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Epitope escape from HIV-1-targeted CD8(+) 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(+) 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(+) 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(+) 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(+) CTL response pressure.

OBJECTIVE: To assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8(+) T lymphocyte (CTL) clones as a mechanism of dysfunction. DESIGN: HIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope. METHODS: In addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A*02-restricted), RPAEPVPLQL (Rev 66-75, B*07-restricted), and KRWIIMGLNK (Gag 263-272, B*27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge. RESULTS: Suboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T cell dysfunction due to anergy, exhaustion, or apoptosis. Pre-exposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells. CONCLUSIONS: Our data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log 10 units) to transduced CD8 + T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. IMPORTANCE While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8(+) T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Although a high level of promiscuity for heterologous epitopes is believed to exist for cellular immunity, limited data explore this issue for human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T lymphocyte (CTL) responses. Here, we found an unexpected degree of heterologous cross-reactivity against HIV-1 epitopes, in addition to the targeted index epitope. Most CTL clones screened cross-reacted against other known HIV-1 epitopes of the same major histocompatibility complex type I (MHC-I) restriction, up to 40% of tested nonindex epitopes in some cases. The observed cross-reactivity was universally lower avidity than recognition of the index epitope when examined for several A*02- and B*57-restricted CTL clones, demonstrating that the high concentrations of exogenous epitope typically used for screening of CTL responses are prone to detect such cross-reactivity spuriously. In agreement with this, we found that these cross-reactive responses do not appear to mediate CTL activity against HIV-1-infected cells. Overall, our data indicate that low-level cross-reactivity is remarkably common for HIV-1-specific CTLs. The role of this phenomenon is unclear, but low-avidity interactions have been shown to foster homeostatic proliferation of memory T cells.IMPORTANCE This study raises two issues related to HIV-1-specific CTL responses. These are key immune responses that retard disease progression in infected persons that are highly relevant to immunotherapies and vaccines for HIV-1. First, we make the novel observation that these responses are promiscuous and that CTLs targeting one epitope may cross-recognize other, completely distinct epitopes in the virus. While these are low-avidity interactions that do not appear to contribute directly to the antiviral activity of CTLs, this raises interesting biologic implications regarding the purpose of the phenomenon, such as providing a stimulus for these responses to persist long term. Second, the data raise a technical caveat to detection of CTL responses against particular epitopes, suggesting that some methodologies may unintentionally detect cross-reactivity and overestimate responses against an epitope.

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

Mutational escape of HIV-1 from HIV-1-specific CD8 + T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of in vivo , our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways. IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8 + T lymphocytes (CTLs) is a major barrier to effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here, we demonstrate that the clonal stability and number of clones for the CTL response against an epitope are inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV-1 escape may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1., Author summary Certain MHC class I types are associated with superior immune containment of HIV-1, underscoring the importance of CD8+ cytotoxic T lymphocytes (CTLs). Epitope escape mutations for these types is limited, indicating reduced immune evasion. Two proposed mechanisms are: 1) CTL targeting of highly sequence-constrained epitopes, or 2) more promiscuous CTLs for epitope variation. However, the in vivo complexity of undefined starting virus, multiple targeted epitopes, polyclonal CTL responses against each epitope, and post-hoc evaluation of the interaction renders examination of mechanisms difficult. Here we approach this question with controlled prospective in vitro experiments using saturation mutagenesis of epitopes in clonal HIV-1, propagated in the absence or presence of CTL clones to define the options for epitope mutation and immune evasion by deep sequencing. We find that two immunodominant epitopes presented by protective MHC types are highly mutation-constrained compared to one presented by a non-protective MHC type, whereas CTL promiscuity for epitope variation is not appreciably different. These results suggest that these protective MHC types are associated with limited HIV-1 escape predominately due to intrinsic constraints on epitope mutation, and underscore the importance of focusing the CTL response on highly conserved epitopes for immunotherapies and vaccines.

The impact of HIV-1 Nef-mediated HLA-I down-regulation on CD8+ cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. In the present study, we investigated the impact of Nef on the antiviral efficiency of HIV-1–specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef was not correlated with the presenting HLA-I type or functional avidity of CTLs, but instead was related directly to the kinetics of infected cell clearance. Whereas Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that whereas Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I down-regulation, this varies depending on the specific epitope and virus inoculum. Reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.

Early HIV-1 infection is marked by rapid evolution of both CD8(+) T lymphocyte (CTL) epitope targeting and viral sequences, while chronic infection demonstrates relative stability of these parameters. To examine the interactions of changing CTL targeting and viremia in early infection, we assessed CTL targeting and viremia levels in persons during early HIV-1 infection (estimated 15-271 days post-infection) who were placed on effective antiretroviral therapy. Pre-therapy, CTL targeting of viral proteins varied between persons depending on time after infection. Across individuals, increasing time after infection was associated with increasing Gag and Pol targeting, suggesting increasing targeting of conserved sequences. The intensity of Gag targeting correlated to lower viremia levels, while Env targeting correlated to higher viremia levels during early infection. This suggested that shifted targeting towards more conserved sequences is involved with the drop of viremia during early infection, consistent with prior observations of correlation between Gag targeting and lower viremia during chronic infection. After suppressive antiretroviral therapy, CTL targeting was generally static, indicating that HIV-1 replication and evolution drives the evolution of CTL targeting in early infection. Overall, these data suggest that early CTL targeting is directed towards more variable epitopes, causing escape and re-targeting until more conserved epitopes are recognized stably in chronic infection. Circumventing this natural history by pre-targeting CTL against more conserved epitopes with a vaccine could minimize the initial period of viral escape and immune damage during acute infection, improving long-term containment of HIV-1.

TCRs mediate CTL specificity, but TCRs recognizing the same epitope often differ between persons due to their stochastic derivation. The role of this variability in the pathogenesis of virus infections and malignancies has been technically difficult to study. We apply an adaptation of TCR spectratyping to study HIV-specific CTLs, defining the clonal breadth and sequences of epitope-specific TCRs from PBMCs without cellular sorting or molecular cloning. Examining 48 CTL responses in 12 persons reveals a mean of 4.5 ± 2.7 clones per response, of both public and private clonotypes. The number of identified epitope-specific TCRs correlates with CTL frequency across epitopes, suggesting that clonal breadth limits the magnitude of the CTL response against HIV-1 in vivo. HLA A- and B-restricted CTLs are similar in their TCR breadth in this small cohort, preliminarily suggesting that qualitative differences may account for their disparate impacts on pathogenesis. Overall, these findings demonstrate that the magnitude of the CTL response in chronic HIV-1 infection is constrained by TCR clonal breadth, suggesting maximal expansion of CTLs in response to chronic antigenic stimulation.

Objective—T cell receptor (TCR) gene therapy is an approach being considered for HIV-1, but epitope mutation is a significant barrier. We assessed whether HIV-specific TCR can be modified to have broader coverage of epitope variants by recombining polymorphisms between public clonotype TCR sequences. Design—Public clonotype TCRs recognizing the same epitope often differ by polymorphisms in their third complementarity determining (CDR3) regions. We assessed whether novel combinations of such polymorphisms could improve TCR recognition of epitope variation. Methods—A TCR recognizing the HLA A*0201-restricted epitope SLYNTVATL (Gag 77-85, SL9) was engineered to have combinations of four polymorphisms in the CDR3 regions compared to another SL9-specific TCR. These novel TCRs were screened for functional avidities against SL9 epitope variants and abilities to mediate cytotoxic T lymphocyte suppression of HIV-1 containing the same epitope variants. Results—The TCRs varied modestly in functional avidities (FAs) for SL9 variants, due to alterations in affinity. This translated to differences in antiviral activities against HIV-1 when FA changes crossed the previously defined threshold required for efficient recognition of HIV-1 infected cells. Higher avidity TCR mutants had generally broader recognition of SL9 variants. Conclusions—These results indicate that rationally targeted increases in FA can be utilized to maximize the antiviral breadth of transgenic TCRs. In contrast to previously reported random mutagenesis to markedly increase FA, tuning through recombining naturally occurring polymorphisms may offer a more physiologic approach that minimizes the risk of deleterious TCR reactivities.

HLA-A*0201-restricted virus-specific CD8+ cytotoxic T lymphocytes (CTLs) do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag19–27), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9-specific CTLs primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA-A*0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro-primed CTLs showed that TV9p6 consistently activated crossreactive and equally functional CTLs as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTLs primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a “public” TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional crossreactive clonotypes that are less readily recruited from the naïve T cell pool by the corresponding wildtype epitope. Mimotope-induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine-induced responses to conserved but poorly immunogenic determinants within the HIV proteome.

Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.

Mucosal immune responses induced by HIV-1 vaccines are likely critical for prevention. We report a Phase 1 safety and immunogenicity trial in eight participants using the vaccinia-based TBC-3B vaccine given subcutaneously to determine the relationship between HIV-1 specific systemic and gastrointestinal mucosal responses. Across all subjects, detectable levels of blood vaccinia- and HIV-1-specific antibodies were elicited but none were seen mucosally. While the vaccinia component was immunogenic for CD8 + T lymphocyte (CTL) responses in both blood and mucosa, it was greater in blood. The HIV-1 component of the vaccine was poorly immunogenic in both blood and mucosa. Although only eight volunteers were studied intensively, the discordance between mucosal and blood responses may highlight mechanisms contributing to recent vaccine failures. © 2008 Elsevier Ltd. All rights reserved.

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4+ T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1gagDNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (gag,pol, andnefconfirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while thegagDNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db β2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8 + T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.

Nef expression is not required for HIV-1 replication and is highly targeted by CD8+ CTL, raising the question of why Nef expression is not lost in order to evade immunity in vivo. We explore whether MHC class I (MHC-I) down-regulation to evade CTL in general is a selective pressure maintaining Nef. HIV-1 with functional Nef (wild type, WT) is compared to virus containing a Nef point mutation (M20A) that selectively ablates MHC-I down-regulation. WT-infected cells are relatively resistant to cytolysis and less suppressed for viral replication by Gag- and RT-specific CTL compared to M20A. These viruses grow similarly in vitro in the absence of CTL, but the presence of Gag- or RT-specific CTL strongly favors WT overgrowth of M20A. Finally, while in vitro selection by Nef-specific CTL readily drives disruption of the nef reading frame, the addition of Gag- or RT-specific CTL markedly limits such escape. These data indicate that MHC-I down-regulation is an important function favoring Nef maintenance due to a net selective advantage in the setting of the general CTL response.

Cytotoxic T lymphocyte (CTL) senescence may be an important mechanism of immune failure in HIV-1 infection. We find that senescence of HIV-1-specific CTL clones causes loss of killing activity, preventable by transduction with telomerase. Furthermore, senescence is associated with reduced expression of the effector molecules granzyme and perforin, suggesting CTL “exhaustion” can result in hypofunction. These results agree with other studies showing that HIV-1-specific CTL exhibit abnormal phenotypes in vivo, and suggest the possibility that chronic turnover is an important mechanism of antiviral failure in HIV-1 infection.

The ability to study HIV-1-specific cytotoxic T cell (CTL) clones in models in vitro or to expand them for immunotherapeutic use is limited by the technical difficulty of propagating these cells. The factors that determine the survival and proliferation of the cells are incompletely understood and could include cytokines provided from feeder cells or serum. We therefore investigated the effects of adding two cytokines reported to have effects on T cell proliferation and function, interleukin (IL)-7 and IL-15. Four HIV-1-specific clones derived from infected persons were cultured under standard conditions with IL-2 compared to IL-7 or IL-15 alone or in combination with IL-2. Proliferation and survival, as reflected by cell numbers after stimulation, were poorly supported by IL-7 or IL-15 alone, and these cytokines appeared to provide no additional benefit when added to IL-2. Similarly, these cytokines alone did not support the functional status of these cells as measured by chromium release assays with peptide-pulsed target cells. Addition of IL-7 or IL-15 to IL-2 did not augment function of the cells. These data suggest that supplementing CTL cultures with these cytokines does not provide improvement of cell growth or function.

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8+ CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.

Telomere length is abnormally short in the CD8+T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8+cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8+cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8 + T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. IMPORTANCE These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia.

Although CD8 + cytotoxic T lymphocytes (CTLs) are protective in HIV-1 infection, the factors determining their antiviral efficiency are poorly defined. It is proposed that Gag targeting is superior because of very early Gag epitope presentation, allowing early killing of infected cells before Nef-mediated downregulation of human leukocyte antigen class I (HLA-I). To study Gag epitope presentation kinetics, three epitopes (SL9 77-85 , KF11 162-172 , and TW10 240-249 ) were genetically translocated from their endogenous location in the Rev-dependent (late) gag gene into the Rev-independent (early) nef gene with concomitant mutation of the corresponding endogenous epitopes to nonrecognized sequences. These viruses were compared to the index virus for CTL-mediated suppression of replication and the susceptibility of this antiviral activity to Nef-mediated HLA-I downregulation. SL9-specific CTLs gained activity after SL9 translocation to Nef, going from Nef sensitive to Nef insensitive, indicating that translocation accelerated infected cell recognition from after to before HLA-I downregulation. KF11-specific CTL antiviral activity was unchanged and insensitive to HLA-I downregulation before and after KF11 translocation, suggesting that already rapid recognition of infected cells was not accelerated. However, TW10-specific CTLs that were insensitive to Nef at the baseline became sensitive with reduced antiviral activity after translocation, indicating that translocation retarded epitope expression. Cytosolic peptide processing assays suggested that TW10 was inefficiently generated after translocation to Nef, compared to SL9 and KF11. As a whole, these data demonstrate that epitope presentation kinetics play an important role in CTL antiviral efficiency, that Gag epitopes are not uniformly presented early, and that the epitope context can play a major role in presentation kinetics.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.

Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57 + ) slow progressors and B57 + rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57 + individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8 + cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro . This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo .

Across several cohorts, human immunodeficiency virus type 1 (HIV-1) Gag- and Env-specific CD8 + T lymphocyte (CTL) responses have demonstrated inverse and positive correlations, respectively, to viremia. The mechanism has been proposed to be superior antiviral activity of Gag-specific CTLs in general. Addressing this hypothesis, we created two HIV-1 constructs with an epitope translocated from Gag (SLYNTVATL, SL9) to Env, thereby switching the protein source of the epitope. A virus expressing SL9 in Env was similar to the original virus in susceptibility to SL9-specific CTLS. This finding suggests that Env targeting is not intrinsically inferior to Gag targeting for CTL antiviral activity.

Infections with live-attenuated viruses within SIV models of HIV-1 pathogenesis have offered the best evidence that a vaccine could generate protective immunity. Desrosiers and colleagues [1] first demonstrated this principle using nef-defective SIV, which established a chronic low-grade asymptomatic infection in macaques associated with protection against subsequent challenge with wild-type SIV. At least in part, this phenomenon appeared to be due to attenuation of viral replication. More recently, Shacklett et al [2] and Hoxie and et al [3] have examined attenuated SIV infection via mutations in the transmembrane domain of gp41, which also subsequently protects from challenge by wild-type SIV. Shacklett et al found that SIV containing multiple (stop and point) mutations disrupting this domain reduced viral replicative capacity in vitro, likely due to effects on gp41 membrane trafficking in infected cells [4, 5]. Hoxie and colleagues more specifically created mutations in the tyrosine-based sorting motif (Y712xxϕ) in the membrane-proximal cytoplasmic domain of gp41 [3]. Despite the role this motif in gp41 trafficking [4, 5], the mutations appeared to have minimal impact on viral fitness, reflected by normal peak viremia during acute infection in vivo and growth kinetics in vitro. Thus the mechanism of attenuation and subsequent immune protection is unclear, but probably not due to markedly reduced viral replication capacity. Interestingly, in vivo CD8 depletion experiments have suggested that CD8+ T lymphocytes (CTLs) may contribute to the protective immunity [6]. To assess whether analogous mutations in HIV-1 might affect viral susceptibility to CTLs, mutations in the Y712xxϕ motif were constructed in HIV-1 NL4-3 [7] by point mutagenesis (QuikChange, Stratagene). These included EnvY712I, EnvY712S and EnvΔGY mutations in the cytoplasmic domain of gp41 (Figure 1A), engineered into the whole genome context of NL4-3.1 containing the clade B consensus sequence at Gag 77-85 (HXB2 a.a. numbering) [8]. These viruses were examined for their ability to replicate in T1 cells [9] (Figure 1B). The EnvY712I and EnvY712S mutants had growth kinetics similar to wild-type NL4-3 (EnvY712), similar to SIVmac239 [3]. In contrast, the EnvΔGY mutant was replication incompetent, suggesting that this mutation more severely impaired HIV-1 than SIV. Figure 1 Susceptibility of Env Y712xxϕ motif mutant viruses to HIV-1-specific CTL To assess the susceptibility of these mutants to CTLs, these viruses were tested in virus suppression assays [10, 11] using HIV-1-specific CTL clones: S1-SL9-3.23T recognizing the HLA A*02-restricted epitope SLYNTVATL in Gag p17 (a.a. 77-85) [12], S31-IV9-10.11T recognizing the HLA A*02-restricted epitope ILKEPVHGV in RT (a.a. 309-317), and S58-PL10-10.8 recognizing the HLA A*02-restricted epitope PLTFGWCYKL in Nef (a.a. 136-145). The HIV-1-permissive target cells were A*02-expressing T1 cells [10]. Comparisons of these viruses showed similar degrees of suppression of the mutant and wild-type viruses by the three CTL clones (Figure 1C and D). Similar results were noted in independent experiments using two other CTL clones recognizing other epitopes (not shown). Overall, these results suggested that directly increased susceptibility to CTL inhibition is not the mechanism of in vivo attenuation of infection with viruses containing these mutations. Because attenuated SIV infection has been the most robust example of protective vaccination in the SIV-macaque model [1, 6, 13–15], understanding how viral attenuation affects antiviral immunity is clearly an important goal. The observation that SIV mutated in the Y712xxϕ motif yields infections with typical high peak viremia followed by chronic low viremia in macaques subsequently protected from wild-type virus challenge [2, 3, 6] suggests that viral replication is not markedly affected by the mutations, but that replication is suppressed during chronic infection after development of CTL responses, which are the major determinant of set-point viremia [16–18]. Furthermore, preliminary data suggests that the low set-point viremia of macaques infected with Y712xxϕ-disrupted SIV may be related to the CTL response [6]. Thus, a simple explanation could be that disruption of this motif somehow renders SIV directly more susceptible to CTL. However, our results suggest that this is not the case for HIV-1; disruption of the Y712xxϕ motif did not appear to increase susceptibility of HIV-1 to CTLs directly. While our in vitro assay may not necessarily predict the interaction of virus and CTLs in vivo, it seems likely that the mechanism of attenuation is either not mediated by CTLs, or indirectly increases the antiviral activity of CTLs. Interestingly, it has been suggested that the HIV-1 Env may facilitate viral escape from CTLs in lymph nodes [19, 20]. Although our data do not address this issue directly, they are compatible with a mechanism whereby reduced levels of Env could reduce viral escape from CTLs. Further work would be required to explore this possibility.

Foxp3 plays a critical role in development of CD4+ regulatory T lymphocytes (Tregs). It was originally proposed as a specific marker for Tregs, but recent studies have shown that Foxp3 can be expressed in proliferating CD8+ and CD4+ T lymphocytes. We further investigated the association between Foxp3 expression and proliferation of peripheral blood CD4+ and CD8+ T lymphocytes and focused on virus-specific memory CD8+ T lymphocytes. We found that resting peripheral blood bulk and cytomegalovirus- or HIV-1-specific CD8+ T lymphocytes do not normally express Foxp3. However, stimulation in vitro triggered these cells to express Foxp3 as well as CD25, and the addition of interleukin-2 possibly enhanced the expression of Foxp3. These data demonstrate that proliferation itself is sufficient to induce the Treg-like phenotype. Given that others have demonstrated Treg functional activity in such "induced Tregs," these results suggest that virus-specific CD8+ T lymphocytes have the capacity to acquire regulatory functions. Although the implications of Foxp3 expression in virus-specific CD8+ T lymphocytes in the immunologic control of persistent HIV-1 viremia remain to be determined, our results are consistent with Foxp3 expression playing an essential role in regulation of cell proliferation and functional outcomes for HIV-1-specific CD8+ T lymphocytes.

The genetic divergence of human immunodeficiency virus (HIV)-1 into distinct clades is a serious consideration for cytotoxic T lymphocyte (CTL)-based vaccine development. Demonstrations that CTLs can cross-recognize epitope sequences from different clades has been proposed as offering hope for a single vaccine. Cross-clade CTL data, however, have been generated by assessing recognition of exogenous peptides. The present study compares HIV-1-specific CTL cross-clade epitope recognition of exogenously loaded peptides with suppression of HIV-1-infected cells. Despite apparently broad cross-clade reactivity of CTLs against the former, CTL suppression of HIV-1 strains with corresponding epitope sequences is significantly impaired. The functional avidity of CTLs for nonautologous clade epitope sequences is diminished, suggesting that CTLs can fail to recognize levels of infected endogenously derived cell-surface epitopes despite recognizing supraphysiologic exogenously added epitopes. These data strongly support clade-specific antiviral activity of CTLs and call into question the validity of standard methods for assessing cross-clade CTL activity or CTL antiviral activity in general.

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. “Functional avidity,” defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD 50 ), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD 50 to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD 50 for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD 50 required for CTL to achieve 50% efficiency of infected cell killing [KE 50 ]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE 50 despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE 50 , demonstrating epitope dependence of KE 50 . Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE 50 . Thus, defining KE 50 values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.

HIV-1 Nef and HIV-1–specific cytotoxic T lymphocytes (CTLs) have important and opposing roles in the immunopathogenesis of HIV-1 infection. Nef-mediated down-modulation of HLA class I on infected cells can confer resistance to CTL clearance, but the factors determining the efficiency of this process are unknown. This study examines the impact of Nef on the antiviral activity of several CTL clones recognizing epitopes from early and late HIV-1 proteins, restricted by HLA-A, -B, and -C molecules. CTL-targeting epitopes in early proteins remained susceptible to the effects of Nef, although possibly to a lesser degree than CTL-targeting late protein epitopes, indicating that significant Nef-mediated HLA down-regulation can precede even the presentation of early protein-derived epitopes. However, HLA-C–restricted CTLs were unaffected by Nef, consistent with down-regulation of cell-surface HLA-A and -B but not HLA-C molecules. Thus, CTLs vary dramatically in their susceptibility to Nef interference, suggesting differences in the relative importance of HLA-A– and HLA-B– versus HLA-C–restricted CTLs in vivo. The data thus indicate that HLA-C–restricted CTLs may have an under-appreciated antiviral role in the setting of Nef in vivo and suggest a benefit of promoting HLA-C–restricted CTLs for immunotherapy or vaccine development.

Many vaccine approaches emphasize producing HIV-1-specific CD8+ T-lymphocyte (CTL) responses. Towards this goal, many studies simply classify vaccinees as “responders” or “nonresponders,” based on arbitrary cutoff criteria. HIV-1-uninfected participants receiving the TBC-3B vaccine were assessed for HIV-1-specific CTL by interferon-γ ELISpot, and compared to HIV-1-infected control subjects not on antiretroviral therapy. Vaccinees also were tested for HIV-1-specific antibody responses and generalized CD8+ T-lymphocyte activation. Different criteria for vaccine “responder” status were applied to the measured CTL values. The vaccinees showed evidence of vaccine exposure by CD8+ T-lymphocyte activation and HIV-1-specific antibodies. Considering any single positive HIV-1-specific CTL measurement a vaccine “response,” all vaccinees could be classified as “responders,” but even slight increases in the stringency of response criteria resulted in a steep decline of the “response” rate. In contrast, HIV-1-infected persons were clearly “responders” against the same proteins by the same criteria. Quantitative assessment of CTL demonstrated low and transient HIV-1-specific CTL compared to natural infection. These analyses emphasize the pitfalls of summarizing vaccine study results using simple cutoff criteria to define response rates, and suggest the utility of more comprehensive descriptions to describe vaccine immunogenicity and persistence of responses.

Gut-associated lymphoid tissue is the major reservoir of lymphocytes and human immunodeficiency virus type 1 (HIV-1) replication in vivo, yet little is known about HIV-1-specific CD8+T-lymphocyte (CTL) responses in this compartment. Here we assessed the breadth and magnitude of HIV-1-specific CTL in the peripheral blood and sigmoid colon mucosa of infected subjects not on antiretroviral therapy by enzyme-linked immunospot analysis with 53 peptide pools spanning all viral proteins. Comparisons of blood and mucosal CTL revealed that the magnitude of pool-specific responses is correlated within each individual (meanr2= 0.82 ± 0.04) and across all individuals (r2= 0.75;P< 0.001). Overall, 85.1% of screened peptide pools yielded concordant negative or positive results between compartments. CTL targeting was also closely related between blood and mucosa, with Nef being the most highly targeted (mean of 2.4 spot-forming cells [SFC[/106CD8+T lymphocytes/amino acid [SFC/CD8/aa]), followed by Gag (1.5 SFC/CD8/aa). Finally, comparisons of peptide pool responses seen in both blood and mucosa (concordant positives) versus those seen only in one but not the other (discordant positives) showed that most discordant results were likely an artifact of responses being near the limit of detection. Overall, these results indicate that HIV-1-specific CTL responses in the blood mirror those seen in the mucosal compartment in natural chronic infection. For protective or immunotherapeutic vaccination, it will be important to determine whether immunity is elicited in the mucosa, which is a key site of initial infection and subsequent HIV-1 replication in vivo.

The variability of HIV-1 sequences within and between persons in vivo complicates immunologic screening with a fixed sequence, and using peptides based on consensus sequences therefore has become a common practice for pathogenesis and vaccine studies. Here, we screen a cohort of HIV-1-infected persons in the United States for CD8+ T lymphocyte (CTL) responses using Gag peptides based on the Clade C primary isolate DU422 and the consensus sequence for Clade B. Surprisingly, the DU422 and Clade B consensus peptides are similar in sensitivity, but many responses are detected only by one set or the other. About equal numbers of discordantly detected responses are specific to consensus Clade B peptides as DU422 peptides. A minority of discordant detection is due to the varying frames of the peptide sets and therefore a technical artifact; the majority is due to sequence differences. This lack of superiority of the Clade B consensus peptides to detect CD8+ T lymphocyte responses is an unexpected finding that suggests that detection of HIV-1-specific cellular immunity with these peptides may be significantly insensitive and raises questions as to whether screening with a single sequence adequately reflects responses to the viral swarm in vivo.

UnlabelledMucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24-180 versus 180-365 days). HIV-1-specific CD8(+) T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17-24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24-180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time.Trial registrationClinicalTrials.gov NCT00076817.