JOURNAL OF VIROLOGY, Apr. 2011, p. 3683–3689
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 7
Simian Immunodeficiency Virus SIVmac239?nef Vaccination Elicits
Different Tat28-35SL8-Specific CD8?T-Cell Clonotypes Compared
to a DNA Prime/Adenovirus Type 5 Boost Regimen
in Rhesus Macaques?
Benjamin J. Burwitz,1Zachary Ende,2Benjamin Sudolcan,3Matthew R. Reynolds,1Justin M. Greene,1
Benjamin N. Bimber,3Jorge R. Almeida,2Monica Kurniawan,4Vanessa Venturi,4Emma Gostick,5
Roger W. Wiseman,3Daniel C. Douek,2David A. Price,2,5and David H. O’Connor1,3*
Department of Pathology, University of Wisconsin—Madison, Madison, Wisconsin 537061; Human Immunology Section,
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 208922; Wisconsin National Primate Research Center, Madison, Wisconsin 537063; Computational Biology Unit,
Centre for Vascular Research, University of New South Wales, Kensington, NSW 2052, Australia4; and
Department of Infection, Immunity and Biochemistry, Cardiff University School of Medicine,
Cardiff CF14 4XN, Wales, United Kingdom5
Received 5 October 2010/Accepted 20 January 2011
Different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccine vectors ex-
pressing the same viral antigens can elicit disparate T-cell responses. Within this spectrum, replicating
variable vaccines, like SIVmac239?nef, appear to generate particularly efficacious CD8?T-cell responses.
Here, we sequenced T-cell receptor ?-chain (TRB) gene rearrangements from immunodominant Mamu-A*01-
restricted Tat28-35SL8-specific CD8?T-cell populations together with the corresponding viral epitope in four
rhesus macaques during acute SIVmac239?nef infection. Ultradeep pyrosequencing showed that viral variants
arose with identical kinetics in SIVmac239?nef and pathogenic SIVmac239 infection. Furthermore, distinct
Tat28-35SL8-specific T-cell receptor (TCR) repertoires were elicited by SIVmac239?nef compared to those
observed following a DNA/Ad5 prime-boost regimen, likely reflecting differences in antigen sequence stability.
Vaccination of nonhuman primates with a live-attenuated
strain of simian immunodeficiency virus (SIV), such as
SIVmac239?nef, consistently protects against pathogenic SIV
challenge (1, 4, 11). Furthermore, evidence from depletion
experiments indicates that this protection is mediated, at least
in part, by CD8?T cells (9, 15, 18). However, the mechanisms
that underlie protective CD8?T-cell immunity in this model
In rhesus macaques, immunodominant SIV-specific CD8?
T-cell responses that are restricted by the same major histo-
compatibility complex (MHC) class I molecule are composed
of T-cell receptor (TCR) repertoires with various levels of
complexity based on the viral antigen being presented. CD8?
T-cell clonotypes specific for the Mamu-A*01-restricted
Gag181-189CM9 epitope, which is biologically constrained and
therefore unable to mutate freely, expand to form diverse TCR
repertoires (13, 14). In contrast, CD8?T-cell clonotypes spe-
cific for the Mamu-A*01-restricted Tat28-35SL8/TL8 epitope
converge on a prevalent TCR? motif and consequently exhibit
limited diversity. This restricted antigen recognition profile,
combined with a tolerance for mutation in this region of the
virus, facilitates immune escape by mutation at potential TCR
contact residues (14). Understanding how different vaccines
affect TCR repertoire formation, therefore, has potential im-
plications for the design of a prophylactic human immunode-
ficiency virus (HIV) vaccine. In particular, vaccines that evolve
within hosts, such as SIVmac239?nef, may elicit different TCR
repertoires than traditional DNA prime-virus boost regimens.
The advent of ultradeep pyrosequencing has provided unpar-
alleled precision in the characterization of viral quasispecies in
HIV-infected humans and SIV-infected nonhuman primates (2,
3, 8, 10, 12, 19). Thus, for the first time, the effects of viral
variation on the cognate CD8?T-cell repertoire can be assessed
quantitatively. Here, we examined the kinetics of Tat28-35SL8
mutation and conducted a comprehensive assessment of the con-
temporaneous Tat28-35SL8-specific CD8?T-cell repertoires in
SIVmac239?nef-vaccinated rhesus macaques.
Four Mamu-A*01?SIVmac239?nef-vaccinated rhesus ma-
caques were selected for this study; details of their vaccination
were published previously (15). Initially, we compared viral
replication levels and magnitudes of Tat28-35SL8-specific CD8?
T-cell responses between this group and a group of four
Mamu-A*01?SIVmac239-infected rhesus macaques (Fig. 1; see
also Table 1 at https://xnight.primate.wisc.edu:8443/labkey/project
.view?) (15, 21). SIVmac239?nef-vaccinated macaques exhibited
lower peak and postpeak viremias, and these viral load
differences were significant at both week 2 and week 3
postvaccination (week 2 medians, 1.1 ? 105versus 4.7 ? 105
viral RNA [vRNA] copies/ml; week 3 medians, 3.6 ? 103
versus 3.5 ? 105vRNA copies/ml [P ? 0.0143 at both time
points; one-tailed Mann-Whitney U test]) (Fig. 1A and B).
* Corresponding author. Mailing address: Department of Pathology,
University of Wisconsin—Madison, 555 Science Dr., Madison, WI
53711. Phone: (608) 265-3389. Fax: (608) 265-8084. E-mail: doconnor
?Published ahead of print on 26 January 2011.
Additionally, all four animals controlled viral replication to
undetectable levels within the first 18 weeks of infection.
Tat28-35SL8 responses also differed between groups, with the
SIVmac239-infectedmacaques exhibiting muchlarger
responses than the SIVmac239?nef-vaccinated macaques (Fig.
1C and D). Three of the SIVmac239-infected macaques
(rh2122, rh2126, and rh2127) were vaccinated with a DNA
vRNA Copies/ml Plasma
05 1015 2025
% Tetramer+CD3+CD8+ T-cells
% Tetramer+CD3+CD8+ T-cells
02468 1012 141618
0510 1520 25
Vaccinated with DNA prime/MVA
boost encoding SIVmacJ5 Tat prior
to challenge with SIVmac239
vRNA Copies/ml Plasma
Week 2 Post-Inoculation
p < 0.05
Week 3 Post-Inoculation
p < 0.05 p < 0.05
FIG. 1. (A) Viral loads (numbers of viral RNA copies/ml plasma) for each macaque following infection with SIVmac239 (unbroken lines) or
SIVmac239?nef-vaccinated and SIVmac239-infected rhesus macaques at weeks 2 and 3 postinoculation. Significance was assessed using a one-tailed
Mann-Whitney test (P ? 0.0143 at both time points). (C) Percentages of tetramer-binding Tat28-35SL8-specific cells within the CD3?CD8?T-cell
population for four rhesus macaques at weeks 2 and 8 after SIVmac239 infection. These results have been published previously (21). (D) Percentages
of tetramer-binding Tat28-35SL8-specific cells within the CD3?CD8?T-cell population for four rhesus macaques at weeks 2 and 8 after SIVmac239?nef
3684NOTES J. VIROL.
prior to intrarectal SIVmac239 infection, thereby explaining the
enhanced magnitude of their week 2 postinfection Tat28-35SL8
Viral variation driven by CD8?T cells in the context of a
live-attenuated SIV vaccine has not been reported previously.
Our group has used ultradeep pyrosequencing to show rapid
and widespread viral variation within the Tat28-35SL8 epitope
by week 3 after SIVmac239 infection, with some macaques
showing low frequency mutations as early as week 2 postin-
fection (2). Therefore, we performed ultradeep pyrose-
quencing of the Tat28-35SL8 epitope at weeks 2 and 3 after
SIVmac239?nef vaccination in the four selected Mamu-A*01?
rhesus macaques by use of previously published methods (2).
Ultradeep pyrosequencing resulted in an average of 2,940
sequence reads per sample, with a range of 2,700 to 3,784
sequence reads. We compared SIVmac239?nef Tat28-35SL8
variation with our previously published sequence data from
the four SIVmac239-infected rhesus macaques (2). Rapid viral
variation was observed within the Tat28-35SL8 epitope in
SIVmac239?nef-vaccinated Mamu-A*01?macaques, despite
their lower acute-phase viral loads and smaller Tat28-35SL8-
specific CD8?T-cell responses relative to those of macaques
infected with SIVmac239 (Fig. 1 and 2; see also raw data at
.view?) (Sequence Read Archive accession no. SRA028442
.1). The pattern of viral variation within the Tat28-35SL8
epitope was similar to our previous findings in SIVmac239-
FIG. 2. Ultradeep pyrosequencing analysis of the Tat28-35SL8 region from four SIVmac239?nef-vaccinated Mamu-A*01?rhesus macaques.
(A) Consensus sequence from each sample. The ultradeep pyrosequencing consensus contains any mutation present in 1% or more of the total
reads. Changes are indicated using the single-letter amino acid code for substitutions where only 1 residue was found at that position with a
frequency of ?1%. All other changes are indicated with the letter X. Nonsynonymous and synonymous mutations are colored according to
prevalence, indicated by the color key on the right. Data from SIVmac239-infected rhesus macaques are shown for comparison and were published
previously (2). (B) Percentage of each variant observed within Tat28-35SL8 at week 2. Only variants present at frequencies of 1% or greater in at
least one macaque are shown. Variants present at less than 1% were summed, and their combined frequency is listed as “Other.” Dashes indicate
that a variant was not present in that sample. (C) Percentage of each variant observed within Tat28-35SL8 at week 3. The details are the same as
those for panel B.
VOL. 85, 2011NOTES 3685
infected macaques (2). Additionally, we observed a similar level
of interindividual variability in terms of the positions and
quantities of specific mutations (Fig. 2A and C). The overall
percentage of viral variants circulating within each macaque
differed at week 3 postvaccination. In r90080, only 31.4% of the
peripheral viral population showed variation within Tat28-35SL8.
In contrast, r88085 and r98037 had higher proportions of viral
variants (81.4% and 79.2%, respectively). Taken together, these
levels of breadth in SIVmac239?nef-vaccinated and SIVmac239-
infected rhesus macaques.
It is possible that SIVmac239?nef epitope variation affects
the protective efficacy of this vaccine, given that coevolution of
the host immune system with the diversifying virus may influ-
ence the TCR repertoire within virus-specific T-cell responses.
In addition, a diversifying live-attenuated virus may elicit im-
mune responses that differ from those generated by nonmo-
saic, static immunogens, such as SIV genes packaged into ad-
enovirus type 5 (Ad5) viral vectors. To investigate these
possibilities, we undertook a comprehensive analysis of all ex-
pressed TRB gene rearrangements within Tat28-35SL8-specific
CD8?T-cell populations by use of an unbiased template-
switch anchored reverse transcription-PCR (RT-PCR) as de-
scribed previously (6, 14).
CD8?T cells specific for Tat28-35SL8 were identified directly
ex vivo using fluorochrome-conjugated SL8/Mamu-A*01 tetra-
meric complexes and sorted at ?98% purity by flow cytometry
(14). At week 2 postvaccination, before substantial variation
occurred within the Tat28-35SL8 epitope, common trends were
repertoires (Fig. 3 and 4A to E). Although these early
SIVmac239?nef Tat28-35SL8-specific CD8?T-cell populations
featured diverse TRBV gene usage (Fig. 4E) and were poly-
clonal, with a median of 21 (range, 12 to 36) unique TCR?
clonotypes per repertoire, many clonotypes conformed to a
common CDR3? motif that comprised a central arginine res-
idue at position 6, the usage of the TRBJ1-5 gene, and an
overall length of 13 amino acids (CDR3-6R). Additionally,
previously described “public” clonotypes were sequenced from
each animal (Fig. 3). These findings are in line with previous
reports and provide further evidence that the characteristics of
the mobilized antigen-specific T-cell repertoire are determined
by the nature of the antigen rather than the context of presen-
tation (13, 14, 16).
Next, we determined the changes that occurred in the
Tat28-35SL8-specific CD8?T-cell repertoires following the emer-
gence of substantial variation within the Tat28-35SL8 epitope
(Fig. 3). Overall, TRBV gene usage remained diverse at 8
weeks after SIVmac239?nef vaccination (Fig. 4E). However,
marked differences in TRBV gene usage over time were appar-
ent. For example, TRBV23-1 was not used at week 2 but com-
prised 59.2% of sequences in r90080 at week 8. The polyclonal-
ity of these responses was greater at week 8 postvaccination
than at week 2, with a median of 31 unique TCR? clonotypes
(range, 24 to 33 clonotypes) per repertoire. Despite this diver-
sification, the CDR3-6R motif remained prevalent and public
clonotypes were observed at week 8 in all four Tat28-35SL8-
specific CD8?T-cell repertoires (Fig. 3 and 4B). These data
suggest that the CDR3? core and TRBJ1-5 play determinative
roles in Tat28-35SL8 antigen recognition, but structural data are
required to elucidate more fully the nature of the TCR/SL8/
It is a widely held belief that vaccine-elicited CD8?T-cell
responses should cross-recognize epitope variants to exert op-
timal efficacy against variable viruses. Such cross-reactivity
could be generated either by incorporating multiple different
clonotypes or by preferential recruitment of specific clonotypes
with highly degenerate antigen recognition profiles (5, 13). To
assess whether TCR repertoires elicited by SIVmac239?nef
vaccination differed from those elicited by a prime-boost vac-
cination, we compared the values for different TCR repertoire
parameters between our cohort and a previously studied co-
hort of rhesus macaques that received a DNA prime-Ad5
boost vaccine containing SIVmac239 Tat (22). For each ma-
caque, we measured TCR diversity, motif frequency, CDR3
length, and TRBJ and TRBV gene usage (Fig. 4A to E); these
features were compared between the two cohorts (Fig. 4F to
J). Estimated for a standard sample size of 70 TCR? se-
quences, the number of unique clonotypes from each macaque
at week 2 after SIVmac239?nef vaccination was similar to the
number of unique clonotypes sequenced at week 2 after Ad5
boost (Fig. 4F). However, there were significant differences
between the two cohorts in the features of the TCR reper-
toires, with the CDR3-6R motif and an overall CDR3 length of
13 amino acids being significantly more prevalent in the Ad5-
boosted cohort than in the SIVmac239?nef-vaccinated cohort
at week 2 postvaccination (P ? 0.008 and P ? 0.014, respec-
tively; Mann-Whitney U test) (Fig. 4G and H). Tat28-35SL8-
specific TCRs often use the TRBV27 gene; indeed, TRBV27
gene expression was observed in all four SIVmac239?nef-vac-
cinated rhesus macaques at both week 2 and week 8 postvac-
cination. Compared to that in the SIVmac239?nef-vaccinated
cohort, TRBV27 gene expression in the Ad5-boosted cohort
had increased complexity, with half of the macaques exhibiting
similar frequencies and the other half exhibiting much higher
frequencies (Fig. 4J). Interestingly, the four macaques in the
Ad5-boosted cohort with the highest frequencies of the
CDR3-6R motif also had the highest frequencies of TRBV27
gene usage; indeed, a median of 81.6% (range, 78.4 to 85.7%) of
all clonotypes in these macaques expressed the TRBV27 gene and
contained the CDR3-6R motif. Structural data will be required to
elucidate the underlying basis of this associative bias.
The kinetics of viral escape within Tat28-35SL8 could af-
fect the emergence of novel CD8?T-cell clonotypes in
SIVmac239?nef-vaccinated rhesus macaques. To assess this
possibility, we compared the frequency of wild-type Tat28-35SL8
sequences at week 3 after SIVmac239?nef vaccination with the
prevalence of the CDR3-6R motif in the TCR repertoires
sequenced from each macaque at week 8 after SIVmac239?nef
vaccination (Fig. 4K). Intriguingly, we observed an association
between the proportion of the TCR repertoire featuring
CDR3-6R clonotypes sequenced at week 8 postvaccination and
the proportion of wild-type Tat28-35SL8 reads observed at week
3 postvaccination within each macaque. Importantly, the dif-
ferences in CDR3-6R frequencies in the week 8 postvaccina-
tion TCR repertoires between the macaques (Fig. 4G) could
not be accounted for by viral sequence differences at week 2
postvaccination (Fig. 2B). This result suggests that continued
presence of wild-type Tat28-35SL8 antigen drives a more fo-
cused CDR3-6R motif-bearing CD8?T-cell response. Such a
3686 NOTESJ. VIROL.
process would be in keeping with previous reports and, by
extension, could help to explain Tat28-35SL8-specific repertoire
diversity patterns in the presence of a complex mixture of
epitope variants, each of which carries the potential to produc-
tively engage distinct TCR structures and expand the corre-
sponding cognate clonotypes (13, 14). However, other factors
clearly affect TCR usage in response to the Tat28-35SL8
epitope, and further investigation is required to confirm this
tentative conclusion given the limited number of macaques and
time points in this study.
FIG. 3. CDR3? amino acid sequences, TRBV and TRBJ gene usage, and relative frequencies of Tat28-35SL8-specific CD8?T-cell clonotypes
for all four macaques at weeks 2 and 8 after SIVmac239?nef vaccination. Clonotypes shared between time points are color coded in the frequency
column, and public clonotypes are color coded in the CDR3 sequence column. Public clonotypes were identified as TCR? amino acid sequences
observed in more than one macaque with reference to an extensive database including data from previous studies (13, 14). Sequences were aligned
against the rhesus macaque TRB genes (7), for which international Immunogenetics (IMGT) information system nomenclature is used. The
asterisk indicates potential allelic variations within the TRBV13-1 (G at nucleotide position 7 from the 3? gene end) and TRBJ1-6 (T at nucleotide
position 20 from the 5? gene end) gene-encoded portions of the CDR3 protein.
VOL. 85, 2011NOTES 3687
Weeks Post-Ad5 Boost
TRBJ1-5 Other 13 a.a. Other
No R in CDR3
TRBJ Gene CDR3 Length
R in CDR3
R in CDR3 at
0 2040 60 80
Number of Clonotypes for a
Standard Sample Size
% TCR Repertoire Conforming
to CDR3-6R Motif
% WT Tat SL8 Sequence at Week 3
p = 0.008
% TCR Repertoire
2 Weeks8 Weeks 2 Weeks
% TCR Repertoire with
CDR3 Length of 13 a.a.
% TCR Repertoire
% TCR Repertoire Conforming
to CDR3-6R Motif at Week 8
FIG. 4. (A) Numbers of unique Tat28-35SL8-specific CD8?T-cell clonotypes in macaques following vaccination with SIVmac239?nef or DNA
prime-Ad5 boost with Tat-encoding vectors, estimated for a standard sample size of 70 TCR? sequences across all samples (20). (B to E)
Frequencies of Tat28-35SL8-specific CD8?T-cell clonotypes in macaques following vaccination with SIVmac239?nef or DNA prime-Ad5 boost
with Tat-encoding vectors that feature particular CDR3 amino acid (a.a.) motif characteristics (B), CDR3 lengths (C), TRBJ gene usage (D), and
TRBV gene usage (E). (F to J) Comparisons of TCR repertoire parameters between macaques at week 2 after SIVmac239?nef vaccination, week
8 after SIVmac239?nef vaccination, and week 2 after Ad5 boost; number of unique Tat28-35SL8-specific CD8?T-cell clonotypes (F), CDR3-6R
motif frequency (G), frequency of CDR3s with lengths of 13 amino acids (H), frequency of TRBJ1-5 gene usage (I), and frequency of TRBV27 gene
usage (J). (K) Relationship between the frequency of Tat28-35SL8-specific CD8?T-cell clonotypes with the CDR3-6R motif at week 8 after
SIVmac239?nef vaccination and the frequency of wild-type (WT) Tat28-35SL8 sequences observed at week 3 after SIVmac239?nef vaccination.
Many HIV vaccines are under investigation, and compre-
hensive analyses of the CD8?T-cell responses that they elicit
will guide future studies. Recently, the idea of vaccinating
macaques with mosaic vaccines containing multiple HIV vari-
ants has been posited and tested, with encouraging results (17).
SIVmac239?nef exhibits viral variation in rhesus macaques
and is replication competent, lending an advantage over mo-
saic prime-boost vaccines. Ultradeep pyrosequencing gives a
nuanced view of such viral variation in live-attenuated SIV-
vaccinated primates, and for the first time, quantitative com-
parisons of viral variation with other measurements of adaptive
immunity are possible. Here, we show that the Tat28-35SL8-
specific TCR? repertoire changes over the course of acute
SIVmac239?nef vaccination, trending toward greater diversity
than for macaques vaccinated with a DNA prime-Ad5 boost
regimen. Ancillary studies are warranted to determine the ex-
tent to which these findings can be generalized. However, the
current study provides both a testable hypothesis, according to
which epitope sequence complexity and cognate repertoire
diversity are intimately linked, and the tools with which to
unravel this interrelationship. Additional studies comparing
prevaccine boost, postvaccine boost, and postinfection TCR
repertoires may further elucidate the complex relationship of
clonotype selection and viral variation.
Constituent TCRs determine the specificity and cross-reac-
tivity profile of a CD8?T-cell population, and it is becoming
abundantly clear that not all TCRs are created equal. Conse-
quently, a detailed understanding of HIV/SIV coevolution with
emerging T-cell responses at the clonotypic level may provide
key information for the design of an effective prophylactic
We thank David Watkins for invaluable advice and for the animal
samples utilized in the study.
This publication was made possible by grant number P51 RR000167
from the National Center for Research Resources (NCRR), a com-
ponent of the National Institutes of Health (NIH), to the Wisconsin
National Primate Research Center, University of Wisconsin—Madi-
son; the research was conducted at a facility constructed with support
from the Research Facilities Improvement Program, grant numbers
RR15459-01 and RR020141-01. Additional funding was provided by
grant numbers 1R01AI084787, R01 AI077376, and 144PRJ23CG from
the NIH. D.A.P. is a Medical Research Council (United Kingdom)
Senior Clinical Fellow; V.V. is an Australian Research Council Future
The contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of the
NCRR or the NIH.
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