Gag-specific Cytotoxic T Lymphocytes Research Articles

The Tat Protein Enhances CTL Responses and Therapeutic Immunity of Gag-Specific Exosome-Targeted T Cell-Based Gag/Tat-Texo Vaccine in Transgenic HLA-A2 Mice

Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.

SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells

Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

Schistosoma mansoni soluble egg antigens enhance Listeria monocytogenes vector HIV-1 vaccine induction of cytotoxic T cells.

Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4(+) T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines.

HIV-1 Gag-specific exosome-targeted T cell-based vaccine stimulates effector CTL responses leading to therapeutic and long-term immunity against Gag/HLA-A2-expressing B16 melanoma in transgenic HLA-A2 mice

Human immunodeficiency virus type-1 (HIV-1)-specific dendritic cell (DC) vaccines have been applied to clinical trials that show only induction of some degree of immune responses, warranting the search of other more efficient vaccine strategies. Since HIV-1-specific CD8+ cytotoxic T lymphocytes (CTLs) have been found to recognize some HIV-1 structural protein Gag conserved and cross-strain epitopes, Gag has become one of the most attractive target candidates for HIV-1 vaccine development. In this study, we generated HIV-1 Gag-specific Gag-Texo vaccine by using ConA-stimulated polyclonal CD8+ T-cells with uptake of Gag-expressing adenoviral vector AdVGag-transfected DC (DCGag)-released exosomes (EXOs), and assessed its stimulation of Gag-specific CD8+ CTL responses and antitumor immunity. We demonstrate that Gag-Texo and DCGag vaccines comparably stimulate Gag-specific effector CD8+ CTL responses. Gag-Texo-stimulated CTL responses result in protective immunity against Gag-expressing BL6-10Gag melanoma in 8/8 wild-type C57BL/6 mice. In addition, we show that Gag-Texo vaccine also induces CTL responses leading to protective and long-term immunity against Gag/HLA-A2-expressing BL6-10Gag/A2 melanoma in 8/8 and 2/8 transgenic HLA-A2 mice, respectively. The average number of lung tumor colonies in mice with 30-days post-immunization is only 23, which is significantly less than that (>300) in control ConA-T-immunized HLA-A2 mice. Furthermore, Gag-Texo vaccine also induces some degree of therapeutic immunity. The average number (50) and size (0.23mm in diameter) of lung tumor colonies in Gag-Texo-immunized HLA-A2 mice bearing 6-day-established lung BL6-10Gag/A2 melanoma metastasis are significantly less than the average number (>300) and size (1.02mm in diameter) in control ConA-T-immunized HLA-A2 mice. Taken together, HIV-1 Gag-Texo vaccine capable of stimulating Gag-specific CTL responses and therapeutic immunity may be useful as a new immunotherapeutic vaccine for viral control in HIV-1 patients.

Epitope targeting and viral inoculum are determinants of Nef-mediated immune evasion of HIV-1 from cytotoxic T lymphocytes

AbstractThe 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.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

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.

Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China

It is generally believed that CD8(+) cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman's rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.

CTL responses of high functional avidity and broad variant cross-reactivity are associated with HIV control.

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication in vivo. However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct ex-vivo samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control.

Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses during primary simian immunodeficiency virus infection in Burmese rhesus macaques

Major histocompatibility complex class I (MHC-I)-restricted CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. In particular, Gag-specific CTL responses have been shown to exert strong suppressive pressure on HIV/SIV replication. Additionally, association of Vif-specific CTL frequencies with in vitro anti-SIV efficacy has been suggested recently. Host MHC-I genotypes could affect the immunodominance patterns of these potent CTL responses. Here, Gag- and Vif-specific CTL responses during primary SIVmac239 infection were examined in three groups of Burmese rhesus macaques, each group having a different MHC-I haplotype. The first group of four macaques, which possessed the MHC-I haplotype 90-010-Ie, did not show Gag- or Vif-specific CTL responses. However, Nef-specific CTL responses were elicited, suggesting that primary SIV infection does not induce predominant CTL responses specific for Gag/Vif epitopes restricted by 90-010-Ie-derived MHC-I molecules. In contrast, Gag- and Vif-specific CTL responses were induced in the second group of two 89-075-Iw-positive animals and the third group of two 91-010-Is-positive animals. Considering the potential of prophylactic vaccination to affect CTL immunodominance post-viral exposure, these groups of macaques would be useful for evaluation of vaccine antigen-specific CTL efficacy against SIV infection.

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.

Amino-Acid Co-Variation in HIV-1 Gag Subtype C: HLA-Mediated Selection Pressure and Compensatory Dynamics

BackgroundDespite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C.Methodology/Principal FindingsIndividuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites.Conclusions/SignificanceClusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints.

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

Gag-Specific Cytotoxic T-Lymphocyte-Based Control of Primary Simian Immunodeficiency Virus Replication in a Vaccine Trial

Gag-specific cytotoxic T lymphocytes (CTLs) exert strong suppressive pressure on human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. However, it has remained unclear whether they can actually contain primary viral replication. Recent trials of prophylactic vaccines inducing virus-specific T-cell responses have indicated their potential to confer resistance against primary SIV replication in rhesus macaques, while the immunological determinant for this vaccine-based viral control has not been elucidated thus far. Here we present evidence implicating Gag-specific CTLs as responsible for the vaccine-based primary SIV control. Prophylactic vaccination using a Gag-expressing Sendai virus vector resulted in containment of SIVmac239 challenge in all rhesus macaques possessing the major histocompatibility complex (MHC) haplotype 90-120-Ia. In contrast, 90-120-Ia-positive vaccinees failed to contain SIVs carrying multiple gag CTL escape mutations that had been selected, at the cost of viral fitness, in SIVmac239-infected 90-120-Ia-positive macaques. These results show that Gag-specific CTL responses do play a crucial role in the control of wild-type SIVmac239 replication in vaccinees. This study implies the possibility of Gag-specific CTL-based primary HIV containment by prophylactic vaccination, although it also suggests that CTL-based AIDS vaccine efficacy may be abrogated in viral transmission between MHC-matched individuals.

Relative dominance of Env-gp41-specific cytotoxic T lymphocytes responses in HIV-1 advanced infection.

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) responses provide an important defense in controlling HIV-1 replication, but they fail to control the progression of AIDS in advanced HIV-1 infection. To uncover the situation of these responses in patients with advanced HIV-1 infection, we assessed HIV-1-specific CTL responses in 20 individuals with advanced HIV-1 infection using 407 overlapping peptides spanning all expressed HIV-1 proteins using a gamma interferon-enzyme-linked immunospot (ELISpot) assay. In comparison to 20 individuals with moderately advanced HIV-1 infection, HIV-1-specific CTL responses were significantly decreased (P=0.044) and less peptides could be recognized (P=0.05) in advanced HIV-1 infection. Weakening of Env-gp120 and Gag-specific CTL responses contributed importantly to the decrease of CTL magnitude (P=0.042 and 0.078, respectively), while Env-gp41-specific CTL responses were relatively stronger during the end-stage of HIV-1 infection (P<0.001). Nef and Env-gp41 represented the most frequently targeted HIV-1 proteins in advanced HIV-1 infection. The entropy scores of peptides targeted in two groups were not significantly different. Only the breadth and magnitude of Env-gp41-specific CTL responses were positively correlated with viral loads in advanced HIV-1 infection (P=0.005 and 0.001, respectively). These findings suggest that progressive HIV-1 infection is associated with a weakening of Env-gp120- and Gag-specific CTL responses, and a simultaneous expansion of Env-gp41-specific CTL which is likely driven by high level viral replication.

Transmission of HIV-1 Gag immune escape mutations is associated with reduced viral load in linked recipients

In a study of 114 epidemiologically linked Zambian transmission pairs, we evaluated the impact of human leukocyte antigen class I (HLA-I)–associated amino acid polymorphisms, presumed to reflect cytotoxic T lymphocyte (CTL) escape in Gag and Nef of the virus transmitted from the chronically infected donor, on the plasma viral load (VL) in matched recipients 6 mo after infection. CTL escape mutations in Gag and Nef were seen in the donors, which were subsequently transmitted to recipients, largely unchanged soon after infection. We observed a significant correlation between the number of Gag escape mutations targeted by specific HLA-B allele–restricted CTLs and reduced VLs in the recipients. This negative correlation was most evident in newly infected individuals, whose HLA alleles were unable to effectively target Gag and select for CTL escape mutations in this gene. Nef mutations in the donor had no impact on VL in the recipient. Thus, broad Gag-specific CTL responses capable of driving virus escape in the donor may be of clinical benefit to both the donor and recipient. In addition to their direct implications for HIV-1 vaccine design, these data suggest that CTL-induced viral polymorphisms and their associated in vivo viral fitness costs could have a significant impact on HIV-1 pathogenesis.

Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination

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-1 gag DNA 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 (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed 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 the gag DNA 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.

Abrogation of AIDS vaccine-induced cytotoxic T-lymphocyte efficacy in vivo due to a change in viral epitope flanking sequences

A current promising AIDS vaccine strategy is to elicit CD8 + cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag 206–216 epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag 206–216-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag 206–216 epitope sequence with SIVmac239. Despite efficient Gag 206–216-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag 206–216 epitope. Further analysis indicated that Gag 206–216-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag 206–216 epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

Long-Term Control of Simian Immunodeficiency Virus Replication with Central Memory CD4 + T-Cell Preservation after Nonsterile Protection by a Cytotoxic T-Lymphocyte-Based Vaccine

Induction of virus-specific CD8(+) cytotoxic T-lymphocyte (CTL) responses is a promising strategy for AIDS vaccine development. However, it has remained unclear if or how long-term viral containment and disease control are attainable by CTL-based nonsterile protection. Here, we present three rhesus macaques that successfully maintained Env-independent vaccine-based control of simian immunodeficiency virus (SIV) mac239 replication without disease progression for more than 3 years. SIV-specific neutralizing antibody induction was inefficient in these controllers. Vaccine-induced Gag-specific CTLs were crucial for the chronic as well as the primary viral control in one of them, whereas those Gag-specific CTL responses became undetectable and CTLs specific for SIV antigens other than Gag, instead, became predominant in the chronic phase in the other two controllers. A transient CD8(+) cell depletion experiment 3 years postinfection resulted in transient reappearance of plasma viremia in these two animals, suggesting involvement of the SIV non-Gag-specific CTLs in the chronic SIV control. This sustained, neutralizing antibody-independent viral control was accompanied with preservation of central memory CD4(+) T cells in the chronic phase. Our results suggest that prophylactic CTL vaccine-based nonsterile protection can result in long-term viral containment by adapted CTL responses for AIDS prevention.

Identification of a cytotoxic T-lymphocyte (CTL) epitope recognized by Gag-specific CTLs in cynomolgus monkeys infected with simian/human immunodeficiency virus

Infection of Macaca fascicularis (cynomolgus monkey) with chimeric simian/human immunodeficiency virus (SHIV) provides a valuable experimental animal model of AIDS and is widely used for the development of human immunodeficiency virus vaccine strategies. In these settings, analysis of CD8(+) T-cell responses during infection represents one of the key parameters for monitoring the evaluation of containment of virus replication. The generation of Gag-specific CD8(+) T cells was reported previously from a cynomolgus monkey infected with SHIV89.6P by taking advantage of a B-lymphoblastoid cell line transduced with a retroviral vector expressing simian immunodeficiency virus (SIV) Gag. Here, it was shown that these cytotoxic T lymphocytes (CTLs) demonstrated specificity for a single 9 aa peptide (NCVGDHQAA) spanning aa 192-200 of the SIVmac239 p55(gag) protein. Furthermore, a positive response was found against the same epitope in one of six other SHIV-infected monkeys. This newly identified SIV Gag CTL epitope in SHIV-infected cynomolgus monkeys will be a useful tool for monitoring and evaluating Gag-specific immune responses during vaccination and infection in the cynomolgus monkey model of AIDS.

Vaginal Protection and Immunity after Oral Immunization of Mice with a Novel Vaccine Strain ofListeria monocytogenesExpressing Human Immunodeficiency Virus Type 1gag

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.