Conditional immune escape during chronic SIV infection Dane Gellerup1, Alexis Balgeman2 ...

31 Pages·2015·0.43 MB·English

JVI Accepted Manuscript Posted Online 21 October 2015
J. Virol. doi:10.1128/JVI.02587-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Conditional immune escape during chronic SIV infection
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3 Dane Gellerup1, Alexis Balgeman2, Chase W. Nelson3, Adam Ericsen2, Matthew Scarlotta2,
4 Austin L. Hughes3, and Shelby O'Connor1,2,#
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6 1Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI a
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7 2Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI d

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8 3Department of Biological Sciences, University of South Carolina, Columbia, SC o
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10 Running title: Conditional immune escape during chronic SIV infection jv
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13 #To whom correspondence should be addressed: a
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14 555 Science Drive y

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15 Madison, WI 53711 0
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18 E-mail: [email protected]
19 Word count Abstract and Importance: 250 and 147
20 Word count Text: 3,819
21 Abstract:
22 Anti-HIV CD8 T cells included in therapeutic treatments will need to target epitopes that do not
23 accumulate escape mutations. Identifying which epitopes do not accumulate variants, but retain
24 immunogenicity, depends on both host MHC genetics and the likelihood for an epitope to
25 tolerate variation. We previously found that immune escape during acute SIV infection is
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26 conditional; the accumulation of mutations in T cell epitopes is limited and the rate of w
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27 accumulation depends on the number of epitopes being targeted. We now test the hypothesis a
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28 that conditional immune escape extends into chronic SIV infection and that epitopes with d

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29 preserved wild-type sequence have the potential to elicit epitope-specific CD8 T cells. We deep o
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30 sequenced SIV from Mauritian cynomolgus macaques (MCMs) that were homozygous and h
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31 heterozygous for the M3 MHC haplotype and had been infected with SIV for about one year. //
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32 When interrogating variation within individual epitopes restricted by M3 MHC alleles, we found a
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33 three categories of epitopes, that we called Categories A, B, and C. Category B epitopes .
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34 readily accumulated variants in M3 homozygous MCMs, but this was less common in M3 /

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35 heterozygous MCMs. We then determined that chronic CD8 T cells specific for these epitopes J
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36 were more likely preserved in the M3 heterozygous MCMs, when compared to M3 homozygous u
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37 MCMs. We provide evidence that epitopes known to escape from chronic CD8 T cell responses y
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38 in animals that are homozygous for a set of MHC alleles are preserved and retain 2
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39 immunogenicity in a host that is heterozygous for the same MHC alleles. 9

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43 Importance:
44 Anti-HIV CD8 T cells that are part of therapeutic treatments will need to target epitopes that do
45 not accumulate escape mutations. Defining these epitope sequences is a necessary precursor
46 to designing approaches that enhance the functionality of CD8 T cells with the potential to
47 control virus replication during chronic infection or after reactivation of latent virus. Using MHC
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48 homozygous and heterozygous Mauritian cynomolgus macaques, we now provide evidence that w
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49 epitopes known to escape from chronic CD8 T cell responses in animals that are MHC a
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50 homozygous are preserved and retain immunogenicity in a host that is heterozygous for the d

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51 same MHC alleles. Importantly, our findings support the conditional immune escape hypothesis, o
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52 such that the potential to present a greater number of CD8 T cell epitopes within a single animal h
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53 can delay immune escape in targeted epitopes. As a result, certain epitope sequences can //
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54 retain immunogenicity into chronic infection. a
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57 Introduction:
58 Conventional cytotoxic CD8 T cells play an essential role in the control of HIV/SIV
59 replication. These potent immune cells recognize MHC class I molecules presenting peptides
60 derived from HIV or SIV on infected cells. Once detected, epitope-specific CD8 T cells secrete
61 cytokines and enzymes, such as granzyme B, to destroy the infected target. Detection of
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62 infected targets depends on the existence of both the presentation of an immunogenic viral w
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63 peptide and an effector epitope-specific CD8 T cell. During the course of infection, HIV/SIV a
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64 accumulates mutations in many, but not all, targeted epitopes (1, 2). These mutations can d

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65 prevent peptide presentation or disrupt recognition by the TCR of epitope-specific CD8 T cells. o
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66 In both cases, these mutations help HIV/SIV evade CD8 T cells and, consequently, allow h
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67 viruses to replicate more efficiently. //
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68 Long-term preservation of immunogenic T cell epitopes is highly relevant to ongoing HIV s
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69 cure and vaccine research. Some have suggested that a functional cure for HIV should activate r
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70 CD8 T cells targeting epitopes that remain immunogenic in the host (3). Others have proposed o
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71 that a successful HIV vaccine will need to elicit CD8 T cells targeting regions that do not a
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72 accumulate escape mutations (4-7). Ultimately, long-term maintenance of CD8 T cell a
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73 responses is important for virus control. Generally, CD8 T cells will not target epitopes that 9
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74 have accumulated escape mutations. As a result, retaining original, immunogenic epitope 1
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75 sequences in HIV/SIV offers the potential to maintain long-term, effective CD8 T cells. b
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76 From a genome-wide association study of HIV elite controllers, there is support that host e
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77 MHC genetics can play a key role in the development of effective antiviral T cell immunity (8).
78 The presence of a single MHC allele, however, does not guarantee virus control, suggesting
79 that other factors are required. Knowing that a single MHC allele is present is sufficient to
80 predict whether specific epitopes will be targeted by CD8 T cells, but this information is not
81 sufficient to predict when each of the targeted epitopes will accumulate escape mutations in a
82 given host.
83 The differential rate of accumulation of escape mutations among animals that share a
84 single MHC allele has been shown during acute SIV infection of MHC homozygous and
85 heterozygous Mauritian cynomolgus macaques (MCMs). We found that the accumulation of
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86 variants in a single epitope targeted by a CD8 T cell response shared by multiple animals was w
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87 delayed when there existed additional acute phase epitope-specific CD8 T cell responses in an a
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88 animal (9). Subsequent sequence changes that accumulate in the second wave of targeted d

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89 epitopes need to be compatible with these initial adaptive mutations. In the previous study, we o
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90 proposed that the SIV genome could tolerate a limited amount of simultaneous variation during h
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91 acute infection, but we did not address whether increasing the number of targeted epitopes //
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92 would affect the likelihood to accumulate point mutations in epitopes targeted during chronic a
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93 infection when the virus population has further diversified. In the current study, we test the .
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94 hypothesis that there is similar conditional immune escape during chronic HIV/SIV infection, a /

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95 time when the CD8 T cell response is thought to be less functional and newly emerging J
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96 mutations need to be compatible within an SIV genome that has undergone further sequence u
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97 evolution (10). y
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98 To test this hypothesis, circulating virus populations were deep sequenced from MCMs 1
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99 who were MHC homozygous and heterozygous for the M3 MHC haplotype and infected with b
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100 clonal SIV. We determined whether increasing the number of potential epitopes presented to u
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101 CD8 T cells would blunt the accumulation of variants in epitope sequences restricted by M3 t
102 MHC molecules. If so, then there remain potentially immunogenic epitopes in MHC
103 heterozygous individuals that could be targeted by CD8 T cells during chronic infection and
104 could be important in long term virus control.
105 Methods:
106 Animals: All animals were used in previous studies (9, 11, 12). They were purchased from
107 Charles River Laboratories or Bioculture Group, and cared for by the Wisconsin National
108 Primate Research Center (WNPRC) according to protocols approved by the University of
109 Wisconsin Graduate School Animal Care and Use Committee. As listed in Supplementary
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110 Table 2, we used samples from nine animals that were homozygous for the M3 MHC genotype,
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111 three animals that were heterozygous for the M1 and M3 MHC genotypes, and two animals that n
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112 were heterozygous for the M5 and M3 MHC genotypes. The time points used for deep d
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113 sequencing are also indicated in Supplementary Table 2. f
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114 h
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115 Deep sequencing SIV: Genome-wide deep sequencing of replicating virus populations was jv
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116 performed as previously described (13). Briefly, viral RNA was isolated from plasma using the s
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117 MinElute Virus spin kit (Qiagen). Viral cDNA was generated and amplified using the o
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118 Superscript™ III One-Step RT-PCR system with Platinum® Taq High Fidelity (Invitrogen) in four o
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119 overlapping amplicons spanning the entire SIV coding sequence. The MinElute Gel Extraction a
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120 Kit (Qiagen) was used to purify PCR products, which were then quantified using the Quant-IT a
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121 dsDNA HS Assay Kit (Invitrogen). Libraries were generated from 1ng of pooled amplicons and 9
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122 then tagged with barcodes using the Nextera XT kit (Illumina). The tagged libraries were 0
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123 quantified with the Quant-IT dsDNA HS Assay Kit and the quality of the library preparation was b
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124 assessed with an Agilent Bioanalyzer. Libraries were pooled and sequenced on an Illumina u
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125 MiSeq using either 2 x 250 or 2 x 300 sequencing kits. t
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127 Generation of sequence alignments: FASTQ reads were imported into Geneious version 7.1
128 (Biomatters, Ltd.). Reads were quality trimmed, paired, and aligned to the SIVmac239
129 reference (Genbank M33262). The frequency of variants present at greater than 1% at each
130 position with at least 20x coverage was determined using the SNP caller in Geneious. The
131 variation at each position and the impact on the amino acid sequence was then exported as a
132 CSV file and used to calculate mean d and d values.
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134 Calculation of mean d and d values at each site: For each within-host virus population, n
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135 SNPGenie was used to analyze SNP call data to determine the mean numbers of synonymous d
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136 substitutions per synonymous site (d ) and of nonsynonymous substitutions per
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137 nonsynonymous site (d ) in comparison with the inoculum sequence (14). These were
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138 estimated by an adaptation of Nei and Gojobori’s (15) method to pooled next-generation tp
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139 sequence data (16, 17). We estimated d and d separately for 14 CD8 T cell epitopes and for jv
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140 the remainders (excluding the epitopes) of each of the seven coding regions including one or s
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141 more of the epitopes analyzed. All reported P-values are two-tailed. r
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142 n

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143 Calculation of variant amino acid epitope sequences: To determine the frequency of each
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144 amino acid sequence, we first annotated the reference genome in Geneious with the 14 CD8 T 9
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145 cell epitopes. The nucleotide sequences spanning each epitope were then extracted from the 0
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146 sequence alignments. Sequences spanning the entire length of epitope were selected. These b
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147 reads were translated, and the number of times each amino acid variant was present was g
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148 calculated using the 'Find duplicates' feature in Geneious. The list of amino acid sequences st
149 and the number of times each was detected was exported from Geneious and a custom python
150 script was used to convert it to a table of sequences. Microsoft Excel was used to quantify the
151 frequency of each amino acid sequence within the replicating virus population.
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153 IFNγ-ELISPOT analysis: Cryopreserved PBMC (peripheral blood mononuclear cells) that had
154 been collected during chronic infection were thawed and then subjected immediately to an IFNγ-
155 ELISPOT assay, as previously described (18) and according to the manufacturer's protocol.
156 The dates when the PBMC were originally cryopreserved are listed in Supplemental Table 4.
157 Briefly, a pre-coated monkey IFNγ-ELISPOTplus plate (Mabtech, Mariemont, OH) was blocked
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158 and peptides corresponding to each of the wild type epitope sequences were added to each o
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159 well at a final concentration of 1μM. Peptides were tested in duplicate. Concanavalin A was lo
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160 used as a positive control at 10μM. Wells were then imaged with an AID ELISPOT reader. e
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161 Samples were considered positive if the number of SFCs (spot-forming cells) per 106 PBMCs o
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162 was greater than the average of four unstimulated samples plus two times the standard h
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163 deviation of the unstimulated samples, or 100 SFCs, whichever was greater. :/
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164 Results:
165 Deep sequencing SIV detects variants within CD8 T cell epitopes previously found to be
166 restricted by M3 MHC molecules in MCMs.
167 The catalog of potential epitopes presented by MHC class I alleles in MCMs who are
168 MHC-identical and infected with clonal SIV can be defined (9, 12, 19). Fourteen SIV epitopes D
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169 that are restricted by MHC class I molecules expressed by the M3 MHC class I haplotype and n
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170 the CD8 T cell responses targeting these epitopes have been identified in M3+ MCMs (9, 12, 18, d
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171 20-22). Some of these 14 epitopes accumulate variants during acute SIV infection, some
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172 accumulate variants during chronic infection, and some rarely accumulate variants.

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173 To characterize the spectrum and frequency of variants that accumulate in these :
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174 epitopes by about one year post infection, we deep sequenced virus populations isolated from i.
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175 plasma present in nine M3 homozygous MCMs during chronic SIV infection. By sequencing
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176 viruses replicating in this MHC-identical population of animals, viral sequence variation g
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177 attributed to T cell escape was limited to those epitopes restricted by M3 MHC alleles. n

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178 Viral RNA was isolated from animals whose viral loads ranged from 104 to 107 copies/ml u
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179 (Table 1 and (9, 11)). Samples were sequenced using the Illumina MiSeq platform as described 9
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180 in the Materials and Methods. Ranges of 230,096 to 2,005,862 paired-end reads were mapped 0
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181 to SIVmac239 (Genbank M33262) for each virus population. The average coverage at each b
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182 nucleotide position for each genome ranged from 2,322 to 27,830 (Table 1). Variants greater g
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183 than 1% were called throughout the coding sequences using the SNP caller in Geneious 7.1. st
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185 Categorizing variant accumulation in 14 SIV epitopes based on the value of d
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186 Evidence for positive selection within CD8 T cell epitopes can be obtained by calculating
187 d (nonsynonymous substitutions per nonsynonymous site) and d (synonymous substitutions
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188 per synonymous site) values within and outside the targeted epitopes. Using the nucleotide
189 frequency at each position, we calculated the mean d and mean d values (Tables 2 and 3)
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190 relative to an SIVmac239 inoculum that was sequenced in a similar manner. We chose a d
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191 value of 0.0168 to distinguish those epitopes that do accumulate mutations from those that do o
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192 not accumulate mutations because it is the maximum dN value for the non-epitope regions of the lo
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193 seven SIV open reading frames evaluated in this study (Table 3). This suggests that SIV d
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194 epitopes with a mean dN value less than 0.0168 are unlikely to accumulate variants under fro
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195 positive selection in M3 homozygous animals. Using this cutoff, we found that 10 epitopes
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196 (Gag HL9, Gag GW9, Pol QP8, Tat QA8, ARF1 QL11, Env RF9, p
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197 Env620-628TL9, Rev59-68SP10, Nef103-111RM9, and Nef196-203HW8) had a mean dN value greater i.
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198 than 0.0168, whereas four epitopes (Gag KA10, Gag PR9, Gag TV9, and Tat m
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199 49CF9) had a mean dN value less than 0.0168. rg
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200 To further subdivide the 14 epitopes by the frequency of amino acid variants, we J
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201 quantified amino acid variation within each epitope of each animal. The frequencies of amino u
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202 acid sequences that were variant in each animal are shown in Tables 4, 5, and 6. We divided y
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203 the 10 epitopes with a mean d > 0.0168 into two groups. We found that the frequency of 2
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204 variant epitope sequences was greater than 95% for the epitopes Gag GW9, ARF1 9
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205 40QL11, Rev59-68SP10, Nef103-111RM9, and Nef196-203HW8 for all nine virus populations and in 8 of g
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206 9 virus populations for Pol QP8 (Table 4). We classified these epitopes as Category A. In s
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207 contrast, we found that the frequency of variation in the other four epitopes (Category B, Table
208 5) ranged from 1 to 98% among all 9 virus populations. For the four epitopes with a mean d <
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209 0.0168 (Gag KA10, Gag PR9, Gag TV9, and Tat CF9), variant sequences were
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