Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques.

Abstract

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8+ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8+ T-cell epitopes to investigate the contribution of epitope-specific CD8+ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08+ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08+ macaques. The five most immunodominant Mamu-B*08-restricted CD8+ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08+ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08+ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8+ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08+ macaques.

Full text

Page 1

1
Infection with “escaped” virus variants impairs control of SIVmac239 replication in Mamu-
1
B*08+ macaques
2

3
Laura E. Valentine1§, John T. Loffredo1§, Alex T. Bean1, Enrique J. León2, Caitlin E. MacNair2,
4
Dominic R. Beal1, Shari M. Piaskowski1, Yann C. Klimentidis3, Simon M. Lank2, Roger W.
5
Wiseman2, Jason T. Weinfurter4, Gemma E. May2, Eva G. Rakasz1,2, Nancy A. Wilson1, Thomas C.
6
Friedrich2,4, David H. O’Connor1,2, David B. Allison3, David I. Watkins1,2*
7

8
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI
9
537061; Wisconsin National Primate Research Center (WNPRC), University of Wisconsin-Madison,
10
Madison, WI 537152; Section on Statistical Genetics, Department of Biostatistics, University of
11
Alabama at Birmingham, Birmingham, Alabama 352943; Department of Pathobiological Sciences,
12
University of Wisconsin School of Veterinary Medicine, Madison, WI 537064
13
§Laura E. Valentine and John T. Loffredo contributed equally to this work.
14
*Address correspondence and reprint requests to Dr. David I. Watkins, Department of Pathology
15
and Laboratory Medicine, University of Wisconsin-Madison, 555 Science Dr., Madison, WI, 53711.
16
Telephone: 1.608.265.3380. Fax: 1.608/265.8084. Email: watkins@primate.wisc.edu
17

18
Running Title: Impaired CTL responses during acute SIV infection
19
Abstract Word Count: 224
20
Text Word Count: 7,732
21

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
J. Virol. doi:10.1128/JVI.01298-09
JVI Accepts, published online ahead of print on 2 September 2009
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 2

2
ABSTRACT
22
Understanding the mechanism(s) by which some individuals spontaneously control
23
HIV/SIV replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that
24
express the MHC class I allele Mamu-B*08 become elite controllers after infection with SIVmac239.
25
Mamu-B*08 has a binding motif very similar to that of HLA-B27, a human MHC class I allele
26
associated with elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08+ animals may
27
be a good model for elite control of HIV. The association with MHC class I alleles implicates
28
CD8+ T cells and/or Natural Killer cells in control of viral replication. We, therefore, introduced
29
point mutations into eight Mamu-B*08-restricted CD8+ T cell epitopes to investigate the
30
contribution of epitope-specific CD8+ T cell responses in the development of control of viral
31
replication. Ten Mamu-B*08+ macaques were infected with this mutant virus, 8X-SIVmac239. We
32
compared immune responses and viral loads of these animals to those of wild-type SIVmac239-
33
infected Mamu-B*08+ macaques. The five most immunodominant Mamu-B*08-restricted CD8+ T
34
cell responses were barely detectable in 8X-infected animals. By 48 weeks post-infection, two of ten
35
8X-infected Mamu-B*08+ animals controlled viral replication to <20,000 vRNA copy eq/ml plasma,
36
while ten of fifteen wild type-infected Mamu-B*08+ animals had viral loads <20,000 vRNA copy
37
eq/ml (p=0.04). Our results suggest that these epitope-specific CD8+ T cell responses may play a
38
role in establishing control of viral replication in Mamu-B*08+ macaques.
39
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 3

3
INTRODUCTION
40
A small number of individuals spontaneously control replication of HIV or Simian
41
Immunodeficiency Virus (SIV) to very low levels. The precise mechanisms underlying this control
42
are of great interest, as a clear understanding of what constitutes a successful immune response may
43
aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which
44
proteins to use as immunogens, the extent to which increasing breadth and magnitude of responses
45
is advantageous, how immunodomination affects T cell responses, and if biasing the immune
46
response towards particular effector profiles is beneficial. Characterization of immune responses
47
made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation
48
and evaluation.
49
HIV elite controllers are generally not infected with grossly unfit viruses (6, 42). Instead,
50
elite control of immunodeficiency virus replication is correlated with the presence of particular
51
MHC class-I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with control
52
of viremia implicates CD8+ T cells as mediators of this immune containment. Several lines of
53
evidence support this hypothesis. These include the correlation between the appearance of CD8+ T
54
cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that
55
alleles associated with viral control restrict dominant acute phase CD8+ T cell responses (3), and
56
that responses directed against epitopes restricted by these alleles frequently select for viral escape
57
variants (4, 27, 38). Perhaps most compelling is the observation that, in a handful of HIV-infected
58
individuals, selection of escape variants by an immunodominant HLA-B27-restricted T cell response
59
temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape
60
variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51),
61
recent data has found normal, high levels of replication in vivo upon transmission of some of these
62
variants (15).
63
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 4

4
The association of control with MHC-I alleles does not, of course, implicate solely CD8+ T
64
cells. MHC-I molecules are also ligands for Killer Immunoglobulin Receptors (KIRs), which are
65
predominantly expressed on Natural Killer (NK) cells. Genetic studies of HIV-infected humans
66
suggest a model in which individuals with particular KIR/HLA combinations are predisposed to
67
control HIV replication more readily than those with other KIR/HLA combinations (36, 37).
68
These data were supported by functional studies of this KIR/HLA pairing in vitro, which
69
demonstrated inhibition of HIV replication by such NK cells (2). The relative contribution of NK
70
and CD8+ T cell responses to control has yet to be elucidated and may be closely intertwined.
71
Previously, experimental depletion of circulating CD8+ cells from SIVmac239-infected ECs
72
resulted in a sharp spike in viremia, which resolved as CD8+ cells repopulated the periphery (19).
73
During re-establishment of control of SIV replication, CD8+ T cells targeting multiple epitopes
74
restricted by alleles associated with elite control expanded in frequency, providing strong
75
circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8-depletion
76
antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication
77
(19). It was therefore difficult to make definitive conclusions regarding separate contributions of
78
these subsets in maintaining control of SIV replication in vivo.
79
Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the
80
macaque MHC-I allele most tightly associated with control of SIVmac239, Mamu-B*08.
81
Approximately 50% of Mamu-B*08+ animals infected with SIVmac239 become elite controllers (32).
82
Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27.
83
Though these two MHC-I genes are dissimilar in domains important for peptide binding, each
84
molecule can bind peptides which are presented by the other molecule (33). This striking similarity
85
suggests that elite control of SIVmac239 in Mamu-B*08+ animals is a good model for elite control of
86
HIV.
87
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 5

5
Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08+
88
rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by
89
Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by two amino acids in the α1 and α2 domains
90
(9, 32)), escape has only been documented in SIV-infected Mamu-B*03+ macaques (16). Variation
91
in these CD8+ T cell epitopes accumulates with different kinetics, starting during acute infection for
92
those targeted by high magnitude responses.
93
In this study, we addressed the question of whether elite control of SIVmac239 in Mamu-
94
B*08+ animals is mediated by the known high frequency CD8+ T cell responses targeting Mamu-
95
B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the
96
goal of reducing or abrogating immune responses directed against these epitopes during acute
97
infection. We hypothesized that Mamu-B*08+ macaques would be unable to control SIV replication
98
without these Mamu-B*08-restricted T cell responses.
99

100
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 6

6
MATERIALS AND METHODS
101
Animals
102
Indian rhesus macaques (Macacca mulatta) housed at the Wisconsin National Primate
103
Research Center, and cared for according to the Guide for the Care and Use of Laboratory Animals
104
of the National Research Council, were used in this study. Protocols were approved by the
105
University of Wisconsin Institutional Animal Care and Use Committee. Animals used in this study
106
were initially typed for the MHC-I alleles Mamu-A*01, -A*02, -A*08, -A*11, -B*01, -B*03, -B*04, -
107
B*08, -B*17, and -B*29 using sequence-specific primers (26, 32). We excluded Mamu-B*17+ animals
108
from this study because that allele is also enriched in elite controllers (55). Comprehensive typing of
109
transcribed MHC-I sequences was determined by massively parallel sequencing of cDNAs (54).
110
Due to the low frequency of Mamu-B*08 in the Wisconsin colony (32), many of the animals used in
111
this study are closely related (Figure 1).
112
Immunological and virological data from four of the SIVmac239-infected animals—r91003,
113
r01027, r00032, and r02019—were published as part of a prior study (30). Historical viral load data
114
from five additional SIVmac239-infected, Mamu-B*17-negative, Mamu-B*08+ macaques were
115
included in the viral load analysis. Animals r96104 and r96113 were controls from a previous
116
vaccine study (1), while r98057 was a control in an unpublished vaccine study. Macaques r00078
117
and r01064 were part of a CD8-depletion experiment (19).
118

119
Primers for site-directed mutagenesis
120
We sequentially introduced mutations into SIVmac239 proviral plasmids using site-directed
121
mutagenesis (Quickchange PCR, Stratagene, La Jolla, CA), following the manufacturer’s protocol.
122
The forward primers used to introduce each mutation follow; the altered codon is bolded and
123
underlined: Vif172-179RL8-R2G: GG AAA CAG TGG AGA GGA GAC AAT AGG AGA GG;
124
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 7

7
Vif123-131RL9-R2K: GCG GGA GAA GTG AGA AAG GCC ATC AGG GG; Nef8-16RL9a-P5Q:
125
CGG TCC AGG CAG TCT GGA GATC; Nef246-254RL9b -L9P: GCA AGA GGC CTT CCT AAC
126
ATG GCT GAC; Nef137-146RL10-A1P: CTG GAA GGG ATT TAT TAC AGT CCA AGA AGA
127
CAT AGA ATC TTAG; Rev12-20KL9 -I6L: CCGA AAA AGG CTA AGG CTA TTA CAT CTT
128
CTG CAT CAAAC; Rev44-51RL8-L7I: G TGG CAA CAG ATC CTG GCC TTG GCA GAT AG;
129
Env573-581KL9-L9M: GAA TTG TTG CGA ATG ACC GTC TGG GG. The reverse-complement
130
of each primer was also used in the mutagenesis PCR reaction. Virus stocks were produced by
131
transfecting Vero cells with proviral DNA, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as
132
recommended by the manufacturer. CEMx174 cells were overlaid onto Vero cells one day post-
133
transfection for expansion of the virus. We then transferred the CEMx174 cells to flasks two days
134
later and maintained them until syncytia were widespread in the culture (about one week). Filtered
135
supernatants from these cultures were stored in the vapor phase of liquid nitrogen until use. Using a
136
commercial ELISA kit (ZeptoMetrix Corporation, Buffalo, NY), we determined the Gag-p27
137
content of the viral stocks. Viral RNA was isolated and sequenced as described below to confirm
138
that only the desired mutations were present. Animals were infected intravenously with 1 ng Gag-
139
p27 equivalent of this virus, resuspended in 0.5 ml PBS. Animals newly infected with wild-type
140
SIVmac239 as part of this study were also infected intravenously with 1 ng Gag-p27 equivalent,
141
resuspended in 0.5 ml PBS.
142

143
Cells
144
EDTA-anticoagulated blood was centrifuged over Ficoll (GE Healthcare Systems,
145
Piscataway, NJ) for 30 minutes at 1860 x g to obtain Peripheral Blood Mononuclear Cells (PBMC).
146
Plasma was removed and clarified by an additional centrifugation. Resulting PBMC were washed
147
once in R10 media (RPMI, 10% FCS, 1% Antibiotic/Antimycotic, 1% L-glutamine (all from
148
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 8

8
Hyclone Laboratories, Logan, UT) and treated with ACK buffer to lyse remaining red blood cells.
149
After washing, the cells were resuspended in R10 media for use in immunological assays. Cells were
150
cryopreserved using Cryostor CS5 (BioLife Solutions, Inc., Bothell, WA). Some tetramer stains on
151
were conducted on cryopreserved PBMC. These cells were thawed at 37 °C, washed once with R10,
152
and used the same day in tetramer stains, as described.
153

154
In vitro viral fitness assays
155
For growth curves, PBMC was isolated from an SIV-negative macaque. We then depleted
156
the PBMC of CD8α-positive cells using non-human primate CD8 Microbeads (Miltenyi Biotec Inc,
157
Auburn, CA) according to the manufacturer’s protocol and activated them using PHA (5 µg/ml) for
158
18-24 hours. One million activated cells were resuspended in 500 µl media and infected with 100
159
pg, 10 pg, or 1 pg of virus. After four hours, the cells were washed once and plated in duplicate
160
(500,000 cells per well in volume of 2 ml R15-50 (RPMI, 15% FCS, 1% Antibiotic/Antimycotic, 1%
161
L-glutamine (all from Hyclone Laboratories) containing 50 Units of interleukin-2/ml (NIH AIDS
162
Research and Reference Reagent Program, Germantown, MD)) in 24 well plates. At each timepoint,
163
500 µl of supernatant was removed and then replaced for viral RNA quantitation.
164
For competition assays, SIVmac239 containing ten synonymous mutations (referred to as
165
∆10s) in gag was constructed using Quickchange PCR. This mutated gag is not detected using our
166
standard primers for quantifying SIV gag, allowing specific, separate quantification of WT and ∆10s
167
sequences from a mixed sample (data not shown), though the amino acid sequence of Gag is
168
identical. The ∆10s virus shows no fitness defect in in vitro competition assays with WT SIVmac239.
169
Target cells were isolated and activated as described for the growth curves. Activated cells
170
were then cultured an additional day in R15-50 media. For the competition assay, one million cells
171
were resuspended in 100 µl media and were infected with the competitor viruses at ratios of 10
172
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 9

9
pg:100 pg Gag-p27, 50 pg:50 pg Gag-p27, or 100 pg:10 pg Gag-p27. After four hours, the cells
173
were washed once and plated in duplicate (500,000 per well in volume of 2 ml R15-50 in 24 well
174
plates). 500 µl of supernatant was removed and replaced for quantitation of viral RNAs.
175
We isolated viral RNA on a Qiagen M48 Biorobot using the M48 Virus Mini Kit (Qiagen
176
Corp. Valencia CA). Quantitation of vRNA for the growth curve samples was performed as
177
described in the “Detection of SIV RNA in plasma” section of the methods. Separate quantitative
178
RT-PCR reactions were performed to quantify wild type and ∆10s virus in each competition assay
179
sample, using sequence-specific amplification primers. Each reaction contained the following
180
reagents: 10 µl Superscript III Platinum One-Step 2x buffer containing 5 mM MgSO4 and 0.2 mM
181
of each dNTP, 0.8 µl Superscript III Platinum One-Step enzyme mix (Invitrogen, Carlsbad CA),
182
forward and reverse primers with a final concentration of 500 nM, probe with a final concentration
183
of 100 nM, 0.5% BSA, 5 µl RNA template, and DEPC water to 20 µl. The probe used in both the
184
WT and ∆10s reactions, and primers to amplify WT gag are described in the “Detection of SIV RNA
185
in plasma” section of the methods. Primers to amplify ∆10s sequence were LifMUT1F 5’- GTG
186
TGC GTG ATC TGG TGT ATAC-3’ and LifMUT3R 5’ – GAC TAG GTG ACT CTG GAC
187
AAT CTG TTT AG-3’ (bold underlining indicates the differences from WT gag). Reactions were
188
run on a Roche Lightcycler 480 (Indianapolis, IN) with the following cycling conditions: cDNA
189
synthesis at 50 °C for 30 minutes; initial denaturation at 95 °C for 2 minutes; 45 amplification cycles
190
of 95 °C for 15 seconds, 68 °C for 30 seconds, analysis mode: quantification, acquisition mode:
191
single. A final cooling step was performed at 40 °C for 30 seconds. Ten-fold serial dilutions of an
192
SIV gag in vitro transcript and ∆10s gag in vitro transcript served as standard curves for each run.
193

194
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 10

10
Cell line recognition of mutant viruses
195
CD8+ T cell lines specific for Mamu-B*08-restricted epitopes were generated from SIV-
196
infected Mamu-B*08+ animals by repeated stimulation of PBMC with irradiated autologous BLCLs
197
pulsed with 1 µM peptide. We maintained the cell lines in R15-50 media using an incubator at 5%
198
CO2 at 37 °C. CD4+ T cell targets were generated, and virus was sucrose-purified by centrifugation
199
through 20% sucrose as described previously (49). Target cells were spinoculated (45) with the virus
200
of interest in order to infect a high percentage of cells. Two days later, infected cells were mixed at a
201
1:1 ratio with cell lines specific for the epitope being tested and incubated at 37 °C for six hours, in
202
the presence of 10 µg/ml Brefeldin A (Sigma) for the last 4.5 hours. Cells were stained for 30
203
minutes at room temperature with anti-CD4 and anti-CD8 monoclonal antibodies, washed twice
204
with FACS buffer (PBS containing 2% FCS and 1% BSA), and fixed with 1% paraformaldehyde
205
diluted in PBS, either for 15 minutes or stored overnight at 4º C. Cells were then washed once with
206
FACS buffer and once with FACS buffer containing 0.1% saponin to permeabilize cells. Anti-IFN-
207
γ and anti-TNF-α antibodies were added to the cells, and they were incubated for 30 minutes at
208
room temperature, washed twice with saponin-containing FACS buffer, and fixed with 1%
209
paraformaldehyde diluted in PBS. Fixed cells were run on a FACSCalibur flow cytometer (Becton
210
Dickinson) and analyzed using FlowJo software (Tree Star, Ashland, OR).
211

212
Detection of SIV RNA in plasma
213
Viral RNA was isolated from EDTA-anticoagulated plasma as described previously, and
214
viral loads were determined using a modification of the previously published protocol (13). Briefly,
215
viral RNA was reverse transcribed and quantified using the SuperScript III Platinum One-Step
216
Quantitative RT-PCR System (Invitrogen, Carlsbad, CA) on a LightCycler 2.0 (Roche, Indianapolis,
217
IN). The final reaction mixtures (20 µl total volume) contained 0.2 mM (each) deoxynucleoside
218
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 11

11
triphosphates, 3.5 mM MgSO4, 150 ng random hexamer primers (Promega, Madison, WI), 0.8 µl
219
SuperScript III reverse transcriptase and Platinum Taq DNA polymerase in a single enzyme mix,
220
600 nM of each amplification primer (forward [SIV1552], 5'-GTCTGCGTCATCTGGTGCATTC-
221
3', and reverse [SIV1635], 5'-CACTAGCTGTCTCTGCACTATGTGTTTTG-3'), and 100 nM
222
probe (5'-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-BHQ1-3').
223
Cycling conditions were 37 °C for 15 minutes, 50 °C for 30 minutes, 95 °C for 2 minutes, followed
224
by 50 amplification cycles of 95 °C for 15 seconds and 62 °C for 1 minute with ramp times set to 3
225
°C/second. Ten-fold serial dilutions of an SIV gag in vitro transcript were quantified during each run
226
and served as a standard curve. We determined copy numbers for samples by interpolation onto the
227
standard curve by using the LightCycler software version 4.0. The lower limit of detection in this
228
assay is 30 vRNA copy equivalents (eq)/milliliter (ml) plasma under standard assay conditions.
229

230
Tetramer and subset staining
231
Tetramers conjugated to either PE or APC were produced by the Wisconsin National
232
Primate Center Tetramer Core. Between 500,000 and 1,000,000 PBMC were stained with tetramers
233
in a volume of 100-200 µl R10 for one hour at 37 °C. Surface stains for CD3 and CD8 were then
234
added, cells were incubated at room temperature for an additional 30-40 min, washed twice and
235
fixed with 1% paraformaldehyde diluted in PBS. Fixed cells were run either on a BD-LSRII or
236
FACSCalibur flow cytometer (Becton Dickinson) and analyzed using FlowJo software version 8.8.6
237
(Tree Star). We gated lymphocytes based on forward and side scatter. Tetramer frequencies are
238
expressed as a percentage of CD3+, CD8+ gated events. Only tetramer staining frequencies of at
239
least 0.05% were considered positive.
240
For determination of CD4 counts, PBMC were stained with titrated anti-CD3, CD4, CD8,
241
CD28, and CD95 antibodies for 30-40 min at room temperature, washed twice and fixed with 1%
242
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 12

12
paraformaldehyde in PBS. Subsets were determined as a percentage of the lymphocyte gate. We
243
then calculated absolute counts using lymphocytes/µl blood, as determined by a complete blood
244
count (CBC) performed on a Pentra 60C+ Hematology analyzer (ABX Diagnostics, Irvine, CA).
245
The following antibodies from BD Biosciences were used over the course of this study: anti-
246
CD3 FITC (clone SP34), APC (clone SP34-2), or Alexa700 (clone SP34-2), anti-CD28 PE (clone
247
CD28.2), anti-CD95 FITC (clone DX2), anti-CD8a Pacific Blue (clone RPA-T8) or PerCP (clone
248
SK1), and anti-CD4 PerCP (clone L200). For assays measuring cell line recognition of mutant
249
viruses, anti-IFN-γ (clone 4S.B3), anti-TNF-α (clone MAb11) and anti-CD4 APC (clone M-T466
250
from Miltenyi Biotec) were used.
251

252
Sequencing of plasma viral RNA
253
Viral RNA was extracted from plasma using the Qiagen MinElute kit or by guanidium
254
thiocyanate extraction. Amplicons containing the fourteen Mamu-B*08-restricted epitopes were
255
amplified for each vRNA sample using the Qiagen One Step RT-PCR kit and SIVmac239 specific
256
primer pairs as previously described (44). In addition, we designed four new primer sets using
257
Primer3 (http://frodo.wi.mit.edu/) to amplify target regions. These primers were: SIV-5557-F (5’-
258
AGG TAG TAC CCA GAA GAA AGG CTA-3’) and SIV-6349-R (5’-ATG TCC TTC CCC TAG
259
ACA TCT ACA-3’), SIV-6465-F (5’-TAG TGG AGG TTC TGG AAG AAC TG-3’) and SIV-
260
7021-R (5’-TGT TCC CCA AGT ATC CCT ATT CT-3’), SIV-8294-F (5’-AAT ATC ACC ATG
261
AGT GCA GAG GT-3’) and SIV-8777-R (5’-ACT CTT GCC AAG TCT CATT GTT C-3’), SIV-
262
8903-F (5’-TTT GAC CTT GCT TCT TGG ATA AA-3’) and SIV-9575-R (5’-TGC TAA TTT
263
TTC TCT CTC TTC AGC-3’). RT-PCR cycling conditions were 50°C for 30 minutes, 95°C for 15
264
minutes, 45 cycles of 94°C for 30 seconds, 53°C for 60 seconds, 72°C for 150 seconds, and then
265
68°C for 20 minutes. Cycling ramp rates were 2°C per second. Unincorporated primers and
266
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 13

13
nucleotides were enzymatically removed from the PCR products using ExoSAP-IT® (USB
267
Corporation, Cleveland, Ohio). Forward and reverse sequencing reactions for each amplicon were
268
carried out the using DYEnamic ET Terminators Sequencing Kit (GE Healthcare, Piscataway, NJ),
269
with the same primers used for RT-PCR. The cycling conditions for sequencing reactions were 30
270
cycles of 95 °C for 20 seconds, 50 °C for 15 seconds, 60 °C for 1 minute. Products were then
271
purified with the Agencourt CleanSEQ® dye-terminator removal kit (Agencourt Bioscience
272
Corporation, Beverly, MA, USA) and run on a 3730xl DNA Analyzer (Applied Biosystems, Foster
273
City, CA). Sequence assembly and base calling were done using CodonCode Aligner version 2.0.6
274
(CodonCode Corporation, Dedham, MA). Bases were called as mixed whenever the secondary peak
275
had an intensity that was greater than 40% when compared to the dominant peak. DNA sequences
276
were conceptually translated and aligned to wild type SIVmac239 (GenBank accession no. M33262)
277
in MacVector 10.6 trial version (MacVector Inc, Cary, NC).
278

279
IFN-γ ELISPOT assay
280
ELISPOT assays were performed using previously described methods (30). Briefly, PBMC
281
were used directly in precoated ELISpotPLUS kits (MABTECH Inc, Mariemont, OH) for the
282
detection of IFN-γ-secreting cells according to manufacturer’s protocols. All tests were performed
283
in duplicate. Wells were imaged and counted with an AID EliSpot reader version 4.0 (AID) and
284
analyzed as described previously (30).
285

286
Statistical Analyses
287
The “set-point” viral load for an animal was calculated as the geometric mean of all available
288
viral loads from each animal between weeks 15 and 48 post-infection. The incidence of “control”
289
(e.g. set point at least one log lower than geometric mean chronic phase viral load for SIVmac239
290
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 14

14
infected rhesus macaques) in WT SIVmac239 (n=15) and 8X-SIVmac239 (n=10) groups was
291
compared using the 2-sided Fisher’s exact test. Set-points for each animal in the 8X and WT-
292
infected groups were log-transformed and the average set points for these groups were then
293
compared using the t-test with Welch correction. The non-parametric Wilcoxon signed-rank test
294
was used to compare the magnitude of epitope-specific CD8+ T cell responses measured by MHC
295
class I tetramer staining, between WT SIVmac239 (n=10) and 8X-SIVmac239 (n=10) infected
296
animals. Additional tests not shown (t-test with Welch correction, following log-transformation)
297
were performed and gave convergent results. Central memory CD4 counts of the groups were
298
compared using the t-test with Welch correction, following log-transformation. Statistical analyses
299
were performed using SPSS v.12.0.
300

301
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from

Page 15

15
RESULTS
302
Construction and Characterization of 8X-SIVmac239
303
We introduced mutations into SIVmac239 with the goal of abrogating immunogenicity of
304
Mamu-B*08-restricted epitopes. We selected mutations for use in this study from mutations which
305
we had observed previously in viral sequences from SIVmac239-infected Mamu-B*08/B*03+
306
animals (16, 30, 31). For some epitopes in which a variety of escape mutations had been observed,
307
we screened viral variants for their effectiveness at reducing recognition of virally infected cells by
308
epitope-specific CD8+ T cells in vitro (Figure 2). The L9M mutation introduced into the Env573-
309
581KL9 epitope was also tested because viral escape has only been observed in SIV-infected Mamu-
310
B*03+ macaques (16). We selected the mutations that were most effective at reducing recognition
311
of virally-infected cells by cell lines specific for the epitope for inclusion in the challenge virus.
312
Thus, the selected mutations are not consensus escape sequences, but rather those least likely to
313
engender T cell responses. There are thirteen defined Mamu-B*08-restricted epitopes (33). Escape
314
variation had not previously been observed in five of the subdominant epitopes.
315
We altered eight epitopes using site-directed mutagenesis (Table 1). These included epitopes
316
targeted by what are typically the four most immunodominant responses during acute SIVmac239
317
infection (Vif172-179RL8, Vif123-131RL9, Nef137-146RL10, and Nef246-254RL9b). We also mutated four
318
epitopes that are typically targeted by lower magnitude responses and/or in a lower frequency of
319
animals. We changed the position two primary anchor residues for both epitopes in Vif. Env573-
320
581KL9 and Nef246-254RL9b were changed at the C-terminal primary anchor residues; these changes
321
also substantially reduce the binding of these peptides to Mamu-B*08. We introduced a proline
322
immediately upstream of the defined minimal epitope for Nef137-146RL10. This mutation is a putative
323
processing escape mutation, as prolines are rarely found at the N-termini or immediately upstream
324
of MHC-I epitopes (50). We mutated the remaining three epitopes, Nef8-16RL9a, Rev12-20KL9, and
325
at Univ of Wisconsin - Mad on December 9, 2009
jvi.asm.org
Downloaded from