CD8+ and CD4+ cytotoxic T cell escape mutations precede breakthrough SIVmac239 viremia in an elite controller
- Benjamin J Burwitz†1,
- Juan Pablo Giraldo-Vela†2,
- Jason Reed1,
- Laura P Newman2,
- Alexander T Bean2,
- Francesca A Nimityongskul2,
- Philip A Castrovinci2,
- Nicholas J Maness3,
- Enrique J Leon1,
- Richard Rudersdorf2 and
- Jonah B Sacha1, 4Email author
© Burwitz et al.; licensee BioMed Central Ltd. 2012
Received: 26 July 2012
Accepted: 14 October 2012
Published: 6 November 2012
Virus-specific T cells are critical components in the containment of immunodeficiency virus infections. While the protective role of CD8+ T cells is well established by studies of CD8+ T cell-mediated viral escape, it remains unknown if CD4+ T cells can also impose sufficient selective pressure on replicating virus to drive the emergence of high-frequency escape variants. Identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure mediated by these cells.
Here, we studied a SIVmac239-infected elite controller rhesus macaque with a 1,000-fold spontaneous increase in plasma viral load that preceded disease progression and death from AIDS-related complications. We sequenced the viral genome pre- and post-breakthrough and demonstrate that CD8+ T cells drove the majority of the amino acid substitutions outside of Env. However, within a region of Gag p27CA targeted only by CD4+ T cells, we identified a unique post-breakthrough mutation, Gag D205E, which abrogated CD4+ T cell recognition. Further, we demonstrate that the Gag p27CA-specific CD4+ T cells exhibited cytolytic activity and that SIV bearing the Gag D205E mutation escapes this CD4+ T cell effector function ex vivo.
Cumulatively, these results confirm the importance of virus specific CD8+ T cells and demonstrate that CD4+ T cells can also exert significant selective pressure on immunodeficiency viruses in vivo during low-level viral replication. These results also suggest that further studies of CD4+ T cell escape should focus on cases of elite control with spontaneous viral breakthrough.
The critical role of CD4+ T cells in directing host immunity to chronic viral infections is well established. CD4+ T cells mediate a myriad of immune functions and are thus characterized into distinct T helper (Th) subsets such as Th1, Th2, Th17, and follicular Th cells depending on their specific effector activity . In addition to coordinating adaptive immunity through their indirect helper functions, CD4+ T cells can also act directly to lyse virally infected cells (reviewed in reference ). Cytolytic CD4+ T cells are associated with control of chronic viral infections such as CMV , EBV , and, more recently, HIV-1 . Furthermore, MHC-II-restricted killing  and CD4+ T cell selected escape mutations  have been observed in vivo in the LCMV mouse model of chronic viral infection.
Whether cytolytic CD4+ T cells exert similar selective pressure in vivo during chronic infection with immunodeficiency viruses like HIV and SIV remains unknown. During HIV infection, the protective role of HIV-specific CD4+ T cells is obfuscated by the preferential infection of these cells . Nevertheless, HIV-specific CD4+ T cells correlate with control of viral replication in both HIV-infected patients  and SIV-infected rhesus macaques . Furthermore, cytolytic CD4+ T cells capable of lysing infected macrophages vigorously expand during acute HIV infection  and establishment of elite control of SIV , suggesting these cells are involved in control of viremia. In agreement with these observations, experimental depletion of CD4+ T cells prior to SIV infection resulted in increased viral replication in macrophages and rapid progression to AIDS . Finally, the modestly protective Env-based RV144 vaccine induced both non-neutralizing antibodies and cytolytic CD4+ T cells, raising the possibility that Env-specific CD4+ T cells contributed to the vaccine effect .
Despite cumulative data suggesting a protective effect of immunodeficiency virus-specific CD4+ T cells, their roles in containing viral replication and driving escape mutations remain controversial. Effective immune responses constrain HIV and SIV replication, resulting in the selection of viral escape variants as previously described for CD8+ T cells, neutralizing antibodies, and recently natural killer cells . Previous studies suggest CD4+ T cells exert little to no selective pressure on HIV-1 [15–19] and that minor viral variants, which fail to stimulate CD4+ T cells, do not gain a replicative advantage within the viral quasispecies . Therefore, identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure against HIV-1. To this end, we followed viral evolution in a unique SIVmac239-infected elite controller (<1,000 viral RNA copies/ml plasma) that experienced breakthrough viremia. We describe the emergence of both CD8+ and CD4+ T cell escape mutations, which preceded loss of SIVmac239 elite control. We demonstrate that Gag-specific CD4+ T cells capable of lysing infected macrophages no longer recognized the post-breakthrough mutant viral sequence, suggesting that CD4+ T cells drove the emergence of this escape variant in vivo. Selection of escape variants that evade immune surveillance and lead to breakthrough viremia is a well-described phenomenon for CD8+ T cells, such as described for the HLA-B*27-bound Gag KK10 epitope in HIV-infected patients [20, 21]. However, to our knowledge, this is the first example of CD4+ T cells selecting for escape mutants in viral breakthrough.
Results and discussion
SIVmac239 breakthrough viremia in an elite controller macaque
Defining effective immune responses that constrain HIV/SIV replication is essential to the development of a prophylactic AIDS vaccine. Rare “elite controllers” (ECs) (HIV-1-infected humans with viral loads below 50 viral RNA (vRNA) copies/ml plasma and SIVmac239-infected rhesus macaques with viral loads below 1,000 vRNA copies/ml plasma in the absence of treatment) offer insight into the necessary components for protective immunity against AIDS viruses. Moreover, breakthrough viruses in ECs contain “footprints” of immune system pressure, yielding further discernment of particularly efficacious immune responses [20, 21].
All seven post-breakthrough substitutions in Env were common in a cohort of fifty-five chronically SIVmac239-infected animals (Additional file 2: Figure S2). In contrast, the four post-breakthrough substitutions in Gag were found only in r00032 (Additional file 3: Figure S3). Because Gag-specific CD8+ T cell responses correlate with lower viral loads  and escape from Gag-specific CD8+ T cells can lead to breakthrough viremia [20, 21, 25], we focused on these unique Gag substitutions present in breakthrough virus from r00032. We longitudinally sequenced these four regions and identified two changes, Gag R276K and V63A, which were first observed at 20 and 70 weeks post-infection, respectively (Figure 1C, left column). In contrast, mutations Gag D205E and K442R immediately preceded breakthrough viremia (Figure 1C, right column). Interestingly, the Gag D205E mutation in SIVmac239 has been shown to abrogate the protective effect of Gag206-216IL11-specific CD8+ T cells in 90-120-Ia haplotype+ Burmese rhesus macaques , and also results in a loss of viral fitness . We utilized ex vivo IFN-γ ELISpot to assess the immune responses targeting these regions of Gag. Ex vivo IFN-γ ELISpot analysis showed that only CD4+ T cells, and not CD8+ T cells, targeted Gag D205 in r00032 at 13 weeks post infection while CD8+ T cells robustly targeted Gag R276 (Figure 1D). CD8+ and CD4+ T cell responses against Gag V63 were equivocally positive, while we detected no responses against Gag K442 at this early time point. No IFN-γ CD8+ or CD4+ T cell responses were detected at 110 weeks post-infection, a post-breakthrough time point at which high-frequency substitutions were present at all four Gag positions (Figure 1C,D). These data suggested that both CD8+ and CD4+ T cells were driving viral evolution during elite control and that escape from these responses may have contributed to viral breakthrough.
Both CD8+ and CD4+ T cells select for escape mutations
CD8+ T cells drove the emergence of the Gag K442R escape mutation as only Gag437-451LQ15-specific CD8+ T cells expanded in culture. Additionally, these CD8+ T cells failed to recognize the mutant sequence in an in vitro recognition assay (Figure 2B). Gag R276 was targeted by both Mamu-B*81:01-restricted Gag271-278LY8-specific CD8+ T cells and DPB1*12-restricted Gag273-287KK15-specific CD4+ T cells. However, the Mamu-B*81:01-restricted Gag271-278LY8-specific CD8+ T cells likely drove the Gag R276K mutation as these cells failed to recognize the post-breakthrough sequence while the DPB1*12-restricted Gag273-287KK15-specific CD4+ T cells efficiently responded to both pre- and post- breakthrough sequences (Figure 2C). Similarly, Gag V63 was targeted by both DRB1*10:07-restricted Gag57-71CG15-specific CD4+ T cells and Gag57-71CG15-specific CD8+ T cells of unknown MHC-I restriction. It is impossible to distinguish the relative importance of the CD8+ and CD4+ T cell responses in driving the Gag V63A substitution as it abrogated recognition by both the Gag57-71CG15-specific CD8+ and CD4+ T cells (Figure 2D). In contrast, Gag D205 was targeted solely by Gag197-211QA15-specific CD4+ T cells restricted by DRB*W4:01 (Figure 1D and Figure 2E). Repeated attempts to measure or isolate CD8+ T cells recognizing this region of Gag were unsuccessful, suggesting the Gag D205E substitution was solely CD4+ T cell driven. Accordingly, the Gag D205E substitution completely abrogated recognition by Gag197-211QA15-specific DRB*W4:01-restricted CD4+ T cells (Figure 2E). This finding, although unexpected, agrees with a recent report that CD4+ T cells are able to drive escape substitutions in HIV-1 in vitro (Philip Norris, personal communication).
Characterization of CD4+ T cell SIV recognition and effector function
Gag197-211QA15-specific CD4+ T cells do not always select for Gag D250E escape variants
CD4+ T cells selected for the escape mutation Gag D205E in r00032. However, whether this was common in animals with CD4+ T cells targeting Gag197-211QA15 remained undetermined. We therefore identified two additional DRB*W4:01+ animals from our cohort of 55 animals. One animal, r95116, was infected with SIVsmE660 as part of a previously described SIV vaccine study . The challenge virus SIVsmE660 contained the Gag D205E substitution, thus precluding the emergence of a Gag197-211QA15-specific CD4+ T cell response. The second animal, r04072, was infected with 8X-SIVmac239, a mutant of SIVmac239 that contains escape mutations with eight Mamu-B*008:01-bound CD8+ T cell epitopes . Despite being Mamu-B*008:01+, r04072 maintained high viral loads throughout infection (Additional file 4: Figure S4A). We longitudinally sequenced virus from r04072 and observed no sequence variation in the Gag197-211QA15 epitope despite a cytotoxic Gag197-211QA15-specific CD4+ T cell response measured directly ex vivo by CD107a cell surface expression  (Additional file 4: Figure S4B,C). Although Gag197-211QA15-specific CD4+ T cells did not select for escape in r04072, this data further support our in vitro results showing cytolytic activity by Gag197-211QA15-specific CD4+ T cells. The data are also in line with recent reports of ex vivo cytolytic CD4+ T cell responses against HIV Gag [2, 36]. Finally, this data suggest that CD4+ T cell escape may only occur in rare cases, such as in ECs with low-level viral replication followed by sudden breakthrough viremia. We therefore also sequenced virus from six ECs, which underwent experimental CD8+ cell depletion, leading to a rapid spike in viremia [11, 37]. These animals all expressed either DRB1*03:06 (the alternate allele restricting Gag197-211QA15-specific CD4+ T cell responses), or the alleles DRB1*06 and/or DRB1*10:07 (which restrict Gag57-71CG15-specific CD4+ T cells) (Additional file 5: Figure S5A). Sequences were acquired at the peak of viral replication following CD8+ cell depletion and following the resolution of viremia after the repopulation of CD8+ cells. We did not observe Gag V63A or D205E escape mutations in any of the animals at either of the time points (Additional file 5: Figure S5B).
The lack of the Gag D205E escape mutation in other SIV infected rhesus macaques expressing DRB*W4:01 or DRB1*03:06 indicates this mutation may be rare and associated with natural spontaneous viral breakthrough in ECs. Thus, we conclude that ECs with viral breakthrough likely provide the best model for the study of CD4+ T cell driven viral escape. Given the paucity of SIV infected rhesus macaques that have been MHC-II typed and shown to express DRB*W4:01 or DRB1*03:06, the phenomenon of CD4+ T cell driven Gag D205E escape will require a larger cohort of infected animals with more extensive MHC-II typing.
CD8+ T cells, neutralizing antibodies, and antiretroviral drugs all drive HIV sequence variation. Here, we provide the first evidence that CD4+ T cells can also drive viral variation. However, we cannot dismiss the possibility that other immune mechanisms may play a role in the escape observed within the CD4+ T cell epitope Gag197-211QA15. For example, a recent study by Chung et. al. indicates that antibody-dependent cellular cytotoxicity (ADCC) can drive HIV escape mutations in Env . ADCC epitopes have been identified in internal proteins including Vpu and Pol, but were not found in Gag in a cohort of 26 HIV-infected individuals . Nonetheless, immune pressure from an unidentified non-CD4+ T cell source could conceivably be responsible for the Gag D205E mutation. Interestingly, Gag197-211QA15 is located in a conserved region of Gag that has been proposed as an ideal target for T cell-based vaccines, and position Gag 205 seems to be under selective pressure as both Gag D205 and Gag E205 are found in HIV clade A/C and B/D consensus sequences, respectively .
Breakthrough viremia in r00032 was temporally associated with the mutations Gag V63A and D205E, implying that increased viral replication was a consequence of immune escape. However, CD4+ T cell dysfunction and exhaustion during chronic HIV and SIV infection are positively correlated with viral load and may also have played a role in breakthrough viremia. Future studies of CD4+ T cell escape in HIV and SIV ECs should focus on the interplay between viral replication, viral variation, and CD4+ T cell dysfunction.
Elite control and long-term non-progression have been associated with CD8+ T cell responses, CD4+ T cell responses, and NK cells [9, 10, 41–45]. Therefore, the CD4+ T cell selected mutation we observed prior to breakthrough viremia is likely only one of many complex determinants in loss of elite control. Indeed, we observed multiple CD8+ T cell selected escape mutations emerging immediately prior to viral breakthrough; thus the contribution of CD8+ and CD4+ T cell responses to viral breakthrough in r00032 requires an assessment of viral variant fitness outside the scope of this study. Nevertheless, the CD4+ T cell-driven mutation at Gag D205E represents the first description of a high-frequency escape mutation in immunodeficiency virus infection and further supports the growing evidence implicating CD4+ T cells as direct suppressors of viral replication.
Uncertainty remains about the importance of CD4+ T cells as effectors against HIV-1, given that expansion of HIV-specific CD4+ T cells may provide additional targets for viral replication . In contrast with other studies [15–19, 46], we show here that CD4+ T cells exert strong immune pressure in vivo in the setting of limited viral replication, evidenced by the emergence of a high frequency CD4+ T cell escape mutation during elite control. Taken together, these data indicate that elicitation of cytolytic CD4+ T cells should be considered in future vaccines against HIV.
Rhesus macaques at the Wisconsin National Primate Research Center were cared for by regulations set by the Guide for Care and Use of Laboratory Animals of the National Research Council and the University of Wisconsin Institutional Animal Care and Use Committee. The infection of r00032 was described previously .
Sequencing and quantification of viral RNA in plasma
Cell-free plasma was obtained from EDTA anti-coagulated whole blood by using Ficoll-Paque Plus (GE Healthcare Bioscience) and density centrifugation. Viral RNA in the plasma was isolated by using the QIAamp MinElute virus spin kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Viral RNA was reverse transcribed and amplified using the One-Step reverse transcription-PCR kit (Qiagen), generating amplicons spanning SIVmac239. Bulk Sanger sequencing was performed on an ABI 3730xl DNA analyzer using ET terminator technology (GE Healthcare). Sequences were assembled against the SIVmac239 reference sequence (GenBank accession M33262) and conceptually translated using CodonCode Aligner version 18.104.22.168 (CodonCode Corporation) and MacVector 11.1 trial version (MacVector,Inc). Plasma SIVmac239 concentrations were monitored by quantitative reverse transcription-PCR as previously described . The limit of detection for this assay is 30 vRNA eq/ml.
Frozen PBMC were used in ELISpot assays for the detection of IFN-γ–secreting cells as previously described . For measuring CD4+ T cell responses, we depleted PBMC of CD8+ cells using a CD8-Microbead kit for nonhuman primates (Miltenyi Biotec) according to manufacturer’s instructions prior to performing the ELISpot assay. Responses were considered positive if the mean number of spot-forming cells (SFC) of duplicate sample wells exceed background plus two standard deviations. Responses of less than 50 SFC per million PBMC were considered negative (below the limit of detection). For IFN-γ ELISpot assays using in vitro expanded cell culture, 5,000 T cells were incubated with 20,000 autologous BLCL and the peptide of interest in each well. Positive responses were determined using a one-tailed t test and an alpha level of 0.05, where the null hypothesis was that the background level would be greater than or equal to the treatment level. If determined to be positive statistically, the values were reported as the average of the test wells minus the average of all negative-control wells. Peptides used in these assays were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
Intracellular cytokine staining (ICS)
ICS was performed as described previously . Briefly, 1 × 105 effector cells were incubated with peptide-pulsed autologous B cells or infected macrophages in the presence of CD107a antibody for 90 min followed by the addition of 5 μg/mL brefeldin A and Golgistop followed by an additional 5 hour incubation. Cells were subsequently stained for CD4 and CD8 surface expression followed by intracellular CD3, IFN-γ, and TNF-α, fixed in 2% PFA, acquired on a BD-LSRII flow cytometer, and analyzed with FlowJo.
T cell and macrophage in vitro culture
T-cell in vitro cultures were initiated with freshly isolated PBMC from animal r00032 co-cultured with irradiated, autologous BLCL pulsed with the three overlapping SIVmac239 15-mer peptides of interest. Following two weekly restimulations, limiting dilution was performed and individual clones were selected from the highest dilutions showing expansion. Cells were maintained throughout in vitro culture in R15-100 media (RPMI media + 15% FCS, supplemented with 100 IU rhIL-2). Specificity was examined in both the bulk T cell line and T cell clones generated by limiting dilution by using each 15-mer peptide individually, followed by truncated versions of the 15-mer peptide as appropriate. To generate macrophages, CD14+ monocytes were isolated via MACS using CD14 microbeads (Miltenyi Biotec) and incubated in 50% fresh R10 media (RPMI + 10% FCS) and 50% KPB-M15-conditioned R10 media supplemented with 10 ng/mL M-CSF (Sigma) for 6 days, feeding every other day. Macropahges were consistently >95% CD14+ and were infected with 100 ng of Gag p27 SIVmac316E (kindly provided by Ronald Desrosiers) or SIVsmE543-3 (kindly provided by Vanessa Hirsch) by spinoculation.
Macrophage 24 hour elimination assay
Macrophages (5 × 104) were infected as described above, the infection was allowed to proceed for 24 h, and they were then cocultured with SIV-specific CD4+ T cells at an E:T of 1:1 at 37°C for 24 h. The culture was then surface stained with antibodies against CD4, CD14, and HLA-DR (BD Biosciences), followed by intracellular staining for Gag p27 using Fix and Perm (Caltag Laboratories) and the 55–2 F12 Gag p27 antibody (National Institutes of Health AIDS Reagent Database) at ≈ 0.7 mg/mL. Events were collected on a BD FACSCaliber and analyzed using FlowJo software. T cell cells were gated out using forward and side scatter dot plots and excluding CD3+ events.
Mapping of MHC restriction and minimal epitope
A cDNA library of r00032 MHC-I and –II alleles was generated as previously described . Either .221 (MHC-I-devoid) or RM3 (MHC-II-devoid) cells were transfected with each allele, pulsed with the peptide of interest, and then used as antigen presenting cells to T cells. Truncated versions of the 15-mer peptide used to culture cell lines were pulsed onto autologous BLCL and used in ICS to determine the minimal epitope by identifying the minimal peptide that resulted in maximal cytokine secretion.
We thank David Watkins, Eva Rakasz, and the Wisconsin National Primate Research Center Elite Controller Resource for PBMC and plasma samples. This work was supported in part by NIH OD 011092. The following reagents were was obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann - La Roche Inc. and SIVmac239 peptides (complete set). We thank Ronald Desrosiers and Vanessa Hirsch for SIVmac316E and SIVsmE543-3 viruses, respectively.
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