Cross-Clade-Specific Cytotoxic T Lymphocytes in HIV-1-Infected Children

We studied cytotoxic T lymphocyte (CTL) cross-reactivity between human immunodeficiency virus type 1 (HIV-1) subtypes within a group of infants infected with either HIV-1 B or non-B clade. Fifteen children were infected with a clade B virus. Nine were infected with non-B virus, including two clade A, four clade D, two clade F, and one clade G. CTL activities fromin vitroactivated peripheral blood mononuclear cells were tested against autologous cell line infected with recombinant vaccinia viruses encoding for Env, Gag, Pol, or Nef proteins from a clade A or B isolate. HIV-1-specific CTL elicited from infection with clade B virus could lyse targets expressing clade A proteins, and vice versa. In infants with positive CTL responses, cross-clade recognition was predominant and was detected within 88% of the Pol, 83% of the Nef, 67% of the Gag, and 55% of the Env responders. Longitudinal studies showed that CTL cross-reactivity to both B and A targets was stable for several years. Elicitation of CTL reactivities capable of elimination of virus-infected cells is an important goal for the development of an efficient AIDS vaccine. The significant cross-reactivity of CTL shown in this study supports the concept that vaccines developed using a single-clade immunogen may be applicable to induce broadly reactive T cell responses.     

HIV-1 Gag、Tat、Rev和Nef蛋白特异性的免疫应答

目的：探讨中国HIV／AIDS患者HIV—1 Gag、Tat、Rev和Nef蛋白特异性CTL应答的特征。方法：应用覆盖HIV-1 B、C亚型Gag、Tat、Rev和Nef蛋白的220个肽段作为抗原，通过ELISPOT方法俭测HIV／AIDS患者HIV特异性CTL应答。结果：无沦HIV—1 B亚型还是HIV-1C亚型所构建肽库的应答强度和频率，主要集中在Gag和Nef蛋白，Tat和Rev蛋白也有不同程度的应答。HIV—1 B、C亚型间应答比较，整体应答强度大致相同，但免疫优势区间存在着一定的差异，B亚型Gagp24亚蛋白的288～313氨基酸区应答最强，而C亚型Gagp24亚蛋白的155～181氨基酸区应答最强；两个亚型免疫优势区应答频率最高的都是Nef蛋白106～143氨基酸区(48．1％)。结论：中国人群CTL应答多集中在Gag和Nef蛋白，B、C业型间略有差异且存在交叉识别，这对设计针对中国人群的HIV疫苗是有重要的意义。     

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-γ 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.     

A Simple Method to Selectively Expand HIV-1 Specific Cytotoxic T Lymphocytes in Vitro

Cytotoxic T lymphocytes (CTL) may play a critical role in controlling the progression of HIV-1 disease. Conventional assays for demonstration of CTL against HIV-1 have used either fresh PBMC or T cell lines and clones generated by non-specific stimulation. These methods are limited in their sensitivity since without specific secondary stimulation in vitro, epitopes recognized at low frequency may not be detected. Moreover, derivation of CTL clones is labor intensive and not practical for studying a large number of patients. We have developed a simple method to enrich HIV-1 specific CTL in vitro. Autologous antigen presenting cells (APC), either adherent macrophages or EBV transformed B-lymphoblastoid cells, are infected with recombinant vaccinia virus encoding individual HIV-1 proteins and after overnight culture the vaccinia virus is inactivated by uv irradiation in the presence of psoralin. The infected APC are then cultured with patient's T cells and CTL activity determined 10–14 days later. We have used this method to stimulate patients' T cells obtained directly from PBMC and also after mitogenic stimulation. In both systems, the HIV-1 specific response could be enhanced up to five to ten fold. This enhancement is comparable to CTL selection by exposure to HIV-1 immunodominant peptide incubated APC. In some patients, viral-specific CTL could be detected after HIV-vaccinia selection even though the mitogen stimulated cultures had no demonstrable antiviral CTL activity. Selective expansion of CTL directed against multiple HIV-1 proteins (env, gag and RT) could be obtained from PBMC as well as from mitogen-stimulated lines from individual patients. As these lines are predominantly CD8+ T cells by flow cytometric analysis and are free of vaccinia virus as ascertained by the lack of cytopathic effect in culture, in vitro vaccinia selection might also be useful to generate CTL lines for adoptive immunotherapy.     

A Simple Method to Selectively Expand HIV-1 Specific Cytotoxic T Lymphocytes in Vitro

Cytotoxic T lymphocytes (CTL) may play a critical role in controlling the progression of HIV-1 disease. Conventional assays for demonstration of CTL against HIV-1 have used either fresh PBMC or T cell lines and clones generated by non-specific stimulation. These methods are limited in their sensitivity since without specific secondary stimulation in vitro, epitopes recognized at low frequency may not be detected. Moreover, derivation of CTL clones is labor intensive and not practical for studying a large number of patients. We have developed a simple method to enrich HIV-1 specific CTL in vitro. Autologous antigen presenting cells (APC), either adherent macrophages or EBV transformed B-lymphoblastoid cells, are infected with recombinant vaccinia virus encoding individual HIV-1 proteins and after overnight culture the vaccinia virus is inactivated by uv irradiation in the presence of psoralin. The infected APC are then cultured with patient's T cells and CTL activity determined 10-14 days later. We have used this method to stimulate patients' T cells obtained directly from PBMC and also after mitogenic stimulation. In both systems, the HIV-1 specific response could be enhanced up to five to ten fold. This enhancement is comparable to CTL selection by exposure to HIV-1 immunodominant peptide incubated APC. In some patients, viral-specific CTL could be detected after HIV-vaccinia selection even though the mitogen stimulated cultures had no demonstrable antiviral CTL activity. Selective expansion of CTL directed against multiple HIV-1 proteins (env, gag and RT) could be obtained from PBMC as well as from mitogen-stimulated lines from individual patients. As these lines are predominantly CD8+ T cells by flow cytometric analysis and are free of vaccinia virus as ascertained by the lack of cytopathic effect in culture, in vitro vaccinia selection might also be useful to generate CTL lines for adoptive immunotherapy.     

Inhibition of HIV-1-specific T-cells and increase of viral load during immunosuppressive treatment in an HIV-1 infected patient with Chlamydia trachomatis induced arthritis.

BACKGROUND: Studies in HIV-1 infected long-term non-progressors could demonstrate a strong HIV-1-specific CTL response, but it is difficult to prove that this strong CTL response actually is the cause of the efficient control of HIV-1 and not the consequence of low HIV-1 replication in these patients. OBJECTIVE: Studies of HIV-1-specific immunity and viral replication in patients undergoing immunosuppressive therapy provide important opportunities to understand the role of HIV-1-specific T-cells. RESULTS: In this report we describe an HLA B27 positive patient with normal CD4 counts and a low viral load of 200 copies/ml without antiretroviral therapy who exhibited a very strong HIV-1-specific CTL response. He had to be treated with steroids, NSAIDS and hydroxchloroquine because of a severe inflammatory reactive arthritis triggered by an acute Chlamydia trachomatis infection. Analysis of HIV-1 specific T-cells by gamma-IFN-ELISPOT revealed a high frequency of HIV-1 gag-specific CTL both in blood and synovial fluid, whereas gag-specific CD4-cells could be detected only in the peripheral blood. Further analysis revealed that the gag-specific T-cells were predominantly targeting the HLA B27-restricted CTL epitope KRWIILGLNK (KK10). Immunosuppressive therapy by prednisone was associated with a moderate increase of HIV-1 viremia and a decrease both in the number and in the gamma-IFN production of KK10-specific CTL indicating that inhibition of CTL function contributed to the increase of viral load. CONCLUSIONS: This study is suggesting that HIV-1 specific CTL play an important role in the control of HIV-1, at least in this patient. Inhibition of CTL function by immunosuppressive therapy with prednisone may enhance viral replication. In addition, we could demonstrate for the first time the migration of HIV-1 specific T-cells into the synovial fluid.     

CD4+ T cell targeting of human immunodeficiency virus type 1 (HIV-1) peptide sequences present in vivo during chronic, progressive HIV-1 disease

We previously detected HIV-1 Gag-specific CD4+ T cells recognizing reference strain viral epitopes in subjects with progressive, chronic infection. To test whether these CD4+ T cells persist in vivo by failing to recognize autologous HIV-1 epitopes, we compared autologous plasma HIV-1 p24 nucleotide sequences with targeted HXB.2 strain Gag p24 CD4+ T cell epitopes in nine chronically infected, untreated subjects. In five responding subjects, 10 of 26 HXB.2 strain p24 peptides targeted by CD4+ T cells exactly matched autologous plasma viral sequences. Four subjects with plasma viral loads >100,000 copies/mL had no measurable p24-specific CD4+ T cell responses despite carrying HIV-1 strains that matched HXB.2 sequences at predicted epitopes. These results show that HIV-1-specific CD4+ T cells can persist in chronic HIV-1 infection despite recognition of epitopes present in vivo. However, with high-level in vivo HIV-1 replication, CD4+ T cells targeting autologous HIV-1 may be non-responsive or absent.     

Nef enhances HIV-1 infectivity via association with the virus assembly complex

The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.     

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC₅₀ V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.     

Suppression of HIV-1 replication by HIV-1-irrelevant CD8+ cytotoxic T lymphocytes resulting in preservation of persistently HIV-1-infected cells in vitro

CD8+ cells of asymptomatic HIV-1 carriers (AC) contain HIV-1-specific cytotoxic T lymphocytes (CTLs) but suppress HIV-1 replication in a class I major histocompatibility complex (MHC-I)-unrestricted manner. In order to selectively investigate the HIV-1-suppressive function of CTLs apart from HIV-1-specific cytotoxicity, HIV-1-irrelevant allo-specific CTLs were established from an HIV-1-uninfected individual and their HIV-1-suppressive activity against autologous CD4+ peripheral blood mononuclear cells (PBMC) was examined. We found that these CTLs significantly suppressed both R5 and X4-HIV-1 replication in either acutely or persistently infected autologous PBMC. Although these CTLs partially killed HIV-1-infected PBMC through Fas ligand, CTLs still suppressed late steps of HIV-1 replication in the presence of neutralizing antibodies to Fas ligand. HIV-1 replication in PBMC that had been suppressed by CTLs was reversible following depletion of CTLs from culture, analogous to the previous observation for CD8+ cell-depleted PBMC of AC. Induction of HIV-1 replication by CTL-depletion was amplified by addition of newly prepared CD4+ cells or activation with staphylococcal enterotoxin B. Our results indicate that CTLs can suppress HIV-1 replication in PBMC in an antigen-nonspecific manner and preserve infected cells in a state capable of restarting HIV-1 replication and transmission.     

Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus-1-infected mothers.

Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV-1-infected mothers. These CTL killed HLA-matched HIV-1-infected H9 target cells or doubly transfected P815-A2-env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA-A1/A3 or HLA-A2, respectively. HIV-1-specific CTL were detected early after birth (less than 2 months) and remained present during the asymptomatic phase of the infection. As in HIV-1-infected adults, HIV-specific CTL declined with disease progression. Surprisingly, HIV-1-specific CTL were detected in the PBMC of three children who subsequently became seronegative.     

Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells

Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication.     

Development of a HIV-1 lipopeptide antigen pulsed therapeutic dendritic cell vaccine

In the search for a therapeutic HIV-1 vaccine, we describe herein the development of a monocyte-derived dendritic cell (DC) vaccine loaded with a mixture of HIV-1-antigen lipopeptides (ANRS HIV-LIPO-5 Vaccine). LIPO-5 is comprised of five HIV-1-antigen peptides (Gag(17-35), Gag(253-284), Nef(66-97), Nef(116-145), and Pol(325-355)), each covalently linked to a palmitoyl-lysylamide moiety. Monocytes enriched from HIV-1-infected highly active antiretroviral therapy (HAART)-treated patients were cultured for three days with granulocyte-macrophage colony-stimulating factor and alpha-interferon. At day 2, the DCs were loaded with ANRS HIV-LIPO-5 vaccine, activated with lipopolysaccharide, harvested at day 3 and frozen. Flow cytometry analysis of thawed DC vaccines showed expression of DC differentiation markers: CD1b/c, CD14, HLA-DR, CD11c, co-stimulatory molecule CD80 and DC maturation marker CD83. DCs were capable of eliciting an HIV-1-antigen-specific response, as measured by expansion of autologous CD4(+) and CD8(+) T-cells. The expanded T-cells secreted gamma-IFN and interleukin (IL)-13, but not IL-10. The safety and immunogenicity of this DC vaccine are being evaluated in a Phase I/II clinical trial in chronically HIV-1-infected patients on HAART (clinicaltrials.gov identifier: NCT00796770).     

酵母表达的HIV-1中国株CN54 Gag蛋白的免疫原性研究

目的评价毕赤酵母表达的HIV-1中国株CN54 Gag蛋白在Balb／c小鼠诱导体液和细胞免疫的能力。方法利用重组Gag蛋白单独及与重组DNA或痘苗联合免疫Balb／c小鼠，检测小鼠产生的特异性结合抗体和IgG1／IgG2a，同时检测T淋巴细胞增殖反应和CTL反应。结果3个蛋白单独免疫组均能诱导小鼠产生抗体和T淋巴细胞增殖反应，其中不加佐剂的免疫组诱导免疫反应低于其它两组。但3组的CTL杀伤活性均不明显。重组蛋白和重组DNA及痘苗联合免疫均诱导产生了较好的体液和细胞免疫反应，而且重组痘苗初始免疫一蛋白加强免疫组还诱导产生了较强的CrL反应。结论酵母表达的HIV-1 Gag蛋白具有良好的免疫原性，在与重组DNA和痘苗疫苗联合使用时具有协同作用，能刺激动物产生更强的HIV-1特异性体液和细胞免疫反应。本研究为构建HIV-1蛋白疫苗和探讨各类疫苗的联合免疫方式提供了有价值的参考数据。     

Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons.

Rare individuals who are repeatedly exposed to HIV-1 through unprotected sexual contact fail to acquire HIV-1 infection. These persons represent a unique study population to evaluate mechanisms by which HIV-1 replication is either prevented or controlled. We followed longitudinally a group of healthy HIV-1 seronegative persons each reporting repeated high-risk sexual activities with their HIV-1-infected partner at enrollment. The volunteers were primarily (90%) male homosexuals, maintaining high risk activities with their known infected partner (45%) or multiple other partners (61%). We evaluated the quantity and specificity of HIV-1-specific T cells in 31 exposed seronegatives (ES) using a IFN-gamma ELISPOT assay to enumerate T cells recognizing epitopes within HIV-1 Env, Gag, Pol and Nef. PBMC from only three of the 31 volunteers demonstrated ex vivo HIV-1-specific IFN-gamma secretion, in contrast to nearly 30% exhibiting cytolytic responses in previous studies. These findings suggest that if T cell responses in ES are induced by HIV-1 exposure, the frequency is at low levels in most of them, and below the level of detection using the ELISPOT assay. Alternative approaches to improve the sensitivity of detection may include use of dendritic cells as antigen-presenting cells in the ex vivo assay and more careful definition of the risk behavior and extent of HIV-1 exposure in conjunction with the evaluation of T cell responses.     

Evasion of cytotoxic T lymphocytes is a functional constraint maintaining HIV-1 Nef expression

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.     

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.     

The Mamu B 17-restricted SIV Nef IW9 to TW9 mutation abrogates correct epitope processing and presentation without loss of replicative fitness

CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.     

Identification of HLA-A*3101-restricted cytotoxic T-lymphocyte response to human immunodeficiency virus type 1 (HIV-1) in patients with chronic HIV-1 infection

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infections. As several HIV-1 CTL epitopes restricted to many HLA types are already known, we aimed at identifying the CTL epitopes restricted by HLA-A*3101 in an effort to expand the epitope repertoire available for the development of potential T cell-mediated therapeutic measures and protective vaccines. Scanning of HIV-1 clade B SF2 strain proteins for the presence of peptides containing HLA-A*3101-binding motifs revealed 88 nine- to 11-mer peptides that had been synthesized and assayed for binding to HLA-A*3101 molecules. Peptides with medium to high HLA-binding affinity were tested for their ability to stimulate a CTL response in the peripheral blood mononuclear cells (PBMCs) from selected HIV-1-infected patients. Two of these binding peptides, Env769-779 (RLRDLLLIAAR) and Nef192-200 (KLAFHHMAR), induced peptide-specific CTLs in PBMCs from at least two of five HIV-1-seropositive individuals. CTL clones specific for the two peptides killed HLA-A*3101-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed HLA-A*3101-restricted CTL epitopes. Identification of T-cell epitopes on HIV-1 proteins will increase our understanding of the role of CD8+ T cells in HIV-1 infections and assist in the design of new protective strategies.