Pol Epitopes Research Articles

Immunological Control of HIV-1 Disease Progression by Rare Protective HLA Allele.

Rare HLA alleles such as HLA-B57 are associated with slow progression to AIDS. However, the evidence for the advantage of rare protective alleles is limited, and the mechanism is still unclear. Although the prevalence of HLA-B*67:01 is only 1.2% in Japan, HLA-B*67:01-positive (HLA-B*67:01+) individuals had the lowest plasma viral load (pVL) and highest CD4 count in HIV-1 clade B-infected Japanese individuals. We investigated the mechanism of immunological control of HIV-1 by a rare protective allele, HLA-B*67:01. We identified six novel HLA-B*67:01-restricted epitopes and found that T cells specific for four epitopes were significantly associated with good clinical outcomes, pVL and/or CD4 count. The wild type or cross-reactive sequences of three protective and immunodominant Pol and Gag epitopes were found in around 95% of the circulating HIV-1, indicating that T cells specific for three conserved or cross-reactive epitopes contributed to good clinical outcomes. One escape mutation (Nef71K) in the Nef protective epitope, which was selected by T cells restricted by either HLA allele in the HLA-B*67:01-C*07:02 haplotype, affected the HLA-B*67:01-restricted RY11-specific T-cell recognition. These results imply that the further accumulation of the Nef71K mutation in the population will negatively affect the control of HIV-1 replication by RY11-specific CD8+ T cells in HIV-1-infected HLA-B*67:01+ individuals. The present study demonstrated that conserved or cross-reactive epitope-specific T cells mainly contribute to control of HIV-1 by a rare protective allele, HLA-B*67:01. IMPORTANCE HLA-B57 is a relatively rare allele around world and the strongest protective HLA allele in Caucasians and African black individuals infected with HIV-1. Previous studies suggested that the advantage of this allele in HIV-1 disease progression is due to a strong functional ability of HLA-B57-restricted Gag-specific T cells and lower fitness of mutant viruses selected by the T cells. HLA-B*57 is a very rare allele and has not been reported as a protective allele in Asian countries, whereas a rare allele, HLA-B*67:01, was shown to be a protective allele in Japan. Therefore, the analysis of HLA-B*67:01-restricted T cells is important to clarify the mechanism of immunological control of HIV-1 by a rare protective HLA allele in Asia. We found that HLA-B*67:01-restricted T cells specific for three conserved or cross-reactive Gag and Pol epitopes are associated with good clinical outcomes in HLA-B*67:01+ individuals. It is expected that T cells specific for conserved or cross-reactive epitopes contribute to a curing treatment.

Control of HIV-1 Replication by CD8+ T Cells Specific for Two Novel Pol Protective Epitopes in HIV-1 Subtype A/E Infection.

ABSTRACTAlthough many HIV-1-specific CD8+ T cell epitopes have been identified and used in various HIV-1 studies, most of these epitopes were derived from HIV-1 subtypes B and C. Only 17 well-defined epitopes, none of which were protective, have been identified for subtype A/E infection. The roles of HIV-1-specific T cells have been rarely analyzed for subtype A/E infection. In this study, we identified six novel HLA-B*15:02-restricted optimal HIV-1 subtype A/E epitopes and then analyzed the presentation of these epitopes by HIV-1 subtype A/E virus-infected cells and the T cell responses to these epitopes in treatment-naive HIV-1 subtype A/E-infected HLA-B*15:02+ Vietnamese individuals. Responders to the PolTY9 or PolLF10 epitope had a significantly lower plasma viral load (pVL) than nonresponders among HLA-B*15:02+ individuals, whereas no significant difference in pVL was found between responders to four other epitopes and nonresponders. The breadth of T cell responses to these two Pol epitopes correlated inversely with pVL. These findings suggest that HLA-B*15:02-restricted T cells specific for PolTY9 and PolLF10 contribute to the suppression of HIV-1 replication in HLA-B*15:02+ individuals. The HLA-B*15:02-associated mutation Pol266I reduced the recognition of PolTY9-specific T cells in vitro but did not affect HIV-1 replication by PolTY9-specific T cells in Pol266I mutant virus-infected individuals. These findings indicate that PolTY9-specific T cells suppress replication of the Pol266I mutant virus even though the T cells selected this mutant. This study demonstrates the effective role of T cells specific for these Pol epitopes to control circulating viruses in HIV-1 subtype A/E infection.IMPORTANCE It is expected that HIV-1-specific CD8+ T cells that effectively suppress HIV-1 replication will contribute to HIV-1 vaccine development and therapy to achieve an HIV cure. T cells specific for protective epitopes were identified in HIV-1 subtype B and C infections but not in subtype A/E infection, which is epidemic in Southeast Asia. In the present study, we identified six T cell epitopes derived from the subtype A/E virus and demonstrated that T cells specific for two Pol epitopes effectively suppressed HIV-1 replication in treatment-naive Vietnamese individuals infected with HIV-1 subtype A/E. One of these Pol protective epitopes was conserved among circulating viruses, and one escape mutation was accumulated in the other epitope. This mutation did not critically affect HIV-1 control by specific T cells in HIV-1 subtype A/E-infected individuals. This study identified two protective Pol epitopes and characterized them in cases of HIV-1 subtype A/E infection.

A longitudinal analysis of immune escapes from HLA-B*13-restricted T-cell responses at early stage of CRF01_AE subtype HIV-1 infection and implications for vaccine design

BackgroundIdentifying immunogens which can elicit effective T cell responses against human immunodeficiency virus type 1 (HIV-1) is important for developing a T-cell based vaccine. It has been reported that human leukocyte antigen (HLA)-B*13-restricted T-cell responses contributed to HIV control in subtype B′ and C infected individuals. However, the kinetics of B*13-restricted T-cell responses, viral evolution within epitopes, and the impact on disease progression in CRF01_AE subtype HIV-1-infected men who have sex with men (MSM) are not known.ResultsInterferon-γ ELISPOT assays and deep sequencing of viral RNAs were done in 14 early HLA-B*13-positive CRF01_AE subtype HIV-1-infected MSM. We found that responses to RQEILDLWV (Nef106–114, RV9), GQMREPRGSDI (Gag226–236, GI11), GQDQWTYQI (Pol487–498, GI9), and VQNAQGQMV (Gag135–143, VV9) were dominant. A higher relative magnitude of Gag-specific T-cell responses, contributed to viral control, whereas Nef-specific T-cell responses were associated with rapid disease progression. GI11 (Gag) was conserved and strong GI11 (Gag)-specific T-cell responses showed cross-reactivity with a dominant variant, M228I, found in 3/12 patients; GI11 (Gag)-specific T-cell responses were positively associated with CD4 T-cell counts (R = 0.716, P = 0.046). Interestingly, the GI9 (Pol) epitope was also conserved, but GI9 (Pol)-specific T-cell responses did not influence disease progression (P > 0.05), while a D490G variant identified in one patient did not affect CD4 T-cell counts. All the other epitopes studied [VV9 (Gag), RQYDQILIEI (Pol113–122, RI10), HQSLSPRTL (Gag144–152, HL9), and RQANFLGRL (Gag429–437, RL9)] developed escape mutations within 1 year of infection, which may have contributed to overall disease progression. Intriguingly, we found early RV9 (Nef)-specific T-cell responses were associated with rapid disease progression, likely due to escape mutations.ConclusionsOur study strongly suggested the inclusion of GI11 (Gag) and exclusion of RV9 (Nef) for T-cell-based vaccine design for B*13-positive CRF01_AE subtype HIV-1-infected MSM and high-risk individuals.

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Effective Suppression of HIV-1 Replication by Cytotoxic T Lymphocytes Specific for Pol Epitopes in Conserved Mosaic Vaccine Immunogens.

Cytotoxic T lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize circulating HIV-1 could be key for both HIV-1 cure and prophylaxis. We recently designed conserved mosaic T-cell vaccine immunogens (tHIVconsvX) composed of 6 Gag and Pol regions. Since the tHIVconsvX vaccine targets conserved regions common to most global HIV-1 variants and employs a bivalent mosaic design, it is expected that it could be universal if the vaccine works. Although we recently demonstrated that CTLs specific for 5 Gag epitopes in the vaccine immunogens had strong ability to suppress HIV-1 replication in vitro and in vivo, it remains unknown whether the Pol region-specific CTLs are equally efficient. In this study, we investigated CTLs specific for Pol epitopes in the immunogens in treatment-naive Japanese patients infected with HIV-1 clade B. Overall, we mapped 20 reported and 5 novel Pol conserved epitopes in tHIVconsvX. Responses to 6 Pol epitopes were significantly associated with good clinical outcome, suggesting that CTLs specific for these 6 Pol epitopes had a strong ability to suppress HIV-1 replication in HIV-1-infected individuals. In vitro T-cell analyses further confirmed that the Pol-specific CTLs could effectively suppress HIV-1 replication. The present study thus demonstrated that the Pol regions of the vaccine contained protective epitopes. T-cell responses to the previous 5 Gag and present 6 Pol protective epitopes together also showed a strong correlation with better clinical outcome. These findings support the testing of the conserved mosaic vaccine in HIV-1 cure and prevention in humans.IMPORTANCE It is likely necessary for an effective AIDS vaccine to elicit CD8+ T cells with the ability to recognize circulating HIV-1 and suppress its replication. We recently developed novel bivalent mosaic T-cell vaccine immunogens composed of conserved regions of the Gag and Pol proteins matched to at least 80% globally circulating HIV-1 isolates. Nevertheless, it remains to be proven if vaccination with these immunogens can elicit T cells with the ability to suppress HIV-1 replication. It is well known that Gag-specific T cells can suppress HIV-1 replication more effectively than T cells specific for epitopes in other proteins. We recently identified 5 protective Gag epitopes in the vaccine immunogens. In this study, we identified T cells specific for 6 Pol epitopes present in the immunogens with strong abilities to suppress HIV-1 in vivo and in vitro This study further encourages clinical testing of the conserved mosaic T-cell vaccine in HIV-1 prevention and cure.

Pol-Driven Replicative Capacity Impacts Disease Progression in HIV-1 Subtype C Infection.

CD8+ T cell-mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a green fluorescent protein (GFP) reporter T cell assay. In recently infected individuals, reverse transcriptase (RT)-integrase-driven RC correlated significantly with viral load set point (r = 0.25; P = 0.03) and CD4+ T cell decline (P = 0.013). Moreover, significant associations between RT integrase-driven RC and viral load (r = 0.28; P < 0.0001) and CD4+ T cell count (r = -0.29; P < 0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (P = 0.05), as was expression of HLA-B*07 (P = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (P = 0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K, and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally described HLA-restricted cytotoxic T-lymphocyte epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC.IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein, but the impact of Pol function on HIV disease progression is not well defined. Here, we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C-infected individuals with recent and chronic infection and measured their ability to replicate in vitro We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.

Similar Epitope Specificities of IgG and IgA Antibodies Elicited by Ad26 Vector Prime, Env Protein Boost Immunizations in Rhesus Monkeys.

ABSTRACTVaccine-elicited immunoglobulin G (IgG) has been shown to be important for protection against simian-human immunodeficiency virus (SHIV) infection in rhesus monkeys. However, it remains unclear whether vaccine-elicited IgA responses are beneficial or detrimental for protection. In this study, we evaluated the kinetics, magnitude, breadth, and linear epitope specificities of vaccine-elicited IgG and IgA responses in serum and mucosal secretions following intramuscular immunization with adenovirus 26 (Ad26) prime, Env protein boost vaccination regimens. The systemic and mucosal antibody responses exhibited kinetics similar to those of the serum antibody responses but lower titers than the serum antibody responses. Moreover, the IgG and IgA responses were correlated, both in terms of the magnitude of the responses and in terms of the antibody specificities against linear human immunodeficiency virus type 1 (HIV-1) Env, Gag, and Pol epitopes. These data suggest that IgG and IgA responses are highly coordinated in both peripheral blood and mucosal compartments following Ad26/Env vaccination in rhesus monkeys.IMPORTANCE Vaccine-elicited IgG responses are important for protection against simian-human immunodeficiency virus (SHIV) infection in nonhuman primates. However, much less is known about the role and function of IgA, despite it being the predominant antibody in mucosal sites. There is debate as to whether HIV-1-specific IgA responses are beneficial or detrimental, since serum anti-Env IgA titers were shown to be inversely correlated with protection in the RV144 clinical trial. We thus assessed vaccine-elicited IgG and IgA antibody responses in peripheral blood and mucosal secretions following vaccination with the Ad26/Env vaccine.

Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

ObjectiveThe specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. DesignCD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. MethodsFrozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. ResultsWe formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. ConclusionsThese results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.

Vaccines for Therapeutic Cellular Immunity that Target HIV Gag, Pol and Nef Epitopes to CD40 and DCIR Elicit T-cell Responses in Rhesus Macaques

AIDS Research and Human RetrovirusesVol. 30, No. S1 Novel Vaccine and Prevention ConceptsVaccines for Therapeutic Cellular Immunity that Target HIV Gag, Pol and Nef Epitopes to CD40 and DCIR Elicit T-cell Responses in Rhesus MacaquesYves Lévy, Sandra Zurawski, Anne-Laure Flamar, Christine Lacabaratz, Cécile Peltekian, Andres Salazar, Rodolphe Thiébaut, and Gérard ZurawskiYves LévyVaccine Research Institute (VRI), Créteil, FranceUniversité Paris-Est Créteil Val de Marne (UPEC), Inserm U 955, Creteil, FranceAssistance Publique-Hôpitaux de Paris, Groupe Henri Mondor-Albert Chenevier, Service d'Immunologie Clinique, Créteil, FranceSearch for more papers by this author, Sandra ZurawskiVaccine Research Institute (VRI), Créteil, FranceBaylor Institute for Immunology Research, Inserm U 955, Dallas, TX, United StatesSearch for more papers by this author, Anne-Laure FlamarVaccine Research Institute (VRI), Créteil, FranceBaylor Institute for Immunology Research, Inserm U 955, Dallas, TX, United StatesSearch for more papers by this author, Christine LacabaratzVaccine Research Institute (VRI), Créteil, FranceUniversité Paris-Est Créteil Val de Marne (UPEC), Inserm U 955, Creteil, FranceSearch for more papers by this author, Cécile PeltekianVaccine Research Institute (VRI), Créteil, FranceUniversité Paris-Est Créteil Val de Marne (UPEC), Inserm U 955, Creteil, FranceSearch for more papers by this author, Andres SalazarOncovir, Washington DC, WA, United StatesSearch for more papers by this author, Rodolphe ThiébautVaccine Research Institute (VRI), Créteil, FranceINSERM U 897 Université Victor Segalen Bordeaux 2, Bordeaux, FranceSearch for more papers by this author, and Gérard ZurawskiVaccine Research Institute (VRI), Créteil, FranceBaylor Institute for Immunology Research, Inserm U 955, Dallas, TX, United StatesSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5544.abstractAboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Vaccines for Therapeutic Cellular Immunity that Target HIV Gag, Pol and Nef Epitopes to CD40 and DCIR Elicit T-cell Responses in Rhesus Macaques." AIDS Research and Human Retroviruses, 30(S1), p. A245FiguresReferencesRelatedDetailsCited byDevelopment of an epitope-based HIV-1 vaccine strategy from HIV-1 lipopeptide to dendritic-based vaccines14 September 2017 | Expert Review of Vaccines, Vol. 16, No. 10 Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Yves Lévy, Sandra Zurawski, Anne-Laure Flamar, Christine Lacabaratz, Cécile Peltekian, Andres Salazar, Rodolphe Thiébaut, and Gérard Zurawski.Vaccines for Therapeutic Cellular Immunity that Target HIV Gag, Pol and Nef Epitopes to CD40 and DCIR Elicit T-cell Responses in Rhesus Macaques.AIDS Research and Human Retroviruses.Oct 2014.A245-A245.http://doi.org/10.1089/aid.2014.5544.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Toward an effective AIDS vaccine development

Toward an effective AIDS vaccine development

Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.

TLR agonists and/or IL-15 adjuvanted mucosal SIV vaccine reduced gut CD4+ memory T cell loss in SIVmac251-challenged rhesus macaques

Adjuvant plays an important role in increasing and directing vaccine-induced immune responses. In a previous study, we found that a mucosal SIV vaccine using a combination of IL-15 and TLR agonists as adjuvant mediated partial protection against SIVmac251 rectal challenge, whereas neither IL-15 nor TLR agonists alone as an adjuvant impacted the plasma viral loads. In this study, dissociation of CD4(+) T cell preservation with viral loads was observed in the animals vaccinated with adjuvants. Significantly higher levels of memory CD4(+) T cell numbers were preserved after SIVmac251 infection in the colons of the animals vaccinated with vaccine containing any of these adjuvants compared to no adjuvant. When we measured the viral-specific CD8(+) tetramer responses in the colon lamina propria, we found significantly higher levels of gag, tat, and pol epitope tetramer(+) T cell responses in these animals compared to ones without adjuvant, even if some of the animals had similarly high viral loads. Furthermore, this CD4(+) T preservation was positively correlated with increased levels of gag and Tat, but not pol tetramer(+) T cell responses, and inversely correlated with beta-chemokine expression. The pre-challenged APOBEC3G expression level, which has previously been shown inversely associated with viral loads, was further found positively correlated with CD4(+) T cell number preservation. Overall, these data highlight one unrecognized role of adjuvant in HIV vaccine development, and show that vaccines can produce a surprising discordance between CD4(+) T cell levels and SIV viral load.

Effective recognition of HIV-1-infected cells by HIV-1 integrase-specific HLA-B∗4002-restricted T cells

HLA-B∗4002 is one of the common HLA-B alleles in the world. All 7 reported HLA-B∗4002-restricted HIV epitopes are derived from Gag, Nef, and Vpr. In the present study we sought to identify novel HLA-B∗4002-restricted HIV epitopes by using overlapping 11-mer peptides of HIV-1 Nef, Gag, and Pol, and found that 6 of these 11-mer Pol peptides included HLA-B∗4002-restricted epitopes. Analysis using truncated peptides of these 6 peptides defined 4 optimal Pol (integrase) epitopes. All epitopes previously reported had Glu at position 2 (P2), suggesting that Glu at P2 is the anchor residue for HLA-B∗4002; whereas only 2 of the integrase epitopes that we here identified had Glu at P2. CTL clones specific for the 2 epitopes effectively recognized HIV-1-infected cells whereas those for other 2 epitopes only weakly recognized them. The antigen sensitivity of the former clones for the epitope peptide was much higher than that of the latter clones, suggesting 2 possibilities: 1) the former T cells have high-affinity TCRs and/or 2) the epitope peptides recognized by the former T cells are highly presented by HLA-B∗4002 in HIV-1-infected cells. These integrase-specific T cells with high antigen sensitivity may contribute to the suppression of HIV-1 replication in HIV-1-infected HLA-B∗4002+ individuals.

Efficacious early antiviral activity of HIV Gag- and Pol-specific HLA-B 2705-restricted CD8+ T cells.

The association between HLA-B 2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B 2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8(+) T cells. In order to better define the mechanisms of the HLA-B 2705 immune control of HIV, we first characterized the CD8(+) T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B 2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B 2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8(+) T-cell response. By comparing inhibitions of viral replication by CD8(+) T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B 2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8(+) T cells but not until 18 h postinfection by VL9-specific CD8(+) T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B 2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B 2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8(+) T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.

P18-11. DALIA: dendritic cell and lipopeptide-induced immunity against AIDS: a phase I trial

Methods In our view, three parameters are critical for the generation of potent ex vivo-generated DC vaccines for HIV patients: 1) antigen; 2) DC type; and 3) activation signal. With this in mind, we designed a clinical trial (IRB#008017; BB-IND #13748) to test the safety and immune efficacy of a therapeutic HIV vaccine consisting of autologous DCs generated ex vivo from monocytes cultured with GMCSF/IFN-alpha and loaded with five lipidated HIV antigens (LIPO5). Our trial exploits a combination of: 1) HIVderived lipopeptides that cover nef, gag and pol epitopes – binding to >90% of HLA molecules and permitting presentation of T cell epitopes and generation of humoral immunity; 2) Interferon (IFN)-DCs, which demonstrate powerful priming functions in vitro; and 3) LPS activation, which enhances priming by IFN-DCs. Such a vaccine is expected to induce strong and diverse HIV-specific immune responses. Results This phase I clinical trial in 19 asymptomatic HIV-infected patients with undetectable viral load while treated with HAART will test the safety of DC vaccination and of analytical treatment interruption (ATI). Patients receive four monthly DC vaccinations in conjunction with antiretroviral therapy. Twelve weeks after the fourth vaccine, patients who meet the pre-specified criteria will stop HAART. ATI will last for up to six months.

P16-04. Cryptic CTL epitopes derived from antisense transcription are frequently recognized in HIV-1 infection

Methods Using MHC-I associated HIV-1 polymorphisms in gag, pol and nef sequences from 730 South Africans with chronic infection; over 957 potential ARF epitopes were predicted. Of these, 25 potential cryptic epitopes (CE) were selected for testing using the standard overnight IFNγ ELISpot assay in PBMC from 61 HIV clade B infected US patients and 14 seronegative controls. In a subset of HIV infected patients: chronic (N = 6) and acute (N = 9), the polyfunctionality of the CE responses was measured using an ICS assay. CTL clones were derived for three antisense cryptic pol epitopes by limiting dilution cloning. The CD8 clones were characterized using 51Cr release and ICS assay.

Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

BackgroundStudies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used.ResultsUsing a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms). The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms.ConclusionWe have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape.

Adenovirus DNA polymerase is recognized by human CD8+ T cells

Donor lymphocytes have potential as a treatment for adenovirus (Ad) disease in haematopoietic stem cell transplant (SCT) recipients, but better understanding of Ad-specific T-cell responses is required. Most healthy adults exhibit memory T-cell responses to hexon, a capsid protein synthesized late after infection. However, since the Ad E3-19k downregulates major histocompatibility complex (MHC) class I molecules, cytotoxic T cells (CTLs) targeted to early viral proteins may be more effective in eliminating Ad-infected cells in vivo. Here we show that Ad-specific CTLs recognize the early region 2 proteins DNA polymerase (Pol) and DNA-binding protein (DBP). Firstly, memory Ad-specific CD8(+) T cells were amplified from healthy donors by in vitro stimulation with Ad-infected dendritic cells and found to exhibit MHC-restricted cytotoxicity to targets expressing Pol and DBP. Secondly, gamma interferon responses to HLA A2-binding motif peptides from Pol and DBP were directly detected in peripheral blood mononuclear cells (PBMCs) from a recently infected normal donor. Peptide-specific CTLs generated to Pol and DBP epitopes were confirmed to exhibit HLA A2-restricted killing of targets expressing Pol or DBP. Lastly, Pol-epitope-specific T cells were detected at similar or higher frequencies than hexon and DBP in three of three SCT recipients recovering from invasive Ad disease. Pol epitopes were well conserved among different Ad serotypes. Therefore, Pol is a promising target for immunotherapy of Ad disease.

Marked Epitope- and Allele-Specific Differences in Rates of Mutation in Human Immunodeficiency Type 1 (HIV-1) Gag, Pol, and Nef Cytotoxic T-Lymphocyte Epitopes in Acute/Early HIV-1 Infection

During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.

Altering Effects of Antigenic Variations in HIV-1 on Antiviral Effectiveness of HIV-Specific CTLs

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.