Download Isolates of Vaccinia Virus Strains, Smallpox Vaccines, and Zoonotic

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Extent of Systemic Spread Determines CD8+
T Cell Immunodominance for Laboratory
Strains, Smallpox Vaccines, and Zoonotic
Isolates of Vaccinia Virus
Inge E. A. Flesch, Natasha A. Hollett, Yik Chun Wong,
Bárbara Resende Quinan, Debbie Howard, Flávio G. da
Fonseca and David C. Tscharke
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Copyright © 2015 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 20 July 2015
http://www.jimmunol.org/content/early/2015/07/18/jimmun
ol.1402508
Published July 20, 2015, doi:10.4049/jimmunol.1402508
The Journal of Immunology
Extent of Systemic Spread Determines CD8+ T Cell
Immunodominance for Laboratory Strains, Smallpox
Vaccines, and Zoonotic Isolates of Vaccinia Virus
Inge E. A. Flesch,* Natasha A. Hollett,* Yik Chun Wong,* Bárbara Resende Quinan,*,†
Debbie Howard,‡ Flávio G. da Fonseca,† and David C. Tscharke*,‡
D8+ T cells are vital effector cells in antiviral immunity
that recognize infected cells displaying antigenic fragments of virus proteins (epitopes) in association with
MHC class I through their TCR (1, 2). Virus infection can result in
many epitopes being presented to CD8+ T cells, but their immunogenicity varies over orders of magnitude (3). Epitopes that elicit
a strong CD8+ T cell response can be considered immunodominant, whereas others that induce a smaller but still detectable
response are subdominant. The two main factors that intersect to
determine immunodominance are the abundance of a given epitope that is presented and the number of T cells in the preimmune
repertoire with a cognate receptor (4, 5). However, these factors do
not completely explain dominance hierarchies. Other factors, such
as recruitment levels of naive CD8+ T cells and immunodomination, also play roles (6–8). Immunodomination is the ability of
T cells responding to dominant epitopes to suppress other responses (9). Understanding these phenomena is important, as they
reflect the basic function of the immune system as well as underpin attempts to explain and then perhaps manipulate the
breadth of immunity to pathogens via vaccination (2).
C
*Research School of Biology, Australian National University, Canberra, Australian
Capital Territory 2601, Australia; †Laboratório de Virologia Básica e Aplicada,
Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil; and ‡John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601,
Australia
Received for publication October 1, 2014. Accepted for publication June 20, 2015.
This work was supported by National Institutes of Health Grants R01 AI067401 and
U19 AI100627, National Health and Medical Research Council (Australia) Grant
APP1023141, and by Australian Research Council Future Fellowship FT110100310.
Address correspondence and reprint requests to Dr. David C. Tscharke, John Curtin
School of Medical Research, Australian National University, 131 Garran Road,
Acton, ACT 2601, Australia. E-mail address: [email protected]
Abbreviations used in this article: FTY720, 2-amino-[2-(4-octylphenyl])-1,3-propanediol
hydrochloride; GP1V, Guarani P1 virus; GP2V, Guarani P2 virus; ICS, intracellular
cytokine staining; i.d., intradermal(ly); IDE, immunodominant epitope; MVA, modified vaccinia Ankara; SDE, subdominant epitope; TK, thymidine kinase; VACV,
vaccinia virus; WR, Western Reserve.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402508
Vaccinia virus (VACV) belongs to the orthopoxvirus family and
was the live vaccine used to eradicate smallpox. In the absence of
smallpox, VACV is being stockpiled in several countries against
a potential nefarious release of variola virus, the causative agent
of this now historic disease. Furthermore, smallpox vaccination
provided protection against a range of orthopoxviruses, some of
which continue to emerge from animal reservoirs and cause human
disease. The most concerning of these is monkeypox virus, but
human cowpox infections are being reported regularly in Europe
and even VACV itself is a cause of human infection in South
America (10–12). Within a species of orthopoxvirus, wide ranges
of virulence have been observed, for example between variola
major and minor (or alastrim) and the two main clades of monkeypox virus (13, 14). Likewise, the reactogenicity of smallpox
vaccines was known to vary and this has been modeled in mice
(15, 16). However, the immunological consequences of differing
virulence have not been explored. Furthermore, there remains
relatively few descriptions of the full virulence range of VACV
strains, which is relevant for policy with regard to vaccination of
laboratory workers and also understanding the risk associated with
emerging VACV, for example in Brazil.
VACV in mice provides an attractive model for understanding
CD8+ T cell responses with well-characterized infections that are
acute and an extensive list of mapped epitopes that have been
ranked in a predictable immunodominance hierarchy (3, 17, 18).
This model has been useful for examining the role of previously
primed T cells, regulatory T cells, TCR diversity, and route of
infection in immunodominance (8, 19–21). In the present study,
we set out to determine whether genetic diversity of the virus is
also a determinant of immunodominance using first a set of vaccine and laboratory strains, and then confirming the results using
two isolates from a recent outbreak of zoonotic VACV in Brazil.
Materials and Methods
Viruses and cell lines
VACV strains were grown and titrated in BHK-21 and BS-C-1 cells, respectively, both of which were maintained in DMEM (Invitrogen) with
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CD8+ T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented
by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the
full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we
show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread
systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid
system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming
and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because
an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus
that occurred in Brazil. The Journal of Immunology, 2015, 195: 000–000.
2
VACCINIA VIRUS SPREAD AND T CELL IMMUNODOMINANCE
glutamine and 10% FBS (D10). VACV strains were Western Reserve (WR,
National Institutes of Health tissue culture adapted [NIH-TC], GenBank
accession no. AY243312, http://www.ncbi.nlm.nih.gov/nuccore/AY243312)
and modified vaccinia Ankara (MVA) (22), B. Moss, National Institutes of
Health; ACAM2000 (23), R. Weltzin, Acambis; Copenhagen, Lister, and
Tian Tan (24), G.L. Smith, University of Cambridge; VACV WR NP-S-GFP
(25), referred to in this study as WR TK2 , J. Yewdell and J. Bennink,
National Institutes of Health; and Guarani P1 virus (GP1V) and Guarani
P2 virus (GP2V) (10), E.G. Kroon, Universidade Federal de Minas Gerais
(Belo Horizonte, Brazil).
Mice and infections
Specific pathogen-free female C57BL/6 mice 8–20 wk of age were obtained from the Animal Resource Centre (Perth, WA, Australia) and from
the Australian National University Bioscience Research Facility. Within
experiments mice were age matched and derived from one source. Mice
were housed and experiments were done according to the relevant ethical requirements and under an approval from the Australian National
University Animal Ethics and Experimentation Committee (approvals
F-BMB-38.8, A2013.037, and A2011.01). For most experiments, mice
were infected i.p. with 1 3 106 PFU VACV strains (see Table I) in 200 ml
PBS. To determine replication in skin, ear pinnae (intradermal [i.d.]) infections were done with 1 3 103 PFU VACV in 10 ml PBS (26, 27).
Stimulations and intracellular staining of IFN-g
Mice were euthanized 7 d postinfection and peritoneal wash, spleens, and
mediastinal lymph nodes were taken for analysis of CD8+ T cell responses
by intracellular cytokine staining (ICS) as described (28, 29). Briefly, cells
were plated at 1–2 3 106 cells/well in D10 into round-bottom 96-well
plates. Synthetic peptides were added to a final concentration of 1027 M
and plates were incubated at 37˚C and 5% CO2. After 1 h, 5 mg/ml brefeldin A (Sigma-Aldrich) was added and plates were incubated for another
3 h. Plates were spun at 4˚C, medium was removed, and cells were
resuspended in 50 ml 1:150 diluted anti–CD8-PE (clone 53-6.7; BioLegend). After 30 min incubation on ice, cells were washed, resuspended
in 50 ml 1% paraformaldehyde, and incubated at room temperature for
20 min before another two washes and staining with 50 ml 1:200 diluted
anti–IFN-g-allophycocyanin (clone XMG1.2; BioLegend) overnight in
PBS with 2% FBS and 0.5% saponin (Sigma-Aldrich) at 4˚C. Cells were
washed three times before acquisition using a FACS LSR II (BD Bio-
Changes in Promoter and Protein across VACV Strains Relative to WR
Namea
Sequence
H-2
Strainb
Promoterc
b
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
C-85T
Nil
C-85T
G-44A, C-85T
Nil
A-41G
Nil
Nil
Nil
Nil
Ins-41GACAATAA
Nil
Del-36
B820
TSYKFESV
K
A8189
ITYRFYLI
Kb
A3270
KSYNYMLL
Kb
A23297
IGMFNLTFI
Db
K36
YSLPNAGDVI
Db
A47138
AAFEFINSL
Kb
L253
VIYIFTVRL
Kb
J3289
SIFRFLNI
Kb
A870
A4288
IHYLFRCV
YAPVSPIVI
Kb
Db
G834
LMYIFAAL
Kb
A1947
VSLDYINTM
Kb
a
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
A2K
Cop
Lister
MVA
K205E
K157E, P160T, V179A, K205E, S213N
K242T
Del95–102, del115–121, trnc241
K247E
K247E
Nil
Nil
Q51K
Nil
Nil
Q51K
P58H
P58H
Nil
P58H
K22N
F36S
K22N, F36S
K22N, F36S
G68E, K71E, M236I, H240Y, E244V,
trnc245
Del-36
K71E, M236I, H240Y, E244V, trnc245
Nil
K71E, H240Y
Del-36
Del29–33, K71E, del153–161, M236I
Nil
T5A, del71-72, A74V
Nil
T5A
Nil
T5A
Nil
T5A
Nil
S227L, I313V, N317D, S318F
Nil
V87M, A229V, S331G
Nil
S227L
A-77G, A-73G
Nil
See above for A8189
InsA-18, A-62G
Nil
InsA-18
Nil
Nil
Nil
InsA-18
Del45–49
Nil
E94K
Nil
Nil
Nil
Nil
Nil
A257T
T-37C
Nil
T-37C
Nil
Nil
Nil
T-37C
Nil
Copenhagen nomenclature; subscript denotes position of the first amino acid in the epitope.
A2K, ACAM2000; Cop, Copenhagen.
c
Changes in the 85 bp upstream of the ATG of the ORF (bp denoted numbering down from A).
b
Protein
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Table I. VACV Ags and their conservation across sequenced strains
The Journal of Immunology
sciences). Analysis was done using FlowJo software (Tree Star). Events
were gated for live lymphocytes on forward scatter by side scatter followed
by CD8+ T cells using CD8 by side scatter and displayed as CD8 by IFN-g.
Data were recorded as IFN-g+, CD8+ cells as a percentage of total CD8+
cells. Backgrounds as determined using irrelevant peptides were usually on
the order of 0.1% and were subtracted from the values presented for test
samples.
Measurement of infectious virus in mouse tissues
Organs were removed after infection, including ovaries, spleen, kidneys,
liver, lungs, heart, brain, and mediastinal lymph nodes for i.p. infected mice
and ear pinnea after i.d. infection. Organs were briefly rinsed with 80%
ethanol and then ground in 1 ml DMEM with 2% FBS in small tissue glass
grinders (no. 358103; Wheaton, Milville, NJ) and then subjected to three
cycles of freezing and thawing. Samples were sonicated before virus was
titrated by plaque assay on BS-C-1 cells (27).
In vivo cytotoxicity assay
i.v. into infected (test) or uninfected (control) C57BL/6 mice. The test
mice were infected 2 or 4 d prior to the injection of CFSE-labeled targets.
In the latter case, mice were injected daily with 1 mg/g 2-amino-[2-(4octylphenyl])-1,3-propanediol hydrochloride (FTY720; Sigma-Aldrich)
starting from the day before infection (30, 31). Mice were euthanized
4 h after injection of targets, and spleens and lymph nodes were examined
for the relative proportions of CFSEhigh and CFSElow populations
amoungst DiD+ events by flow cytometry. To calculate specific lysis, the
following formula was used: ratio = percentage CFSElow/percentage
CFSEhigh. The percentage of target cell killing was: [1 – (ratio uninfected
[control] recipients/ratio infected [test] recipients)] 3 100.
Statistical analyses
Statistical comparisons were done using an unpaired t test with a Welch
correction for unequal variance, or ANOVAs with Tukey pairwise posttests
(GraphPad Prism, GraphPad Software, La Jolla, CA).
Results
VACV strains fit into two distinct patterns of
immunodominance
To determine how virus strain affected CD8+ T cell responses, we
tested a wide range of VACV strains, including WR, the most
common laboratory strain; ACAM2000, the current United States
smallpox vaccine, which is a clone derived from Dryvax (23, 32,
FIGURE 1. CD8+ T cell responses to VACV epitopes after immunization of mice with different strains of VACV. Groups of C57BL/6 mice were infected
i.p. with 106 PFU VACV strains WR, Copenhagen (Cop), Tian Tan (TT), Lister (Lis), MVA, or ACAM2000 (A2K). Seven days later, numbers of splenic
CD8+ T cells that produce IFN-g in ex vivo stimulations with the indicated individual peptides were measured by ICS. (A) Results for each of the peptides
and strains, showing the total numbers of epitope-specific CD8+, IFN-g+ cells. (B) The same as in (A), but simplified by showing only the response to the
IDE (B820) and the sum of responses to the SDE based on numbers of CD8+, IFN-g+ cells. Virus strain was found to be a statistically significant source of
variation in this experiment (p = 0.002; ANOVA), and pairwise significant differences were found for SDE responses between ACAM2000, Lister, and
MVA compared with WR and Tian Tan and for ACAM2000 and MVA compared with Copenhagen (p , 0.05, Tukey multiple comparisons test). (C)
Relative proportion of the total measured CD8+ T cell responses that are accounted for by the IDE and sum of SDE responses, based on (A) and (B).
Comparisons between WR, TT, and Cop or between ACAM2000, Lis, and MVA are not significant, but all comparisons across these sets of three viruses
(e.g., WR and ACAM2000) are significant (p , 0.001). (D) Response to the IDE and the sum of responses to the SDE based on the percentage of CD8+
cells that are IFN-g+. Data shown are means and SEM of groups of 6–12 mice.
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As published (29), splenocytes from uninfected C57BL/6 mice were labeled with 5 mM Vybrant DiD cell labeling solution (Molecular Probes) in
DMEM for 1 h at 37˚C, washed, and split into two populations. One
population was pulsed with 1027 M B820 peptide for 1 h at 37˚C and
labeled with a high concentration (5 mM) of CFSE for 8 min at 37˚C
(CFSEhigh cells). The second population was left without peptide and was
labeled with a low concentration (0.5 mM) of CFSE (CFSElow cells). Cells
were mixed in equal proportions and a total of 4 3 107 cells were injected
3
4
VACCINIA VIRUS SPREAD AND T CELL IMMUNODOMINANCE
FIGURE 2. Virus growth in ear pinnae after i.d. infection with different
VACV strains. Groups of C57BL/6 mice were i.d. infected into the ear
pinnae with 1 3 103 PFU VACV strains as shown. Five days later, ears
were ground and virus was titrated. Data shown are individual titers for
each mouse, and the line denotes the mean for each group.
Sequence differences in Ags and promoters do not correlate
with immunodominance
The epitopes themselves were all conserved across the VACV
strains, but it was possible that changes in promoter sequences or
the wider protein altered expression or processing of epitopes,
respectively. If a pattern of change was consistent for the strains
grouped by immunodominance, this would offer an easy explanation for the CD8+ T cell data above. High-quality full-genome
sequences were available for WR, Copenhagen, ACAM2000,
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33); Lister, the most widely used smallpox vaccine; Copenhagen
and Tian Tan, which were used in Europe and China (34); and
MVA, a replication-deficient VACV strain used as a vaccine vector
in many clinical trials (35–39). For each virus, mice were infected
with 1 3 106 PFU by i.p. injection and splenic CD8+ T cell responses to a panel of 12 peptides were measured by a standard
assay that detects IFN-g–secreting cells after a brief in vitro
stimulation with peptides in the presence of brefeldin A (referred to as IFN-g–ICS). The peptides used correspond to epitopes
conserved across all strains (Table I), and, as shown recently, this
assay allows highly accurate enumeration of epitope-specific
CD8+ T cells in this model (28). For all strains, the overall hierarchy of the 12 epitopes was similar, with B820 being the
immunodominant epitope (IDE) and the other 11 peptides being
subdominant epitopes (SDE) (Fig. 1A). However, the total size of
the responses (when summed across all epitopes) varied. WR,
Tian Tan, and Copenhagen elicited responses of .4 3 106 CD8+
T cells per spleen, whereas ACAM2000, Lister, and MVA induced
around half that number. Furthermore, the difference in responses
across the strains was due to significantly lower responses to the
SDE in the cases of ACAM2000, Lister, and MVA compared with
WR and Tian Tan and for ACAM2000 and MVA compared with
Copenhagen (p , 0.05, ANOVA with Tukey multiple comparisons
test; Fig. 1B). We have recently shown that infection of mice with
WR by different routes alters the ratio of IDE/SDE responses (8),
and this can be most easily seen when showing B820-specific responses as a fraction of the sum of responses to all epitopes.
Shown this way, the six VACV strains fall into two groups with
respect to the ratio of IDE/SDE responses (Fig. 1C): the IDE either accounts for ∼42% (WR, Tian Tan, Copenhagen) or 61%
(ACAM2000, Lister, MVA) of the total measured responses to
VACV. This was supported statistically by pairwise comparisons.
For all pairs across the groups (e.g., WR and ACAM2000), the
difference in the IDE/SDE ratio was significant (p , 0.001), but
within each group of viruses (e.g., WR and Copenhagen or
ACAM2000 and Lister), no pair had a significant difference.
When data are viewed for the IDE and sum of SDEs as a percentage of CD8+ T cells, the same pattern in immunodominance is
seen, but the differences in the total size of responses are less
apparent (Fig. 1D). In summary, significant variations in both the
size of the total CD8+ T cell response and the extent to which
these are dominated by B820 are seen across these strains of
VACV, but interestingly the level of domination by B820 appeared
to be bimodal, rather than being graded across strains.
FIGURE 3. Virus growth and spread in organs of mice infected with
different VACV strains. Groups of C57BL/6 mice were i.p. infected with
106 PFU VACV strains as shown on each chart. At days 2, 5, and 8
postinfection, virus was titrated from spleen, mediastinal lymph nodes
(LN), heart, serum, brain, lungs, kidneys, liver, and ovaries. Data shown
are individual titers for each mouse per organ, and the line denotes the
mean for each group; the dashed line shows the limit of detection (10 PFU/
organ).
The Journal of Immunology
Lister, and MVA, so assuming that our stocks of these strains were
similar, we looked for changes in the predicted amino acid
sequences of Ags and in the 85 bp immediately upstream of each
of their open reading frames. These are shown in Table I using the
WR sequence as the reference; whereas at least one change was
found in at least one region for all 12 Ags across the strains, the
pattern of these never corresponded with the immunodominance
data in Fig. 1.
Virus replication across VACV strains
simple differences in levels of replication might not fully explain
the immunodominance phenomenon.
Immunodominance correlates with virus spread
Our immunodominance profiles across strains were obtained from
mice infected by the i.p. route, which unlike i.d. injection allows
VACV spread. Previously we have shown that the lack of spread
after inoculation by i.d. injection and other peripheral sites is
associated with increased immunodominance (8). Therefore, we
wondered whether differences in dissemination might help explain
the two immunodominance profiles across VACV strains. Mice
were infected i.p. with the VACV strains, and virus loads were
measured in spleen, mediastinal lymph node, heart, serum, brain,
lungs, kidneys, liver, and ovaries at days 2, 5, and 8 postinfection
(Fig. 3). WR showed surprisingly broad spread with very substantial levels of virus across all organs except serum and brain.
Tian Tan and Copenhagen were also able to spread to multiple
sites, but levels of virus were very much lower than for WR, and
detection at most sites was sporadic across the groups. In contrast,
Lister and ACAM2000 did not demonstrate convincing spread.
These experiments revealed very large differences in virus loads
and spread across the strains. Furthermore, they demonstrate that
the ability of VACV to spread to multiple organs after i.p. injection correlates well with immunodominance profile.
To draw out distinctions between ability to replicate and virus
spread further, we took advantage of a recombinant WR virus that
is less able to replicate in vivo owing to loss of thymidine kinase
(TK) function, but is otherwise identical to WR. Inactivation of TK
FIGURE 4. Immunodominance profile is more closely linked to virus spread than replication alone. (A–C) Groups of C57BL/6 mice were infected i.p.
with 1 3 106 PFU WR or WR TK2 (TK2) and the number and percentages of splenic CD8+ T cells that produce IFN-g in ex vivo stimulations with the
indicated peptides were measured by ICS. (A) Results for each of the peptides, showing the total numbers of epitope-specific CD8+, IFN-g+ cells. (B)
Relative proportions of the total measured CD8+ T cell responses that are accounted for by the IDE and sum of SDE responses, based on (A). (C) The
response to the IDE and the sum of responses to the SDE shown as the percentage of CD8+ cells that are IFN-g+. (D) Virus titers in ear pinnae 5 d after i.d.
infection with 1 3 103 PFU VACV strains Copenhagen (Cop), WR TK2, or Lister. (E) Mice were injected with 1 3 106 PFU WR TK2 i.p. and amounts of
virus in the organs shown were measured 2, 5, and 8 d later. (F) Mice were injected with 1 3 106 PFU WR Copenhagen, WR TK2, or Lister i.p. and
amounts of virus in the organs shown were measured 2 d later. In (A)–(C), data are means and SEM of three and eight mice for WR and WR TK2, respectively, over two experiments; in (D)–(F) results for individual mice are shown with lines denoting means and the dashed line showing the limit of
detection (10 PFU/organ).
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We noticed that the VACV strains where immunodomination was
more marked were those found previously to be less virulent in an
i.d. model of infection in mice (16). A feature of this i.d. model is
very restricted spread of infection (8, 26, 40), so measurement of
virus loads in ears provides a simple assay for virus growth
in vivo. We used this model to determine whether virus growth
across the strains correlated with the immunodominance observations. Virus loads were measured in the ears of mice 5 d postinfection with 1 3 103 PFU VACV strains (Fig. 2). Levels of
infectious WR, Copenhagen, and Tian Tan were significantly
higher than Lister and ACAM2000. However, whereas the first
three strains had very similar levels of virus, Lister and
ACAM2000 appeared to differ from each other. Furthermore,
MVA (not tested here) does not replicate in mice at all (41). If
a dose effect were in play, ACAM2000 and MVA would be expected to show even sharper dominance than Lister, but instead we
found two discrete IDE dominance patterns. This suggested that
5
6
VACCINIA VIRUS SPREAD AND T CELL IMMUNODOMINANCE
Virus spread correlates with priming sites of VACV-specific
CD8+ T cells
A potential link between virus spread and CD8+ T cell immunodominance is the number or nature of lymphoid organs where
immune responses can be primed. Furthermore, a precedent for
this has been shown for VACV WR when administered by different routes (8). For this reason we wanted to examine priming
sites for a representative of each of the two immunodominance
groups of VACV strains, that is, WR (less dominance) and
ACAM200 (more dominance). To do this we used an in vivo
cytotoxicity assay 2 d postinfection. This approach has been
shown to reflect sites of priming after HSV (43) and VACV (8)
infection of mice and is based on the observation that primed
CD8+ T cells rapidly acquire cytotoxic function but remain at their
original priming site for 3–4 d before entering circulation (see
supplemental figure 1 in Ref. 8). Therefore, mice were infected
i.p. with 1 3 106 PFU WR or ACAM2000, and B820-specific
in vivo cytotoxicity assays were done 2 d later looking at results
obtained across a variety of lymphoid organs (Fig. 5A). B820specific killing was highest in mediastinal lymph nodes, which
drain the peritoneal cavity, but it was also well above background
for all sites in WR-infected mice. After ACAM2000 infection,
cytotoxicity was also seen in multiple sites, but it was significantly
less than for WR in spleen, mediastinal, and cervical lymph nodes.
Furthermore, the greatest difference between the strains was for
the spleen, where the average killing for ACAM2000 was marginal as shown by some mice having more B820-labeled than
unlabeled cells remaining (generating a specific killing value of
,0). To address concerns that there might be some recirculation
of primed CD8+ T cells, we repeated this experiment in mice
given FTY720, doing the in vivo killing assay 4 d postinfection to
increase the numbers of primed CD8+ T cells that could be detected (Fig. 5B). As expected, the average B820-specific killing
was higher in all sites in these mice, but it was always higher for
FIGURE 5. Early and FTY720-blocked B820-specific cytotoxicity
shows different levels of CD8+ T cell priming in spleens and lymph nodes
of WR- and ACAM2000-infected mice. (A) Spleen cells from naive mice
were pulsed with B820 peptide and labeled with CFSE to give a high level
of fluorescence intensity (CFSEhigh). Unpulsed spleen cells were labeled
with CFSE to give low fluorescence intensity (CFSElow). A 1:1 mixture of
1 3 107 cells of each cell population was injected i.v. into naive mice or in
mice that had been infected i.p. with 1 3 106 PFU VACV-WR or
ACAM2000 2 d previously. After 4 h, mice were sacrificed and lymph
nodes and spleens were analyzed for CFSEhigh and CFSElow target cells. To
quantify in vivo cytotoxicity, the elimination of B820-pulsed CFSEhigh
-labeled cells relative to unpulsed CFSElow-labeled cells was monitored.
(B) As for (A), but mice were administered FTY720 daily starting 24 h
prior to infection and in vivo cytotoxicity measured 4 d postinfection.
Results of individual mice and means of groups are shown. *p , 0.05. LN,
lymph node.
WR-infected compared with ACAM2000-infected mice, and this
difference was again statistically significant in the spleen. Taken
together, these data suggest that priming occurs across multiple
secondary lymphoid organs for both viruses, with the second experiment allowing more time for the primed cells to build up and
be detected. However, this priming was clearly more limiting for
ACAM2000 than WR, and priming in the spleen was always
significantly lower for this strain of VACV.
Immunodominance is sharper at the site of infection than in the
spleen
To determine whether the immunodominance differences in the
spleen might have an effect on the repertoire of T cells that are able
Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017
increases the lethal dose of WR virus by several orders of magnitude after intracranial infection of mice and limits virus loads
after high-dose i.p. infection (42). Using the same experimental
methods as above, we compared CD8+ T cell immunodominance
(Fig. 4A–C). This showed that WR TK2 had a similar immunodominance profile as the parental WR virus, with the IDE accounting for ∼40% of the total measure CD8+ T cell response.
Next we compared WR TK2 growth in ear pinnae with that of
Copenhagen (reduced immunodominance) and Lister (increased
immunodominance), but in this experiment there was no significant difference between these three strains, again confirming that
growth at a site that restricts further spread does not predict
immunodominance (Fig. 4D). After i.p. infection, WR TK2 was
able to disseminate to multiple organs when examined at multiple
times, but this spread was sporadic (Fig. 4E). To be certain that
WR TK2 could spread as well as another strain that had demonstrated reduced immunodominance, the experiment was repeated, but this time Copenhagen and Lister were included
(Fig. 4F). This confirmed that strains Copenhagen and WR TK2,
but not Lister, spread to multiple organs. Taken together, these
data suggest that it is virus spread, rather than simple replication
differences, that determine CD8+ T cell immunodominance patterns. Finally, these data place the attenuation of TK2 WR viruses
into the context of other VACV strains and demonstrate that even
without TK, this virus remains more virulent than traditional
vaccine strains such as Lister, when judged by ability to spread.
We also noted that similar to Lister and not TK2 WR, ACAM2000,
the current clonal smallpox vaccine used in the United States, was
never seen to spread (Fig. 3).
The Journal of Immunology
7
FIGURE 6. Splenic CD8+ T cell immunodominance
profiles are maintained at the site of infection. Groups
of C57BL/6 mice were infected i.p. with 106 PFU WR
or ACAM2000 and 7 d later CD8+ T cells responses
were determined in peritoneal exudate cells. (A) Results
for each of the peptides, showing the total numbers of
epitope-specific CD8+, IFN-g+ cells. (B) Relative proportion of the total measured CD8+ T cell response that
are accounted for by the IDE and sum of SDE responses, based on (A). (C) The response to the IDE and
the sum of responses to the SDE shown as the percentage of CD8+ cells that are IFN-g+. Data shown are
means and SEM of groups of six to nine mice from
three experiments.
Differences in immunodominance are a feature of VACV
isolates from outbreaks
One of the concerns with any studies using laboratory or vaccine
strains of VACV is the extent to which their artificial maintenance
on animals and in culture over long periods have endowed phenotypes that are not relevant to survival in nature. Isolates of VACV
from zoonotic outbreaks in Brazil provide an opportunity to examine strains that have the ability to persist in the environment and
cause human disease. In one such outbreak, two strains of VACV
were isolated, namely GP1Vand GP2V, and further investigation of
these found that the former strain had much greater virulence,
spread, and replication than did the latter strain in mice (10, 44).
We used these isolates to test whether the replication and virulence differences between recent isolates of VACV would deter-
mine their CD8+ T cell immunodominance profiles as was seen in
the laboratory and vaccine strains. Mice were infected i.p. and the
response to the set of 12 peptides was determined (Fig. 7). Results
for these strains fell into the same two groups based on size of
total response and especially immunodominance, with the IDE
accounting for 45 and 63% of the measured response for virulent
GP1V and avirulent GP2V, respectively.
Discussion
In this study, we explored the apparent difference in CD8+ T cell
responses to epitopes conserved across VACV strains that were
noted when the first minimal determinants were identified (3). We
confirmed that for MVA, responses to SDE are more compromised
than those recognizing the IDE and extend this to a wider set of
less virulent strains. This group of viruses that elicits responses
with a greater focus on the IDE is notable in that it includes strains
considered safer for use in humans as smallpox or recombinant
vaccines. We have not tested whether the shift in immunodominance and thus narrowing of specificity for these might compromise protection against subsequent challenge, but this is worth
further consideration. On the one hand, poorer responses elicited
by the less virulent strains have been investigated previously and
shown to correlate with reduced engagement of costimulators such
as OX40 and CD27 (45). On the other hand, requirement of OX40
was equal both for IDE and SDE, suggesting that this does not
underlie the immunodominance changes we report (46). Also, in
considering the impact of changes in immunodominance on protection, it is noteworthy that immunization with single CD8+
T cell epitopes can protect mice against lethal VACV and ectromelia virus challenge (3, 47, 48). We found in the present study
that B820-specific (IDE) responses differ less across the strains,
and thus if adequately primed, the response to this epitope alone
would provide significant protection irrespective of the vaccine.
FIGURE 7. Zoonotic isolates of VACV
fit into two patterns of immunodominance.
Groups of C57BL/6 mice were infected i.p.
with 106 PFU GP1V or GP2V and 7 d later
CD8+ T cells responses were determined in
the spleen. (A) Results for each of the peptides, showing the total numbers of epitopespecific CD8+, IFN-g+ cells. (B) Relative
proportion of the total measured CD8+ T
cell response that are accounted for by the
IDE and sum of SDE responses, based on
(A). (C) The response to the IDE and the
sum of responses to the SDE shown as the
percentage of CD8+ cells that are IFN-g+.
Data shown are means and SEM of groups
of eight mice from two experiments.
Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017
to combat infection, we examined CD8+ T cell responses to our
panel of peptides in cells from the peritoneal cavity of i.p. infected
mice (Fig. 6). Approximately 10-fold more CD8+ T cells were
detected in the peritoneal cavity of WR-infected compared with
ACAM2000-infected mice (5 3 107 versus 5 3 106 CD8+ T cells,
respectively; naive mice have 1 3 106 CD8+ T cells). Additionally, responses were more heavily skewed toward the IDE in the
peritoneal cavity than the spleen for both strains of VACV
(compare Figs. 1 and 6). However, there remained a difference
between the strains with the IDE accounting for 53 and 73%
of the measured response for WR and ACAM2000, respectively
(Fig. 6B). When viewed as a percentage of CD8+ T cells, responses to ACAM2000 appear to be stronger than those to WR,
emphasizing the importance for taking total numbers into account
in these experiments (Fig. 6C). Therefore, all CD8+ T cell responses were more skewed toward the IDE at the site of infection,
but differences in immunodominance between VACV strains were
maintained.
8
VACCINIA VIRUS SPREAD AND T CELL IMMUNODOMINANCE
Table II. VACV host modulating proteins and their conservation across sequenced strains
Protein/Function
a
Cop
A2K
Lister
MVA
Y
N
Y
N
N
N
N
Y
N
N
Y
Y
Y
N
N
Y
Y
Y
Y
N
Y
Y
Y
N
N
N
N
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
N
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
N
Y
Y
N
N
Y
N
N
Y
N
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Yb
N
N
N
Y
Y
Y
Y
Yb
N
N
Y
a
Present in the Brazillian VACV strains GP1V and GP2V but with at least one coding change.
Present but with small inframe deletions, function unknown.
N, no; Y, yes.
b
Finally, Ab responses will remain, and we have not addressed any
possible differences in the specificity of these here. Alternatively,
the comparison between virulent VACV strains causing systemic
infection and those that remain limited might be a model of the
difference between smallpox vaccination (with VACV) and disease. It has been argued strongly that smallpox survivors had
superior immunity to those vaccinated against the disease (49).
Although there are many possible reasons for this (including those
noted above), it is possible that in the face of smallpox, all aspects
of immunity need to be optimal to provide protection, and that
includes breadth of cellular responses.Although it was beyond the
scope of this investigation, it would be of interest to know whether
breadth of humoral immunity was also greater in mice that experience systemic, compared with local, VACV infections.
Among the VACV strains we examined, several are used in
people or are in clinical trials, and these are all from the less
virulent group and responses were characterized by increased
immunodominance. This could be of potential importance for
recombinant vaccines where immunodominance of responses to the
vector are thought to be a problem (50). Of greatest direct importance, it impacts the design of preclinical work and suggests
clearly that all such work should be done with a strain suitable for
using in humans, rather than a virulent laboratory strain such as
WR. This is needed to avoid complications that might be caused
by changes in immunodominance rather than overall immunogenicity of vectors or immunization strategies (8, 51).
Our data examining priming sites should be interpreted with
a recent publication examining immunodominance changes after
infection with WR by different routes of infection (8). Taken together, these suggest that differences in systemic spread of virus
drive the changes in immunodominance. After i.d. immunization
compared with i.p. or i.v. immunization, spread of WR was limited, resulting in reduced priming of CD8+ T cells in the spleen
and increased domination by the IDE. Additionally, greatly reducing the dose of WR given by the i.p. route such that priming
was restricted to the mediastinal lymph node similarly increased
immunodomination (8). The large difference in ability of the
various strains of VACV to spread was reflected in the level of
priming seen in different lymphoid organs and especially the
spleen. Therefore, a similar mechanism probably underpins both
observations. In the case of the different routes we found that
competition for costimulation was a driver for increased immunodomination where priming was confined to local draining
lymph nodes (8). This is likely to be occurring in this case where
less virulent viruses are unable to spread beyond a local draining
lymph node.
Studies with VACV are sometimes regarded as irrelevant, owing
to the unknown origin and incomplete passage history of the
laboratory strains. For this reason we included viruses that were
derived from a VACV zoonotic outbreak in Brazil (10). The two
VACVs have previously been reported to have higher (GP1V) and
lower (GP2V) virulence. We found that this virulence difference
influenced the size of immune responses and the immunodominance profile in the same way as it did across the laboratory and
vaccine strains. This suggests that our findings are relevant to
viruses that are maintained in an ecological niche, which we note
for similar isolates of VACV from Brazil has been shown to include rodents (52). Although we did not test the virulence of these
strains again, our study is the most comprehensive examination of
VACV spread and virus loads across laboratory and vaccine
Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017
Secreted type I IFN-binding protein
Secreted CC chemokine-binding protein
(35K)
Secreted type II IFN-binding protein
Secreted IL-1b–binding protein
Secreted IL-18–binding protein
Secreted TNF-binding protein CrmC
Secreted TNF-binding protein CrmE
A41 secreted chemokine-binding protein
SPI-1 serpin
SPI-2/CrmA serpin
SPI-3 serpin
A39 semaphorin
A44 3-b-hydroxysteroid dehydrogenase
A52 intracellular TLR and IL-1 signal
inhibitor
A46 Toll–L1 receptor-like protein
B14 NF-kB inhibitor
A49 NF-kB inhibitor
K7 immune modulator
F1 antiapoptotic
M2 NF-kB inhibitor
A55 Kelch BTB
C2 Kelch BTB
F3 Kelch BTB
A35 MHC class II inhibitor
K3 IFN resistance, eIF-2a homolog
E3 IFN resistance, dsRNA binding proteina
N1 virulence factor
O1 ERK1/2 pathway inhibitor
C2 complement control protein (VCP)
C7 host range gene
WR
The Journal of Immunology
Acknowledgments
We thank Drs. J.W. Yewdell, J. Bennink, and B. Moss (National Institute
of Allergy and Infectious Diseases, National Institutes of Health),
Prof. G.L. Smith (University of Cambridge), Dr. R. Weltzin (Acambis), and
Prof. E.G. Kroon (Universidade Federal de Minas Gerais) for provision
of viruses. We also thank Stewart Smith for general laboratory assistance
and Research School of Biology animal services for husbandry of mice.
Disclosures
The authors have no financial conflicts of interest.
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