Enhancement of viral escape in HIV-1 Nef by STEP vaccination.
ABSTRACT: Properly priming cytotoxic T-lymphocyte (CTL) responses is an important task in HIV-1 vaccination. However, the STEP trial showed no efficacy even though the vaccine elicited HIV-specific CTL responses. Our study is to investigate whether or not the STEP vaccine enhanced viral escape in infected volunteers.The signature of viral escape, the presence of multiple escape variants, could be falsely represented by the existence of multiple founder viruses. Therefore, we use a mathematical model to designate STEP study patients with infections from a single founder virus. We then conduct permutation tests on each of 9988 Gag, Pol, and Nef overlapping peptides to identify epitopes with significant differences in diversity between the vaccine and placebo groups using previously published STEP trial sequence data.We identify signatures of vaccine-enhanced viral escape within HIV-1 Nef from the STEP trial. Vaccine-treated patients showed a greater level of epitope diversity in one of the immunodomiant epitopes, EVGFPVRPQVPL (Nef65-76), compared with placebo-treated patients (P?=?0.0038). In the other three Nef epitopes, there is a marginally significant difference in the epitope diversity between the vaccine and placebo group (P?
Project description:A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef????? epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A-H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.
Project description:Epitope escape from HIV-1-targeted CD8<sup>+</sup> cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8<sup>+</sup> T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8<sup>+</sup> T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8<sup>+</sup> T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8<sup>+</sup> CTL response pressure.
Project description:T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1.ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.
Project description:Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT), Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of "escape maps," which identify known and putative cytotoxic T lymphocyte (CTL) epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4(+) T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other genes investigated indicates differential HLA class I-driven evolution in different viral genes. The relationship between HLA class I-associated polymorphisms and lower CD4(+) cell count suggests that immune escape correlates with disease status, supporting an essential role of maintenance of effective CTL responses in immune control of HIV-1. The design of preventative and therapeutic CTL-based vaccine approaches could incorporate information on predictable escape pathways.
Project description:BACKGROUND:Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n?=?5) and (2) elite controllers (ECs; n?=?6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (VE) and latest (VL) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes. RESULTS:Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were?<?1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (<?1%); all VCs showed a high number of mutations, with significant frequency changes between VE and VL visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure. CONCLUSIONS:The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.
Project description:Human immunodeficiency virus (HIV) possesses a major threat to the human life largely due to the unavailability of an efficacious vaccine and poor access to the antiretroviral drugs against this deadly virus. High mutation rate in the viral genome underlying the antigenic variability of the viral proteome is the major hindrance as far as the antibody based vaccine development is concerned. Although the exact mechanism by which CTL epitopes and the restricting HLA alleles mediate their action towards slow disease progression is still not clear, the important CTL restricted epitopes for controlling viral infections can be utilized in future vaccine design. This study was designed for the characterization the HIV-1 optimal CTL epitopes and their corresponding HLA alleles. CTL epitope cluster distribution analysis revealed only two HIV-1 proteins, namely, Nef and Gag, which have significant cluster forming capacity. We have found the role of specific HLA supertypes such as HLA B?07, HLA B?58, and HLA A?03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins, to be presented as epitopes. The analyses revealed that the clusters of optimal epitopes for Nef and p24 proteins of HIV-1 could potentially serve as a source of vaccine.
Project description:Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.
Project description:The live attenuated simian immunodeficiency virus (LASIV) vaccine SIV?nef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIV?nef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIV?nef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIV?nef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIV?nef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.
Project description:CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.
Project description:The influence of major histocompatibility complex class I (MHC-I) alleles on human immunodeficiency virus (HIV) diversity in humans has been well characterized at the population level. MHC-I alleles likely affect viral diversity in the simian immunodeficiency virus (SIV)-infected pig-tailed macaque (Macaca nemestrina) model, but this is poorly characterized. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIV(mac251) genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIV(mac251) at 4 to 10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms at both individual mutations and groups of mutations spanning 10-amino-acid segments were linked using in-house bioinformatics pipelines, since cytotoxic T lymphocyte (CTL) escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays, we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described to be CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and strategies for treatment of HIV infection.Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV, and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV type 1-infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing nearly identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaques to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques, and this enables a more refined level of analysis of future vaccine design and strategies for treatment of HIV infection.