Caroline S. Fernandez

Rapid Viral Escape at an Immunodominant Simian-Human Immunodeficiency Virus Cytotoxic T-Lymphocyte Epitope Exacts a Dramatic Fitness Cost

ABSTRACT Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.

Analysis of Pigtail Macaque Major Histocompatibility Complex Class I Molecules Presenting Immunodominant Simian Immunodeficiency Virus Epitopes

ABSTRACT Successful human immunodeficiency virus (HIV) vaccines will need to induce effective T-cell immunity. We studied immunodominant simian immunodeficiency virus (SIV) Gag-specific T-cell responses and their restricting major histocompatibility complex (MHC) class I alleles in pigtail macaques (Macaca nemestrina), an increasingly common primate model for the study of HIV infection of humans. CD8+ T-cell responses to an SIV epitope, Gag164-172KP9, were present in at least 15 of 36 outbred pigtail macaques. The immunodominant KP9-specific response accounted for the majority (mean, 63%) of the SIV Gag response. Sequencing from six macaques identified 7 new Mane-A and 13 new Mane-B MHC class I alleles. One new allele, Mane-A*10, was common to four macaques that responded to the KP9 epitope. We adapted reference strand-mediated conformational analysis (RSCA) to MHC class I genotype M. nemestrina. Mane-A*10 was detected in macaques presenting KP9 studied by RSCA but was absent from non-KP9-presenting macaques. Expressed on class I-deficient cells, Mane-A*10, but not other pigtail macaque MHC class I molecules, efficiently presented KP9 to responder T cells, confirming that Mane-A*10 restricts the KP9 epitope. Importantly, naïve pigtail macaques infected with SIVmac251 that respond to KP9 had significantly reduced plasma SIV viral levels (log10 0.87 copies/ml; P = 0.025) compared to those of macaques not responding to KP9. The identification of this common M. nemestrina MHC class I allele restricting a functionally important immunodominant SIV Gag epitope establishes a basis for studying CD8+ T-cell responses against AIDS in an important, widely available nonhuman primate species.

Comparative Efficacy of Subtype AE Simian-Human Immunodeficiency Virus Priming and Boosting Vaccines in Pigtail Macaques

ABSTRACT Vaccination against AIDS is hampered by great diversity between human immunodeficiency virus (HIV) strains. Heterologous B-subtype-based simian-human immunodeficiency virus (SHIV) DNA prime and poxvirus boost vaccine regimens can induce partial, T-cell-mediated, protective immunity in macaques. We analyzed a set of DNA, recombinant fowlpox viruses (FPV), and vaccinia viruses (VV) expressing subtype AE HIV type 1 (HIV-1) Tat, Rev, and Env proteins and SIV Gag/Pol in 30 pigtail macaques. SIV Gag-specific CD4 and CD8 T-cell responses were induced by sequential DNA/FPV vaccination, although lower FPV doses, VV/FPV vaccination, and DNA vaccines alone were not as consistently immunogenic. The SHIV AE DNA prime, FPV boost regimens were significantly less immunogenic than comparable B-subtype SHIV vaccination. Peak viral load was modestly (0.4 log10 copies/ml) lower among the AE subtype SHIV-immunized animals compared to controls following the virulent B subtype SHIV challenge. Protection from persistent high levels of viremia and CD4 T-cell depletion was less in AE subtype compared to B subtype SHIV-vaccinated macaques. Gag was highly immunodominant over the other AE subtype SHIV vaccine proteins after vaccination, and this immunodominance was exacerbated after challenge. Interestingly, the lower level of priming of immune responses did not blunt postchallenge Gag-specific recall responses, despite more modest protection. These studies suggest priming of T-cell immunity to prevent AIDS in humans is possible, but differences in the immunogenicity of various subtype vaccines and broad cross-subtype protection are substantial hurdles.

MAIT cells are depleted early but retain functional cytokine expression in HIV infection

Mucosal‐associated invariant T (MAIT) cells home to mucosal sites and exert antimicrobial activity against bacteria and other microorganisms. HIV infection leads to early depletion of gut T cells and translocation of bacterial products. There are reports that MAIT cells, defined by coexpression of Vα7.2 and CD161, are depleted during HIV infection and residual MAIT cells are functionally impaired. However, one study suggested that MAIT cells might remain after HIV infection but evade detection through CD161 downregulation. Thus, the impact of HIV infection on MAIT cells is unclear. We studied longitudinal blood samples from 31 HIV‐infected subjects for MAIT cell numbers, phenotype and function using both standard Vα7.2/CD161 surface markers and an MR1 tetramer. We found that MAIT cells were depleted early during HIV infection, and although there was a concomitant rise in Vα7.2+CD161– cells, these were MR1 tetramer negative, indicating that these are unlikely to be altered MAIT cells. Antigen‐mediated activation of residual MAIT cells showed that they remained functional out to 2 years following HIV infection. Although MAIT cells are depleted in HIV infection, residual and functionally active MAIT cells persist and may still be able to assist in controlling bacterial translocation during HIV infection.

The pigtail macaque MHC class I allele Mane-A*10 presents an immundominant SIV Gag epitope: identification, tetramer development and implications of immune escape and reversion.

Abstract:  The pigtail macaque (Macaca nemestrina) is a common model for the study of AIDS. The pigtail major histocompatibility complex class I allele Mane‐A*10 restricts an immunodominant simian immunodeficiency virus (SIV) Gag epitope (KP9) which rapidly mutates to escape T cell recognition following acute simian/human immunodeficiency virus infection. Two technologies for the detection of Mane‐A*10 in outbred pigtail macaques were developed: reference strand‐mediated conformational analysis and sequence‐specific primer polymerase chain reaction. A Mane‐A*10/KP9 tetramer was then developed to quantify CD8+ T lymphocytes primed by multigenic DNA vaccination, which have previously been difficult to detect using standard interferon‐γ‐based T cell assays. We also demonstrate mutational escape at KP9 following acute SIV infection. Mane‐A*10+ animals have lower set point SIV levels than Mane‐A*10− animals, suggesting a significant fitness cost of escape. These studies pave the way for a more robust understanding of HIV vaccines in pigtail macaques.

MHC class I allele frequencies in pigtail macaques of diverse origin

Pigtail macaques (Macaca nemestrina) are an increasingly common primate model for the study of human AIDS. Major Histocompatibility complex (MHC) class I-restricted CD8+ T cell responses are a critical part of the adaptive immune response to HIV-1 in humans and simian immunodeficiency virus (SIV) in macaques; however, MHC class I alleles have not yet been comprehensively characterized in pigtail macaques. The frequencies of ten previously defined alleles (four Mane-A and six Mane-B) were investigated in detail in 109 pigtail macaques using reference strand-mediated conformational analysis (RSCA). The macaques were derived from three separate breeding colonies in the USA, Indonesia and Australia, and allele frequencies were analysed within and between these groups. Mane-A*10, an allele that restricts the immunodominant SIV Gag epitope KP9, was the most common allele, present in 32.1% of the animals overall, with similar frequencies across the three cohorts. Additionally, RSCA identified a new allele (Mane-A*17) common to three Indonesian pigtail macaques responding to the same Gag CD8+ T cell epitope. This broad characterization of common MHC class I alleles in more than 100 pigtail macaques further develops this animal model for the study of virus-specific CD8+ T cell responses.

Control of Viremia and Prevention of AIDS following Immunotherapy of SIV-Infected Macaques with Peptide-Pulsed Blood

Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIVmac251 replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably ∼10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.

Vaccine-Induced T Cells Control Reversion of AIDS Virus Immune Escape Mutants

ABSTRACT Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8+ T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8+ T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8+ T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common.

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

ABSTRACT NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4+ (on average 67%) with smaller populations being CD8+ (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4+ NKT cells occurred in all six macaques infected with CXCR4-tropic SHIVmn229 early after infection, with a concomitant rise in CD8+ NKT cells in some animals. The depletion of CD4+ NKT cells was tightly correlated with the depletion of total CD4+ T cells. R5-tropic SIVmac251 infection of macaques resulted in a slower and more variable decline in CD4+ NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4+ NKT cells. An inverse correlation between the depletion of total and CD4+ NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4+ NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.

Screening and confirmatory testing of MHC class I alleles in pig-tailed macaques

Pig-tailed macaques (Macaca nemestrina) are a commonly studied primate model of human AIDS. The Mane-A1*084:01 MHC class I allele (previously named Mane-A*10) is important for the control of SIV infection by CD8+ T cells in this model. Validated methods to detect this allele in large numbers of macaques are lacking. We studied this MHC allele using sequence-specific PCRs in 217 pig-tailed macaques and identified 75 (35%) positive animals. We then performed massively parallel pyrosequencing with a universal 568-bp MHC class I cDNA-PCR amplicon for 50 of these 75 macaques. All 50 animals expressed Mane-A1*084:01 or closely related variants of the Mane-A1*084 lineage. Mane-A1*084 transcripts accounted for an average of 20.9% of all class I sequences identified per animal. SIV infection of a subset of these macaques resulted in the induction of SIV-specific CD8+ T cell responses detected by Mane-A1*084:01 tetramers. An average of 19 distinct class I transcripts were identified per animal by pyrosequencing. This analysis revealed 89 new Mane class I sequences as well as 32 previously described sequences that were extended with the longer amplicons employed in the current study. In addition, multiple Mane class I haplotypes that had been inferred previously based on shared transcript profiles between unrelated animals were confirmed for a subset of animals where pedigree information was available. We conclude that sequence-specific PCR is useful to screen pig-tailed macaques for Mane-A1*084:01, although pyrosequencing permits a much broader identification of the repertoire of MHC class I sequences and haplotypes expressed by individual animals.