. Lymph node histology was
comparable between vitamin A-supplemented and non-supplemented
monkeys at these days.
Levels of vitamin A
Three monkeys (14Y, 24Y, and 50Y) were supplemented with vitamin
A (100,000 IU) on days 10 and 11 post-infection. Vitamin A levels were
monitored through day 21 post-infection (Figure 9). At this point the serum
retinol levels in the vitamin A-supplemented and non-supplemented groups
of monkeys begin to diverge (Figure 9b). It is expected that the vitamin A
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levels in the non-supplemented group of monkeys (17Y, 31Y, and 46Y) will
continue downward until approximately day 50, as this was previously
observed in two non-supplemented rhesus macaques following measles virus
infection (Figure 2). The limited data obtained demonstrated a statistically
significant decrease in serum retinol at day 21 in the non-supplemented
group of monkeys. This was not observed in the vitamin A supplemented
group. Plasma retinol levels at subsequent times will be determined when all
samples have been collected at the end of the study period (day 180).
Comprehensive blood counts
Comprehensive blood counts were done on day 10 and on every bleed
thereafter to assess changes in leukocyte populations. Cell counts were
reported for white blood cells (WBCs), lymphocytes, and neutrophils, as well
as the lymphocyte:neutrophil ratio (Figure 10). There was a high level of
individual variation in WBCs among the monkeys (Figure 10a). Monkey 50Y
(VA+) exhibited high WBC and lymphocyte counts, that remained above the
normal range for most time points (Figure 10a, 10c). However, monkey 14Y
(VA+) had below normal WBC and lymphocyte counts (Figure 10a, 10c).
Overall, the average WBC and lymphocyte counts remained in the normal
range (Figure 10b, 10d), with individual increases after the rash (Figure 10a,
10c). There was considerable variation in neutrophil counts (Figure 10e), and
the average number showed a transient increase post-rash (Figure 10f). The
lymphocyte:neutrophil ratios were elevated above the expected value of 1, for
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most monkeys and for all days following rash (Figure 10g, 10h). Differences
between vitamin A-supplemented and non-supplemented monkeys were not
identified.
Frequency of CD4+ and CD8+ cells
CD4+ and CD8+ T cell frequencies were reported as the percentage of
the live, non-monocyte (CD14-), non-B cell (CD20-) population after gating
on lymphocyte and single cell populations (Figure 11). The frequency of CD4+
cells for most monkeys were approximately 50% during the first month postinfection, but decreased approximately 10% for vitamin A-supplemented and
non-supplemented groups of monkeys at day 39 (Figure 11a, 11b). The
average CD4+ frequency increased in the vitamin A-supplemented group at
day 56 (not significant), and returned to a similar frequency as the nonsupplemented group by day 84, though this difference was not statistically
significant. All monkeys, except monkey 46Y, maintained similar frequencies
of CD8+ cells throughout 3 months post-infection. Monkey 46Y exhibited a
high frequency of CD8+ and low frequency of CD4+ cells for the first month.
The CD4:CD8 ratios of monkeys are typically 2. These ratios increased for
some monkeys early after measles virus infection, but were not identifiably
different between the groups (Figures 11e, 11f).
IFN-γ T cell response
ELISPOT assays were used to detect IFN-γ-producing T cells specific
for measles virus proteins H, N, and F (Figure 12). At day 21 post-infection,
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all six monkeys had developed a measles virus H-specific IFN-γ response
(Figure 12a, 12b), and monkeys 14Y, 46Y, and 50Y showed N- and F-specific
IFN-γ responses (Figures 12c, 12e). Though individual differences were
evident on the robustness of each response, the vitamin A-supplemented
group and non-supplemented groups had similar average responses for each
measles virus protein (Figures 12b, 12d, 12f). At day 35, the measles-specific
IFN-γ-producing T cell response decreased to baseline, with the exception of
an H-specific IFN-γ response detected for monkey 14Y at day 52.
Th17 cell characterization by Flow Cytometry
To determine if vitamin A supplementation impacted the Th17 effector
cell population, the levels of IL-17 and IL-21 cytokine expression were
determined from gated CD4+ T cell populations using ICS and flow cytometry
(Figure 13, 14). Frequencies of IL-17+ and IL-21+ CD4+ T cells differed
depending on the stimulation condition. Measles virus peptides from the H
and N proteins were used to detect a measles virus-specific response, and
staphylococcus enterotoxin B (SEB) was used to stimulate all PBMCs as a
positive control.
Frequencies of measles virus H-specific CD4+ IL-17+ cells increased in
all monkeys from less than 0.2% at days 0 and 10, to 0.5%-3.25% at day 18
and then remained below 1% from day 28 (Figure 15a, 15b). Measles virus
N-specific IL-17+ T cells were detected in monkey 31Y of approximately 3%
86
at day 18, and increased in monkey 17Y to over 1% between day 18 and day
56 (Figure 15c, 15d).
The measles virus H-specific IL-21+ frequencies increased from an
initial level below 0.15% on days 0 and 10, to between 1-4% for all monkeys
at day 18 (Figure 16a, 16b). Subsequently, the response fluctuated with a
decrease at day 28 for all monkeys and an increase at day 39 for half of the
monkeys. The vitamin A-supplemented group demonstrated a higher Hspecific IL-21+ frequency at day 39 compared to the non-supplemented group
of monkeys. At day 56, frequencies of H-specific IL-21+ cells were still
elevated in some monkeys compared to their naïve state. These H-specific IL21+ frequency changes remained above their baseline frequencies through all
time points, but there were notable differences between the magnitude and
days of responses in individual monkeys, that were not specific to vitamin Asupplemented or non-supplemented monkeys.
N-specific IL-21+ frequencies of CD4+ T cells varied between monkeys
(Figure 16c). Monkey 50Y increased to 0.8% at day 10, and monkeys 31Y and
14Y exhibited a more robust increase at day 18, ranging between 4-5.5%. The
remaining monkeys exhibited N-specific IL-21+ frequencies above their naïve
states, but were variable and remained below 2%. However, monkey
averages indicated a clear biphasic increase in N-specific IL-21+ frequencies
at day 18 and day 39 (Figure 16d). A difference between vitamin Asupplemented and non-supplemented monkeys was not observed.
87
To further investigate Th17 cells and their response in vitamin Asupplemented and non-supplemented monkeys, transcription factor
expression and cytokine production were investigated by comparing Th17
and non-Th17 populations. Th17 cells were defined as CD4+ IL-17+ T cells in
this panel, and non-Th17 cells were CD4+ IL-17- T cells. These populations
were plotted together as histograms of RORγt transcription factor expression,
at days 0, 18, and 56 (Figure 17). At day 0, prior to infection, IL-17- and
IL17+ populations had very low levels of RORγt expression. However, by day
18, both IL-17- and IL-17+ populations increased in RORγt expression, but
the Th17 cells exhibited a more positive RORγt signal. At day 56, two distinct
populations were evident and stimulated and unstimulated Th17 populations
expressed higher levels of the transcription factor RORγt.
Due to the rarity of double positive IL-17+IL-21+ cells, IL-17+ cells
versus IL-17- cells were also plotted at days 0, 18, and 56 to compare the
amount of IL-21 production between these two populations (Figure 18). At
day 0, IL-17+ cells produced similar levels of IL-21 compared to the IL-17cells. However, by day 18, IL-17+ cells exhibited a slight increase in IL-21
production, but there was still considerable overlap with the IL-17- cells. By
day 56 IL-17+ cells produced much higher levels of IL-21 than the IL-17cells.
Detection of IL-17-producing T cells in response to measles virus
infection was further explored with an ELISPOT assay (Figure 19). The
88
unstimulated response was not further stimulated ex vivo, so IL-17 spot
forming cells in the unstimulated condition were due to in vivo stimulation in
the infected monkey (Figure 19a, 19b). Measles virus-specific IL-17 T cell
responses that were present in wells stimulated by measles virus infected
Vero cell lysate (ABI) (Figure 19c, 19d). To provide a picture of the measles
virus-only specific IL-17-producing T cell response the unstimulated
responses were subtracted from the measles-specific responses (Figure 19e,
19f). In all monkeys, small measles virus-specific IL-17+ T cells were detected
at day 14, but was more apparent at day 52 and decreased by day 71/72.
There were no significant differences between the vitamin A-supplemented
and non-supplemented monkey groups.
To detect IL-17A cytokine in the monkey plasma, IL-17A ELISAs were
performed (Table 3). Values were deemed significant if they were two times
the background. Monkeys 17Y and 50Y exhibited higher and more consistent
levels of IL-17A compared to the other monkeys, with 14Y and 31Y showing
little to no detectable IL-17A in the plasma, despite the robust Th17
responses that were detected in other assays.
Antibody response
The measles-specific IgG binding antibody response was assayed by
EIA using measles virus-infected Vero cell lysate (ABI) and anti-monkey
HRP conjugated IgG antibody (Figure 20). Five of the monkeys developed a
measles virus-specific antibody response, although monkey 24Y did not,
89
despite the presence of viremia and detection of measles virus shedding in
nasal secretions indicating an infection occurred (Table 2, Figure 4). Data are
reported as the reciprocal of the highest dilution with a value two times the
background. For each monkey, the highest measles virus-specific IgG
response was observed at day 52 (Figure 20a). There was no significant
difference observed in the antibody response to measles virus infection in the
vitamin A-supplemented and non-supplemented monkeys, however, there
was a high standard deviation in the vitamin A-supplemented group as
monkey 24Y did not have a detectable response (Figure 20b).
The number of antibody secreting cells (ASCs) in PBMCs and bone
marrow were measured. Total antibody-secreting cells increased in
circulation early in response to measles virus infection, with a distinct peak
at day 14 (Figure 21a, 21b), but few were identified as producing antibody to
measles virus (Figure 21c, 21d). Measles virus-specific ASC were detected in
blood in large numbers at day 52 (Figure 21c, 21d).
Bone marrow (BM) was collected twice a month for the first two
months, and once near the end of the third month. Total numbers of IgM,
IgA, and IgG-ASCs from BM remained relatively stable until day 56, when
monkeys displayed an increase in BM ASCs, which plateaued by day 84
(Figure 22a, 22b). At day 28, measles virus-specific ASCs were unable to be
counted because of a signal that exceeded the countable threshold in monkeys
24Y and 50Y, though they were increased in monkeys 14Y, 17Y, 31Y, and
90
46Y. They then decreased at day 40, and subsequently increased again at day
56. Peaks of measles virus-specific ASCs were found at day 28 and day 56-84
(Figures 22c, 22d). For the two monkeys (17Y, 24Y) with data at day 84, this
upward trend of measles virus-specific bone marrow ASCs continued. The
non-supplemented group displayed higher measles virus-specific ASC in BM
at day 28 and day 84. However, this was not significant and some points were
unable to be calculated due to too many spots, or background levels that were
too high to count the spots on the plate. Therefore, missing data points may
change averages significantly.
Neutralizing antibodies were detected by PRNTs in five of the six
monkeys (Figure 23a). Monkey 24Y did not mount a neutralizing antibody
response. All other monkeys showed a dramatic increase in neutralizing
antibodies at day 14 and day 18, that remained elevated through day 84
(Figure 23b).
Discussion
Vitamin A supplementation has been associated with improved
clinical outcomes in patients with measles when given during the acute rash
phase (27-29, 122, 123). There are several theories for the potential ways in
which vitamin A may improve the health of patients with measles. This study
of vitamin A-supplementation of rhesus macaques was done to elucidate the
effects of vitamin A on the immune response to measles infection.
Measurement of T cell (Th1, Th17) and antibody (EIA, ASC, PRNT) through
91
day 84 has not shown a difference between vitamin A-supplemented and nonsupplemented groups. However, none of the hypotheses have been discounted
due to small groups and missing data. In our study, differences in plasma
retinol levels begin to diverge at day 21 between the vitamin A-supplemented
and non-supplemented monkeys (Figure 9), and so differences observed due
to vitamin A treatment are hypothesized to occur between days 21-50, or
slightly thereafter, as this is when plasma retinol levels previously showed a
decrease in non-supplemented monkeys (Figure 2). We also have yet to
confirm that the amount of vitamin A supplementation given prevents this
decrease after measles.
Vitamin A and lymphopenia
Measles virus-induced lymphopenia is transient, and lymphocytes
return to normal or elevated levels in both vitamin A-supplemented and nonsupplemented monkeys shortly after rash subsides (Figure 10b). This occurs
before the divergence of vitamin A levels is observed between the two monkey
groups at day 21, and therefore, vitamin A supplementation will not affect
measles virus-induced lymphopenia. However, the frequencies of CD4+ T
cells dropped approximately 10% at day 39 for unknown reasons. The vitamin
A-supplemented monkeys recovered more quickly (by day 56) than the nonsupplemented monkeys, who do not recover from this until day 84. This
finding approached statistical significance (p < 0.1). It is possible that nonsupplemented monkeys have a vitamin A deficiency-induced lymphopenia
92
between day 39 and 84 and that larger groups of monkeys are needed to
identify this difference (137).
Vitamin A, measles virus replication, and clearance of measles virus
persistent infection
Vitamin A has also been previously suggested to inhibit measles virus
replication in vitro (110, 111). However, viremia is established prior to
vitamin A supplementation and non-supplemented monkeys (Figure 4), and
is cleared by day 21 when retinol levels begin to drop. Future studies will
determine whether vitamin A enhances the clearance of measles virus RNA.
An increase in IL-21 producing T cells observed at day 39, was higher
in the vitamin A-supplemented monkeys than the non-supplemented
monkeys, and approached statistical significance (p < 0.1). This increase in
IL-21-producing T cells in vitamin A-supplemented monkeys could increase
Th17 cell differentiation (77, 103). This idea is supported by the increase in
measles virus-specific IL-17-producing T cells at day 52 (Figure 19). IL-21
could also function to control persistent measles virus infection (171, 172). If
this is the case, vitamin A may play a role in clearance of persistently
infected cells. This mechanism still needs to be explored further in larger
groups of monkeys and with quantitation of measles virus RNA.
Vitamin A and the measles virus-specific antibody response
Another theory of vitamin A function that may mitigate the severity of
disease and death due to measles is that it plays a role in enhancing the
93
antibody response. However, monkey 24Y was supplemented with vitamin A,
and over the course of infection did not appear to have a detectable IgG or
neutralizing antibody response (Figure 20, 23), but did have detectable ASCs
(Figure 21, 23). For EIA, this discrepancy may be due to lack of crossreactivity of the anti-human Ab and monkey IgG. Interestingly, the shedding
of measles virus was only detected on one time point from monkey 24Y (Table
2). It is possible that no antibody class switching occurred, and any measles
virus-specific antibody response mounted by monkey 24Y was a different
isotype, though this has not been confirmed. Trends of ASCs in the peripheral
blood and bone marrow show that 24Y has a decreased number of ASCs,
though the variation in numbers over time follows the same trends as
monkeys in both vitamin A-supplemented and non-supplemented groups
(Figure 21, 22). Further investigation is needed to resolve this issue. Vitamin
A supplementation in a randomized clinical trial in African children two
years or younger showed higher measles virus-specific IgG antibodies
compared to the non-treated group, as well as a significant reduction in
mortality (29). At present, our data do not support this, but technical issues
with detecting monkey IgG and with counting large numbers of ASCs need to
be resolved.
Measles virus-specific IFN-γ response
The measles virus-specific IFN-γ response was predominantly Hspecific, and developed around day 21 post-infection along with small
94
numbers of N- and F-specific IFN-γ producing T cells. This response, along
with cytotoxic CD8+ T cells, likely contributes to the rash and clearance of
infectious virus (75, 79, 138), and the response wanes quickly. It appears that
there is potential for a transient reappearance of this response later in the
course of infection, as monkey 14Y exhibited a recurrence of H-specific IFN-γproducing T cells at day 52. Future studies will determine whether this is
associated with an increase in viral RNA. Vitamin A had no identifiable effect
on altering this response.
Th17 biphasic response to measles virus infection
Measles virus-specific IL-17-producing T cells were detected by
ELISPOT with peaks at day 18 and day 56 following infection. This biphasic
IL-17 producing T cell response was also identified by intracellular cytokine
staining and flow cytometry, which confirmed increases in CD4+IL-17+ T
cells at these days. Th17 cells were further characterized as having increased
RORγt expression and higher levels of IL-21 production, compared to nonTh17 cells.
Conditions, limitations, and future directions of vitamin A analysis
No statistically siginificant differences in immune responses to
measles virus infection were observed between the vitamin A-supplemented
and non-supplemented monkey groups. Observed differences between the
vitamin A-supplemented and non-supplemented groups had large,
overlapping standard deviations among the averages for each group. The
95
vitamin A group included monkey 24Y, which did not mount detectable
antibody or Th17 immune responses as well as monkey 14Y, which
consistently had very high immune responses compared to other monkeys.
These studies will be repeated to increase the numbers of monkeys in each
group. It may be of interest to challenge 24Y again.
Conclusion
Thus far, the specific role of vitamin A in host response to measles
virus infection still remains unclear. Vitamin A does not play a role in
measles virus-induced lymphopenia, but may prevent later formation of
lymphopenia that is induced by vitamin A-deficiency. Vitamin A may play a
role in enhancing clearance of measles virus RNA from persistently infected
cells and samples have been collected to determine this. We did not confirm a
correlation between increased vitamin A levels and increased measles-specific
antibody titers, and vice versa (29, 113, 130). Major differences were not
observed in the IFN-γ-producing T cell response between vitamin Asupplemented and non-supplemented monkeys. Furthermore, this study has
allowed the characterization of the Th17 response up to three months
following measles virus infection in vivo, with major peaks of Th17 cells
present around day 18 and day 56.
Further analysis of samples from this study will explore the Th1 and
Tfh responses, and other potential differences due to vitamin A
supplementation. Three months of the study remain, and samples have been
96
archived so there is much data to be collected and analyzed yet. Additionally,
qualitative imaging should be done using immunohistochemistry on the skin
biopsies to confirm and localize measles virus in rash biopsy samples. The
evaluation of measles virus RNA clearance will include studies to detect
measles virus RNA in PBMCs, bone marrow, and lymph node cells (B cell, T
cell, and monocyte populations), to determine the type of cell harboring
measles virus RNA.
This study is the first step towards elucidation of the role of vitamin A
in providing better health outcomes following measles virus infection.
Overall, the immune responses to measles virus infection varied between
individual monkeys, as it does in the human population. The number of
monkeys used needs to be larger to identify small differences due to vitamin
A supplementation.
97
Table 1. PCR primers, target and cycling conditions for detection of
the measles virus N gene.
Primers for Detection of Measles Virus N Gene (IDT)
Cycling
Target
Primer and Sequence
Conditions
50°C, 30 min
94°C, 2 min
350 bp
MV 41
94°C, 30 s; 55°C, 30
amplicon of 5’- CATTACATCAGGATCCGG -3’
s; 68°C, 45 s for 40
the measles MV 42
cycles
virus N gene 5’- GTATTGGTCCGCCTCATC -3’
68°C, 5 min; 4°C,
hold
Table 2. Measles virus shedding in respiratory secretions. Virus
shedding was monitored by RT-PCR detection of measles virus N gene in
RNA isolated from nasal swabs. (+) indicates a positive result, (-) indicates
there was no amplified PCR product. (VA+) indicates monkeys supplemented
with vitamin A at the time of rash (day 10, 11).
Presence of Measles Virus RNA
Day
14Y
(VA+)
17Y
24Y
(VA+)
31Y
46Y
50Y
(VA+)
7
-
+
-
-
-
+
10
+
+
-
+
+
+
14
+
+
+
+
+
+
18
-
+
-
+
-
+
21
-
+
-
-
-
+
39
-
-
-
-
-
-
98
Table 3. IL-17A ELISAs. IL-17A ELISAs (Mabtech) were done on the
plasma samples. Wells were considered to be positive if the absorbance was
greater than two times the background absorbance levels. (+) plasma only,
(++) 1:30 dilution, (-) no IL-17A detected. (VA+) indicates monkeys
supplemented with vitamin A at the time of rash (day 10, 11).
IL-17A ELISA Results
Day
14Y
(VA+)
17Y
24Y
(VA+)
31Y
46Y
50Y
(VA+)
0
-
-
-
-
+
-
7
-
+
-
-
-
-
10
-
+
-
+
+
+
14
-
+
-
-
-
+
18
-
+
+
-
+
-
21
-
+
-
-
-
+
28
-
+
-
-
-
++
35
-
+
-
-
-
-
40
-
+
-
-
-
-
52
-
+
-
-
-
++
56
-
+
-
-
-
+
71/72
-
-
-
-
-
-
84
-
-
-
-
-
-
99
Figure 1. Vitamin A (retinol) status and usage is impaired during
infection. During infection, decreased food intake can lead to lower vitamin
A uptake. During infection, less retinol is absorbed and more is excreted in
urine. Other hypotheses leading to vitamin A impairment include impaired
transport of stores from liver to peripheral tissues or by increasing
requirements at sites of inflammation [137].
Figure 2. Plasma retinol levels averaged between two rhesus
macaques after measles virus infection [Lin, W.H, and Griffin, D.E.,
unpublished].
100
Figure 3. Time course of measles virus clearance. Infectious virus
shown in blue, viral RNA outlined by the dashed black line, in reference to
the characteristic rash (red box) that occurs around day 10 [1].
Figure 4. Viremia is present by day 7, and is cleared in all animals by
day 18. Viremia was measured by tissue culture infectious dose 50 (TCID50)
assays, by co-cultivation of PBMCs from each animal on a monolayer of
Vero/hSLAM cells. (a) Individual monkeys and (b) averages between the
vitamin A-supplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y; solid line) were compared using a
two-way ANOVA and a Bonferroni multiple comparison correction. There
were no statistically significant differences between the two monkey groups.
101
Figure 5. Change in total body weight over course of measles virus
infection. (a) Changes in weight reported as the percent change from day 0
for each monkey. (b) Averages between the vitamin A-supplemented monkeys
(14Y, 24Y, 50Y; dashed line) and the non-supplemented monkeys (17Y, 31Y,
50Y; solid line) were compared using a two-way ANOVA and a Bonferroni
multiple comparison correction. There were no statistically significant
differences between the two monkey groups.
Figure 6. Maculopapular rash was very robust on monkey 50Y on day
10 post-infection. The upper limbs (a) and trunk (b) of 50Y showed the most
extensive rash.
102
Figure 7. Rash histology of skin biopsies. Hematoxylin and eosin
staining of skin punch biopsies. Rash severity was graded and monkeys were
ordered from most severe to least severe: 17Y (a), 14Y (b), 50Y (c), 46Y (not
pictured), 31Y (not pictured), and 24Y (d). Yellow boxes highlight areas of
cellular debris and edema (a, c). White arrows point out eosinophils (a, b),
grey arrows point out lymphocytes (a, b), and black arrows point out
macrophages (a, b). Scale bars are 100 um in (a, b, d) and 50 um in (c). 400X.
103
a
b
c
d
Figure 8. Histology of lymph node biopsies. Lymph node biopsies are
pictured for vitamin A-supplemented monkeys 14Y (day 71) at 2X (a) and 20X
(b) and monkey 50Y (day 78) at 2X (c) and 20X (d). Lymph nodes are reactive,
with other cells in addition to lymphocytes seen at 20X. Lymph node
reactivity was assessed by Tori Baxter, DVM and Diane Griffin, MD, PhD.
Scale bars are 1 mm (a, c) and 100 um (b, d).
104
Figure 9. Vitamin A levels begin to drop at day 21 in the nonsupplemented group of monkeys (17Y, 31Y, 46Y) but remain more
stable in vitamin A-supplemented monkeys (14Y, 24Y, 50Y). (a) Retinol
concentrations were determined for each monkey by HPLC analysis of
plasma, by the laboratory of Dr. Richard Semba. (b) Averages between the
vitamin A-supplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y’ solid line) were compared using a
two-way ANOVA and a Bonferroni multiple comparison correction. Results
were significant at day 21. (**) p < 0.0
105
Figure 10. Comprehensive blood counts and differential leukocyte
counts following rash. Space between the horizontal/dashed lines in each
graph indicates the normal range of cell counts for rhesus macaques between
3-4 years old: 7,700-13,300 WBCs/ul, 2,671-8,350 lymphocytes/ul, and 2,6715,147 neutrophils/ul [54], and the normal lymphocyte:neutrophil ratio is 1 (a,
c, e, g). Averages between the vitamin A-supplemented monkeys (14Y, 24Y,
50Y; dashed line) and the non-supplemented monkeys (17Y, 31Y, 50Y; solid
line) were compared using a two-way ANOVA and a Bonferroni multiple
comparison correction (b, d, f, h). There were no statistically significant
differences between the two monkey groups.
106
Figure 11. Frequency of CD4+ and CD8+ cells within the CD14-CD20live cell population, and CD4:CD8 cell ratio. CD4+ and CD8+ T cell
frequencies were determined by flow cytometry. Cells were first gated by
lymphocytes and single cells, and frequencies were assessed in the nonmonocyte, non-B cell populations (a, c, e). Averages between the vitamin Asupplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y; solid line) were compared using a ttest for data on each day (b, d, f). There were no statistically significant
differences between the two monkey groups.
107
Figure 12. Measles virus H, N, and F protein-specific IFN-γ secreting
T cells peak at 21 days post-infection. IFN-γ secreting T cells were
detected by ELISPOT (a, c, e). Averages between the vitamin Asupplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y; solid line) were compared using a
two-way ANOVA and a Bonferroni multiple comparison correction (b, d, f).
There were no statistically significant differences between the two monkey
groups.
108
Figure 13. Intracellular staining for IL-17A. Gating scheme for IL-17
from CD4+ cells on day 0 (a), 18 (b) and 56 (c) post-infection is shown. On
days 0 and 18 CD4+ cells were defined as CD14-CD20-CD3+CD8- and from
day 28 onward as CD14-CD20-CD3+CD4+. Plots are shown for one vitamin
A-supplemented monkey (50Y) in the top row, and one non-supplemented
monkey (46Y) in the bottom row. Cells were stimulated with SEB. IL-17+
populations are the top square in each plot, and IL-17- populations are in the
bottom square. Percentages of IL-17+ and IL-17- from CD4+ populations are
indicated in their respective boxes.
109
Figure 14. Intracellular staining for IL-21. Gating scheme for IL-21 from
CD4+ cells on day 0 (a), 18 (b) and 56 (c) post-infection is shown. On days 0
and 18 CD4+ cells were defined as CD14-CD20-CD3+CD8- and from day 28
onward as CD14-CD20-CD3+CD4+. Plots are shown for one vitamin Asupplemented monkey (50Y) in the top row, and one non-supplemented
monkey (46Y) in the bottom row. Cells were stimulated with SEB. IL-21+
populations are selected in each plot, with percentages IL-21+ from CD4+
cells indicated in their respective boxes.
110
Figure 15. Frequency of IL-17+ cells as a percentage of total CD4+ T
cells peaked at day 18. IL-17+ frequencies were determined by
intracellular staining and flow cytometry (a, c, e). Averages between the
vitamin A-supplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y; solid line) were compared using a ttest for data on each day (b, d, f). There were no statistically significant
differences between the two monkey groups.
111
Figure 16. Frequency of IL-21+ cells as a percentage of total CD4+ T
cells showed peaks at day 18 and day 39 post-infection. IL-21+
frequencies were determined by intracellular staining and flow cytometry (a,
c, e). Averages between the vitamin A-supplemented monkeys (14Y, 24Y,
50Y; dashed line) and the non-supplemented monkeys (17Y, 31Y, 50Y; solid
line) were compared using a t-test for data on each day (b, d, f). There were
no statistically significant differences between the two monkey groups.
112
Figure 17. RORγt expression was upregulated in CD4+ T cells by day
18 post-infection, and is higher in IL-17+ cells than IL-17- cells. The
level of RORγt expression was detected using intracellular staining and flow
cytometry and was evaluated on IL-17+ and IL-17- CD4+ T cells on day 0 (a),
day 18 (b), and day 56 (c). CD4+IL-17- populations are in red and CD4+IL17+ cell populations are in blue. For a, b and c, RORγt shift data from nonsupplemented monkey 46Y is shown in the left column, and vitamin Asupplemented monkey 50Y is shown on the right for each day. The top row is
from the DMSO negative control condition, followed by measles virus Hstimulated, N-stimulated and the bottom row depicts SEB-stimulated
conditions.
113
Figure 18. IL-21 production begins to increase by day 18, and is much
greater by day 56 post-infection in IL-17+ cells than IL-17- cells. The
level of IL-21 expression was detected using intracellular staining and flow
cytometry and was evaluated on IL-17+ and IL-17- CD4+ T cells on day 0 (a),
day 18 (b), and day 56 (c). CD4+IL-17- populations are in red and CD4+IL17+ cell populations are in blue. A shift was used to compare IL-21 expression
between IL-17+ and IL-17- populations, rather than looking at double
positive populations, because the number of events were low for IL-17+ and
IL-21+ populations. For a, b and c, IL-21 shift data from non-supplemented
monkey 46Y is shown in the left column, and vitamin A-supplemented
monkey 50Y is shown on the right for each day. The top row is from the
DMSO negative control condition, followed by measles virus H-stimulated, Nstimulated and the bottom row depicts SEB-stimulated conditions.
114
Figure 19. IL-17A-secreting T cells are present in a biphasic
response, with an early peak at day 14 and a late peak at day 52. IL17A secreting T cells were detected by ELISPOT assays (a, c, e).
Unstimulated condition was not stimulated ex vivo (a, b). Measles virusspecific IL-17A T cells were detected by ex vivo stimulation with measles
virus-infected Vero cell lysate (ABI) (c, d). The unstimulated response was
subtracted from the measles virus-specific response to remove background
and observe the measles virus-stimulated response only (e, f). Averages
between the vitamin A-supplemented monkeys (14Y, 24Y, 50Y; dashed line)
and the non-supplemented monkeys (17Y, 31Y, 50Y; solid line) were
compared using a t-test for data on each day (b, d, f). There were no
statistically significant differences between the two monkey groups.
115
Figure 20. Measles virus-specific IgG as detected by ELISA. Plasma
from each monkey was used for an EIA detecting IgG that bound measles
virus antigen. Results are plotted at the highest consecutive dilution two
times above the background (a). Averages between the vitamin Asupplemented monkeys (14Y, 24Y, 50Y; dashed line) and the nonsupplemented monkeys (17Y, 31Y, 50Y; solid line) were compared using a ttest for data on each day (b). There were no statistically significant
differences between the two monkey groups.
116
Figure 21. Total antibody- and measles virus-specific antibodysecreting cells in PBMCs as detected by ASC assays. ASCs detected to
(a) total antibody or (c) measles virus-specific antibody in PBMCs. Averages
between the vitamin A-supplemented monkeys (14Y, 24Y, 50Y; dashed line)
and the non-supplemented monkeys (17Y, 31Y, 50Y; solid line) were
compared using a t-test for data on each day (b, d). There were no
statistically significant differences between the two monkey groups.
117
Figure 22. Total antibody- and measles virus-specific antibodysecreting cells in BM as detected by ASC assays. ASCs detected to (a)
total antibody or (c) measles virus-specific antibody in bone marrow
mononuclear cells. At day 28, measles virus-specific ASCs (c) were unable to
be counted because of a signal that exceeded the countable threshold in
monkeys 24Y and 50Y. Averages between the vitamin A-supplemented
monkeys (14Y, 24Y, 50Y; dashed line) and the non-supplemented monkeys
(17Y, 31Y, 50Y; solid line) were compared using a t-test for data on each day
(b, d). There were no statistically significant differences between the two
monkey groups.
118
Figure 23. Neutralizing antibody response as detected by PRNTs.
Reciprocal titers of neutralizing antibodies were measured by 50% plaque
reduction of a DI-free strain of Edmonston infection on Vero cells (a).
Averages between the vitamin A-supplemented monkeys (14Y, 24Y, 50Y;
dashed line) and the non-supplemented monkeys (17Y, 31Y, 50Y; solid line)
were compared using a t-test for data on each day (b). There were no
statistically significant differences between the two monkey groups.
119
Chapter Four:
Discussion of the immune responses to measles virus infection
in vitro and in vivo
120
The innate immune response to measles virus infection
Understanding the immune response to measles virus infection in
humans has been a difficult process. Individual differences in the immune
response, as well as immune response differences due different strains of
measles virus have complicated analysis and synthesis of an overall picture.
The innate immune response was analyzed using several measles virus
strains for in vitro infections of monocyte-derived dendritic cells.
Dendritic cells play an important role in the innate immune response,
but also provide the proper environment for the development of adaptive
immune responses. The role of measles virus C and V proteins were explored
using an infection model of moDCs to examine their role in type I interferon
induction and how that may affect ISG/VSIG expression
Type I interferon
In these experiments, no type I interferon was detected after infection
of moDCs by DI-free measles vaccine or Wt strains. Because the V and C
proteins inhibit type I interferon induction and responses, the Wt C KO and
Wt V KO strains of measles virus were expected to induce type I interferon
expression. This was not observed in our studies. However, a previous study
was able to detect interferon α and β following Wt C KO and Wt V KO
measles virus infection in vivo (97). A more sensitive assay should be
explored to provide definitive data on whether or not type I interferons are
induced by the measles virus infections of moDCs.
121
The role of measles virus and its C and V proteins on interferonstimulated gene (ISG) and viral stress-induced gene (VSIG)
expression
Measles virus C- and V-KO viruses exhibited the ability to enhance the
induction of IFIT1, with the V protein KO virus also enhancing Mx1
transcription. Under the assumption that the Wt C KO and Wt V KO viruses
have produced low biologically active type I interferons, that are below our
limit of detection, it is likely that these measles virus KO strains were able to
activate the transcription of these genes through multiple mechanisms,
including the viral stress-induced and interferon stimulated pathways.
Contrastingly, the Wt measles virus, with the C and V proteins intact, were
likely only to activate the viral stress-induced pathway alone. IFIT1 mRNA
appears to be more efficiently induced than Mx1 in the first 48 hours
following measles virus infection. Differences in ISG/VSIG expression showed
that Edm vaccine strain exhibited a much greater upregulation of expression
of these antiviral protein genes than the Wt strains. In the future, it would be
interesting to include a comparison with a double C- and V-protein KO and to
expand the analysis to Edm and its DI-free C- and V-deleted vaccine strains.
The early adaptive immune response to measles virus infection
Measles virus infection of rhesus macaques has provided the
opportunity to evaluate development of the adaptive immune processes to
infection. All monkeys displayed hallmark characteristics of measles virus
122
infection, with development of viremia and rash. All monkeys had mounted a
measles virus-specific IFN-γ-producing T cell response around day 21 postinfection. The IFN-γ response correlated with clearance of infectious measles
virus from blood between days 10 and 18. After viremia had been cleared,
most IFN-γ-producing T cells disappeared from circulation by day 35 postinfection. These IFN-γ-producing T cells, and their role in the Th1 response,
will be confirmed by flow cytometry analysis and intracellular cytokine
staining to detect IFN-γ, IL-2, and TNF-α in CD4+ T cells. Data from in vitro
measles virus infection show a significant upregulation of IL-27 transcripts in
moDCs at 24 hours post-infection, which would support the differentiation of
Th1 T effector cells.
Th17 regulatory cytokine expression to measles virus infection
Production of cytokines by moDCs that influence the adaptive immune
response, favor the inhibition of Th17 cell differentiation. Cytokines that lead
to a favorable environment for Th17 cell differentiation and survival are IL-6,
IL-1β, and IL-23A, and although a non-significant trend of upregulation of
pro-inflammatory cytokine transcripts (IL-1β and IL-6) was present, a
significant downregulation of IL-23A mRNA and upregulation of IL-27
mRNA are predicted to prevent long-term differentiation of Th17 cells early
in infection. Although mRNA expression of Th17 regulatory cytokines favors
the moDC inhibition of Th17 cell differentiation within the first 48 hours
following measles virus infection, a robust Th17 response was detected in
123
vivo at later time points. In the future it would be interest to test for the
presence of these cytokines and examine APC function from samples in vivo,
to cover more extensive time points because it is clear that a Th17 response is
mounted in all monkeys.
The Th17 response to measles virus infection
The Th17 response to measles virus infection in monkeys is biphasic,
with a small peak between weeks 2 and 3 post-infection, and a much larger
response between weeks 5 and 6 post-infection. Detection of the early IL-17
response did not depend on ex vivo stimulation with measles virus antigens
and this returns to baseline. These cells were likely stimulated in vivo. A
second wave of the measles virus-specific Th17 response detected by ex vivo
stimulation was established by day 52. Flow cytometry analysis of IL-17+
cells by intracellular cytokine staining showed that they also expressed
RORγt and IL-21. The role of this response in maturation of the antibody
response requires further study.
The measles virus antibody response
All monkeys had a large increase in circulating ASCs at day 14, but
most were not producing detectable antibody to measles virus. However,
large numbers of measles virus-specific ASC were present in the bone
marrow at day 28. These decreased by day 40, and increased again by day 56.
Measles virus-specific ASCs in PBMCs were detected at day 52, which likely
trafficked to the bone marrow to establish a measles virus-specific, long-lived
124
plasma cell population. Measles virus-specific IgG was detected in plasma
beginning at day 14, and continued to increase until day 52 when this
response peaked. Avidity of this antibody has not yet been measured.
The role of vitamin A on the immune response to measles virus
infection
Vitamin A was supplemented at the time of the rash, at day 10 and 11.
Plasma retinol levels in the non-supplemented group of monkeys were first
decreased at day 21 so it is expected that the major effects of vitamin A
supplementation would not manifest until day 21 or later. It is likely that
vitamin A plays no role in the prevention of the measles virus-induced
viremia or lymphopenia that occurs early following infection, before vitamin
A levels diverge. Furthermore, data from this study does not currently
support previous findings that vitamin A correlates with a higher measles
virus-specific IgG antibody response.
The vitamin A-supplemented group did exhibit some changes that
approached statistical significance. Monkeys that were supplemented with
vitamin A recovered from a decrease in CD4+ T cells more quickly than nonsupplemented monkeys. In addition, higher numbers of IL-21-producing T
cells were present at day 39 in the vitamin A-supplemented monkeys
function to control persistent measles virus infected cells (171, 172). These
observations and mechanism needs to be further explored.
125
Three months of this study remain and many samples have been
archived, so much data has yet to be collected and analyzed. Vitamin A plays
a role in differentiation of CD4+ T effector cell subsets, and data that has yet
to be analyzed through flow cytometry and intracellular cytokine staining
will further explore development of the Th1 and Tfh cells lineages. This
experiment will also be repeated to increase the number of monkeys in each
group.
126
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Curriculum vitae
Nicole E. Putnam
(920) 216-6835 - [email protected]
EDUCATION
Master of Science in Molecular Microbiology & Immunology
Expected May 2014
Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
Thesis: The innate and adaptive immune response to measles virus
Advisor: Dr. Diane E. Griffin
Certificate in Public Health Preparedness
Expected May 2014
Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
Bachelor of Science in Biochemistry and Psychology
December 2010
University of Wisconsin-La Crosse, La Crosse, WI
RESEARCH EXPERIENCE
ScM Thesis Research
2012-2014
Dr. Diane Griffin, Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD
Determination of innate immunological effects in vitro of the V and C protein
in wild type and vaccine strains of measles virus using virus growth, plaque
assays, plaque purification, PCR and agarose gel electrophoresis, qPCR, RTPCR, interferon bioassays, isolation of PBMCs, and generation of monocyte
derived dendritic cells. Determination of effects of vitamin A supplementation
on wild-type measles infection in rhesus macaque models using TCID50,
EIAs, ASCs, ELISPOTs, PRNTs, and multiparameter flow cytometry.
Pharmaceutical Research and Development Intern
2010
Dr. Raghavan Rajagopalan, Covidien, St. Louis, MO
Used synthetic chemistry to make novel photosensitive compounds,
bioconjugation of photosensitive and fluorescent compounds to antibodies and
peptides to target ovarian and colon cancer cell lines using LC-MS, NMR,
ESR, bioconjugation of compounds to peptides and antibodies, limited
confocal microscopy, and in vitro cytotoxicity assays.
Laboratory Manager
2009-2010
Dr. Alex O’Brien, Dr. Bart VanVoorhis, La Crosse, WI
Scheduled lab assistants for data collection and experiment times, managed
informed consent paperwork, while maintaining duties of research assistant.
144
Research Assistant
2008-2009
Dr. Alex O’Brien, Dr. Bart VanVoorhis, La Crosse, WI
Assisted in experiment development using SuperLab program, collected data
biweekly, and participated in journal club.
Undergraduate Researcher
2008-2009
Dr. Aaron Monte, La Crosse, WI
Assisted in the organic synthesis of the beta-alkaloid compound
tetrahydroharmine.
HONORS AND AWARDS
MSCI Scholarship 2013-2014
SOURCE Service Scholar in community outreach 2013-2014
SOURCE Recognition award 2014
First Place in the 2010 Covidien Intern Poster Symposium
High Honor Award 2010 from Psi Chi International Honor Society
Officer 2009 for Eta Phi Alpha Honors Fraternity
Society Memberships: Golden Key International Honor Society, Psi Chi
International Honor Society, Eta Phi Alpha Honors Fraternity, American
Chemical Society, American Society for Microbiology
PUBLICATIONS AND PRESENTATIONS
Rajagopalan, R., A. R. Poreddy, A. Karwa, B. Asmelash, N. E. Putnam, L.
Chinen, M. Nickols, J. J. Shieh, and R. B.Dorshow. (2011, January 22-27).
Folate receptor targeted Type 1 photosensitizer bioconjugates for tumor
visualization and phototherapy. Presented at the ―Optical Methods for Tumor
Treatment and Detection: Mechanisms and Techniques in Photodynamic
Therapy XX‖ conference, part of the SPIE BiOS: Biomedical Optics
Symposium. http://dx.doi.org/10.1117/12.875166.
Rajagopalan, R., T. Lin, A. Karwa, A. Poreddy, B. Asmelash, N. Putnam, D.
Lin, and R. Dorshow. (2011, May 10-14). Discovery and development of novel
thiaza and thioxa type 1 photosensitizers. ―13th World Congress of the
International Photodynamic Association‖ conference.
145