Impact of antigen specificity on CD4+ T cell activation in chronic HIV-1 infection.

BACKGROUND: HIV infection induces chronic immune activation which is associated with accelerated disease progression; the causes of this activation, however, are incompletely understood. We investigated the activation status of CD4+ T cells specific for chronic herpes viruses and the non-persistent antigen tetanus toxoid (TT) in HIV positive and HIV negative donors to assess whether persistent infections contribute to chronic CD4+ T cell activation. METHODS: Untreated HIV+ patients and healthy, aged matched controls were recruited and activation levels assessed and compared between cells specific for persistent and non-persistent antigens. Activation levels on antigen-specific CD4+ T cells were measured by intracellular cytokine staining following in vitro stimulation with various recall antigens (CMV, EBV, HSV, VZV and TT) in conjunction with cell surface phenotyping. RESULTS: Activation levels of herpes virus-specific CD4+ T cell populations, assessed by co-expression of CD38 and HLA-DR, were significantly elevated in HIV+ individuals compared to normal controls and compared to TT-specific responses. In contrast, we found similar levels of activation of TT-specific CD4+ T cells in HIV+ and HIV- donors. CONCLUSIONS: These results show a disparate distribution of immune activation within CD4+ T cell populations depending on their specificity and suggest that the elevated level of immune activation that characterizes chronic HIV infection may be influenced by the persistence of other antigens.

Limited maintenance of vaccine-induced simian immunodeficiency virus-specific CD8 T-cell receptor clonotypes after virus challenge.

T-cell receptors (TCRs) govern the specificity, efficacy, and cross-reactivity of CD8 T cells. Here, we studied CD8 T-cell clonotypes from Mane-A*10(+) pigtail macaques responding to the simian immunodeficiency virus (SIV) Gag KP9 epitope in a setting of vaccination and subsequent viral challenge. We observed a diverse TCR repertoire after DNA, recombinant poxvirus, and live attenuated virus vaccination, with none of 59 vaccine-induced KP9-specific TCRs being identical between macaques. The KP9-specific TCR repertoires remained diverse after SIV or simian-human immunodeficiency virus challenge but, remarkably, exhibited substantially different clonotypic compositions compared to the corresponding populations prechallenge. Within serial samples from individual pigtail macaques, only a small subset (33.9%) of TCRs induced by vaccination were maintained or expanded after challenge. Most (66.1%) of the TCRs induced by vaccination were not detectable after challenge. Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the "fitness" of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.

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 SIV(mac251) 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 approximately 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.

Safety, immunogenicity and efficacy of peptide-pulsed cellular immunotherapy in macaques.

BACKGROUND: Simple and effective delivery methods for cellular immunotherapies are needed. We recently published on the effectiveness of using ex vivo pulsing of overlapping SIV Gag 15mer peptides onto fresh peripheral blood cells in 32 SIV(mac251)-infected pigtail macaques. METHODS: We now report on the safety of this approach, analysis of a novel assay for immunogenicity, the effect of an MHC allele, Mane-A*10, on CD8 T cell escape occurring and disease outcome. RESULTS: The vaccine strategy was safe, with no perturbations in weight or hematological profiles in comparison to controls. The high levels of SIV-specific T cell immunogenicity of this approach was confirmed using a novel assay measuring upregulation of surface CD134 of CD4 T cells. A substantial effect of the Mane-A*10 allele in reducing SIV viral load of pigtail macaques was observed in both vaccinees and controls; the virologic efficacy of the immunotherapy in comparison to controls was greatest in Mane-A*10- animals. Escape mutations at several new CD8 T cell epitopes throughout the SIV proteome were observed, primarily in animals with poorer virologic control. CONCLUSIONS: In summary, we provide further information that peptide-pulsed PBMC are a safe, immunogenic and effective immunotherapy. The observed influence of MHC alleles and immune escape allows us to design more insightful future immunotherapy studies.

Killing kinetics of simian immunodeficiency virus-specific CD8+ T cells: implications for HIV vaccine strategies.

Both the magnitude and function of vaccine-induced HIV-specific CD8+ CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-gamma were simultaneously studied in SIV Gag(164-172) KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-gamma and with limited (<5%) degranulation and granzyme B release. Vaccination with live-attenuated SIV resulted in a rapid cytolytic profile of SIV-specific CTLs with rapid (<0.5 h) and robust (>50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIV(mac251) challenge of prime-boost-vaccinated macaques, albeit with less IFN-gamma expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure.

Vaccine-induced T cells control reversion of AIDS virus immune escape mutants.

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.

Comparative efficacy of subtype AE simian-human immunodeficiency virus priming and boosting vaccines in pigtail macaques.

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.

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.

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-gamma-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.

Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost.

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.

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, naive 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.