HIV-seropositive Donors Research Articles

HIV Genotypes and Primary Drug Resistance Among HIV-Seropositive Blood Donors in Brazil

There are few surveillance studies analyzing genotypes or primary (transmitted) drug resistance in HIV-infected blood donors in Brazil. The aim of this study was to characterize patterns of HIV genotypes and primary resistance among HIV-seropositive donors identified at 4 geographically dispersed blood centers in Brazil. All HIV-infected donors who returned for counseling at the 4 REDS-II Hemocenters in Brazil from January 2007 to March 2011 were invited to participate in a case-control study involving a questionnaire on risk factors. Viral sequencing was also offered to positive cases to assign genotypes and to detect and characterize primary resistance to reverse transcriptase and protease inhibitors according to World Health Organization guidelines. Of the 341 HIV-seropositive donors who consented to participate in the risk factor and genetics study, pol sequences were obtained for 331 (97%). Clade B was predominant (76%) followed by F (15%) and C (5%). Primary resistance was present in 36 [12.2%, 95% confidence interval (CI) 8.2 to 15.5] of the 303 individuals not exposed to antiretroviral therapy, varying from 8.2% (95% CI: 2.7 to 13.6) in Recife to 19.4% in São Paulo (95% CI: 9.5 to 29.2); there were no significant correlations with other demographics or risk factors. Although subtype B remains the most prevalent genotype in all 4 areas, increasing rates of subtype C in Sao Paulo and F in Recife were documented relative to earlier reports. Transmitted drug resistance was relatively frequent, particularly in the city of Sao Paulo which showed an increase compared with previous HIV-seropositive donor data from 10 years ago.

Genetic Characterization of HIV-1 Strains Circulating in Kazakhstan

To determine the HIV-1 genetic diversity in Kazakhstan, 85 blood samples from HIV-seropositive donors were collected between 2001 and 2003. The study population consisted of 91.8% injecting drug users (IDUs); the remainder was infected sexually or iatrogenically. A genomic region that included part of the polymerase gene was sequenced for all 85 samples, and from these, 6 samples were randomly selected for nearly full genome sequencing. Subtype A was the most common genetic form (94.1%), followed by CRF02_AG (4.7%) and subtype C (1.2%). All subtype A sequences clustered closely with samples from countries of the former Soviet Union (FSU). From these sequences, 47 (58.8%) presented the secondary protease inhibitor mutation V77I that has been linked to a genetic lineage in the FSU epidemic. In addition, most had the other 2 mutations that characterize the "V77I haplotype." All 6 nearly full-length sequences were subtype A and clustered with other FSU strains. The CRF02_AG strains from this population clustered with strains from Uzbekistan, reflecting the spread of the CRF02_AG epidemic in Central Asia. The HIV epidemic in Kazakhstan is predominantly in IDUs and is indigenous to the geographic region, and most of the strains are genetically similar to those circulating in the FSU and other parts of Central Asia.

Detection of HIV Vaccine-Induced Cell-Mediated Immunity in HIV-Seronegative Clinical Trial Participants Using an Optimized and Validated Enzyme-Linked Immunospot Assay

An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.

Cross-Clade CD8 T-Cell Responses to HIVIIIB and Chinese B??? and C/B??? Viruses in North American and Chinese HIV-Seropositive Donors

HIV variation presents an obstacle to a global AIDS vaccine. Viral diversity and host variations in MHC expression both affect vaccine responses. Whether CD8 T cells from HIV-infected donors in 1 part of the world cross-recognize isolates from other regions will provide guidance about whether country-specific vaccines are needed. We compared recognition of HIV(IIIB) and representative B' (Thai B) and recombinant C/B' virus strains endemic in China by CD8 T cells from 7 HIV-infected North American donors and 4 Chinese donors. IFN-gamma production in response to HIV(IIIB) or the Chinese viruses was comparable. Although 1.6 +/- 0.8% of American donor CD8 T cells produced IFN-gamma above the background level in response to IIIB virus, 1.5 +/- 0.8% responded to B' virus, and 1.4 +/- 0.7% responded to C/B' virus. Responses to adherent cells infected with vaccinia viruses expressing B' and C/B' virus gag and env were also comparable in magnitude with responses to IIIB virus. Cytolysis of CD4 T cells infected with B' virus was comparable with lysis of cells infected with IIIB virus, but lysis of the more divergent C/B' virus was somewhat reduced. T cells, selected for IFN-gamma production to IIIB virus, also efficiently lysed cells infected with Chinese viruses. Therefore, cross-clade CD8 T-cell responses to IIIB virus and prevalent Chinese viral strains are common.

Cross-reactivity between HLA-A2-restricted FLU-M1:58-66 and HIV p17 GAG:77-85 epitopes in HIV-infected and uninfected individuals.

BackgroundThe matrix protein of the influenza A virus and the matrix and capsid proteins of the human immunodeficiency virus (HIV) share striking structural similarities which may have evolutionary and biological significance. These similarities led us to hypothesize the existence of cross-reactivity between HLA-A2-restricted FLU-M1:58–66 and HIV-1 p17 GAG:77–85 epitopes.MethodsThe hypothesis that these two epitopes are cross-reactive was tested by determining the presence and extent of FLU/GAG immune cross-reactivity in lymphocytes from HIV-seropositive and seronegative HLA-A2+ donors by cytotoxicity assays and tetramer analyses. Moreover, the molecular basis for FLU/GAG cross-reactivity in HIV-seropositive and seronegative donors was studied by comparing lymphocyte-derived cDNA sequences corresponding to the TCR-β variable regions, in order to determine whether stimulation of lymphocytes with either peptide results in the expansion of identical T-cell clonotypes.ResultsHere, we report evidence of cross-reactivity between FLU-M1:58–66 and HIV-1 p17 GAG:77–85 epitopes following in vitro stimulation of PBMC derived from either HIV-seropositive or seronegative HLA-A2+ donors as determined by cytotoxicity assays, tetramer analyses, and molecular clonotyping.ConclusionThese results suggest that immunity to the matrix protein of the influenza virus may drive a specific immune response to an HLA-A2-restricted HIV gag epitope in HIV-infected and uninfected donors vaccinated against influenza.

Comparison of human immunodeficiency virus antigens as stimulants for lymphocyte proliferation assays.

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm(3), and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.

CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection

AbstractCD8 T cells are classified as naı̈ve, effector, or memory cells on the basis of CD45RA, CD62L, and CCR7 expression. Sequential engagement of cell-surface CD62L and CCR7 receptors is required for efficient trafficking to lymphoid tissue by means of high endothelial venules. Naı̈ve CD8 T cells are CCR7+CD62L+ CD45RA+, whereas long-term memory cells are CCR7+CD62L+CD45RA−. Effector cytotoxic T cells are thought to be CCR7−CD45RA+. The distribution of CD8 subsets and cytolytic protein expression in healthy donors and donors seropositive for human immunodeficiency virus (HIV) were compared. In HIV-infected subjects, CCR7− CD8 T cells expanded at the expense of naı̈ve and long-term memory cells. In both healthy donors and HIV-infected donors, CCR7+ CD8 T cells were uniformly negative for perforin. In all subsets, perforin and granzyme A were not coordinately expressed, with perforin expression being more tightly regulated. The properties of CD8 T cells specific for cytomegalovirus, Epstein-Barr virus (EBV), and HIV were studied by staining with major histocompatibility complex peptide tetramers. Antigen-specific cells for chronic infections with these viruses were uniformly CCR7− and predominantly CD62L−. In 2 HIV-seropositive donors, 3- to 4-fold fewer EBV-tetramer–positive cells were present in lymph nodes compared with blood. Antigen-specific CD8 T cells are therefore preferentially excluded from lymphoid sites, even when infection is primarily in lymphoid tissue. This may protect lymphoid tissues from immunopathological changes but compromise immune defense against viruses, such as HIV and EBV, that target lymphocytes. HIV-specific CD8 T cells do not express CD45RA, whereas EBV- and CMV-specific CD8 T cells are heterogeneous in CD45RA+expression. Lack of CD45RA expression may indicate incomplete differentiation of HIV-specific CD8 T cells to cytotoxic T cells.

Mechanisms of impaired anticryptococcal activity of monocytes from donors infected with human immunodeficiency virus.

The mechanisms by which monocytes from patients infected with human immunodeficiency virus (HIV) have reduced growth inhibitory activity against Cryptococcus neoformans was examined. Monocyte-enriched peripheral blood mononuclear cells from 12 HIV-seropositive donors with CD4 cell counts of 10-210 cells/mm3 (median, 85) and HIV-seronegative donors were compared in assays to determine the binding and phagocytosis of C. neoformans and the respiratory burst and degranulation in response to C. neoformans and zymosan. Monocytes from HIV-infected and uninfected persons bound and ingested C. neoformans equally well; however, generation of hydrogen peroxide and specific release of beta-glucuronidase in response to C. neoformans was significantly reduced in monocyte-enriched cells from the HIV-infected donors. The impaired anticryptococcal activity of monocytes from persons with HIV may be related to defects in both oxidative and nonoxidative effector pathways that occur after the binding and internalization of the organism.

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.

Priming with IFN-gamma restores deficient IL-12 production by peripheral blood mononuclear cells from HIV-seropositive donors.

Production of IL-12 is deficient in PBMC from HIV-infected individuals. Because of recent studies demonstrating that IFN-gamma priming increases the production of IL-12 in normal PBMC, we examined the role of IFN-gamma in the production of IL-12 in PBMC from HIV-seropositive donors. In response to Staphylococcus aureus, production of IFN-gamma and IL-12 was reduced in PBMC from HIV-seropositive compared with that from HIV-seronegative donors. Priming with IFN-gamma, through increases in both IL-12 p40 and p35 mRNA levels, caused a significant increase in IL-12 release by PBMC from both HIV-seropositive and HIV-seronegative donors. However, the increase was greater for PBMC from HIV-seropositive donors, largely restoring the deficit in IL-12 production seen in unprimed cells. In response to Cryptococcus neoformans, Candida albicans, and Mycobacterium tuberculosis, three pathogens that frequently cause opportunistic infections in persons with AIDS, IFN-gamma production was also reduced in PBMC from HIV-seropositive compared with seronegative donors. When primed with IFN-gamma, PBMC from both HIV-seropositive and seronegative donors released substantial and similar quantities of IL-12 in response to these organisms. Taken together, these results demonstrate that IFN-gamma can restore the deficit in IL-12 production seen in HIV infection.

Role of IL-12 in peripheral blood mononuclear cell responses to fungi in persons with and without HIV infection.

Clinical trials of IL-12 in persons infected with HIV have been proposed based on recent evidence suggesting IL-12 plays a critical role in the development of protective immune responses, and the HIV infection is associated with a deficiency of IL-12. As fungal infections are among the most common opportunistic infections associated with AIDS, we examined whether IL-12 p40 gene expression and p70 release in response to Cryptococcus neoformans and Candida albicans were deficient in monocyte-enriched PBMC from HIV-seropositive donors and whether rIL-12 could augment the proliferation of PBMC from HIV-seropositive donors in response to these fungi and to Pneumocystis carinii. PBMC from HIV-seronegative donors expressed IL-12 p40 mRNA in response to C. neoformans, C. albicans, and the positive control Staphylococcus aureus Cowan strain 1 (SAC), although the induction of IL-12 p40 mRNA was later and more prolonged with C. neoformans as the stimulus. Expression of IL-12 p40 mRNA in response to the three stimuli was similar in cells from HIV-seropositive and HIV-seronegative donors. However, when stimulated with SAC, cells from HIV-seropositive donors released significantly less IL-12, suggesting HIV infection induces a post-transcriptional defect in IL-12 release in response to SAC. While PBMC from HIV-seropositive donors had impaired proliferative responses to the three fungi tested, addition of rIL-12 did not enhance proliferation. These studies do not lend further support for the therapeutic use of IL-12 to prevent or treat fungal infections in persons infected with HIV.

Genetic modification of human peripheral blood lymphocytes with a transdominant negative form of Rev: safety and toxicity.

A transdominant mutant form of the rev gene, M10, confers resistance to infection by the human immunodeficiency virus (HIV) in vitro and is currently under investigation as a potential intervention in acquired immunodeficiency syndrome (AIDS). In this report, we examine three issues relevant to the safety of autologous transfer of human T cells genetically modified with Rev M10. First, the potential for malignant transformation was assessed in vitro using interleukin-2 (IL-2) dependence and fibroblast transformation assays, and tumorigenicity was evaluated in severe combined immunodeficient (SCID) mice. Possible toxicity was evaluated by pathologic analysis following adoptive transfer of genetically modified human T cells into SCID mice. Second, methods were developed that permit T cell activation required for gene transfer but do not allow replication of endogenous HIV. Third, T cell function was evaluated in peripheral blood lymphocytes (PBL) of HIV-seropositive donors transduced with Rev M10 and compared to a negative control mutant, delta Rev M10. By all criteria, no oncogenicity or toxicity was observed. Human T cells transduced with these vectors did not grow in the absence of IL-2 in vitro, and no tumors were observed following transplantation of genetically modified human cells into recipient SCID mice. Histopathological analysis of heart, lung, liver, spleen, and kidney of animals 1-21 weeks following adoptive transfer of gene-modified human T cells revealed no significant abnormalities. Additionally, no differences were observed in the pattern of cytokine secretion in enriched human PBL expressing Rev M10 compared to delta Rev M10. (ABSTRACT TRUNCATED AT 250 WORDS)

Different patterns of HIV-1-specific cytotoxic T-lymphocyte activity after primary infection

To analyse the HIV-1-specific cytotoxic T-lymphocyte (CTL) responses of nine HIV-seropositive subjects in relation with primary infection. Anti-HIV CTL were generated by in vitro stimulation of peripheral mononuclear cells obtained from HIV-seropositive donors at various times after primary infection. They were tested against several structural or regulatory HIV-1 proteins, using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1LAI proteins. An important CTL activity was found during the first month following seroconversion only in those donors who showed clinical symptoms during primary infection. The temporal evolution of this response differed for each subject; one remained a non-responder even 30 months after seroconversion. The structural proteins were recognized particularly early, while the antigenicity of regulatory proteins appeared later. Different patterns of HIV-specific CTL response can be observed after primary infection. The evolution of infection in these different HIV-seropositive subjects will be particularly interesting to analyse.

Comparison of two assays for detection of HIV antibodies in saliva

The presence of HIV antibodies was screened in 241 paired samples of serum and saliva from seronegative subjects with risk factors for human immune deficiency virus (HIV) infection (n = 99), asymptomatic and symptomatic HIV-seropositive patients (n = 104) and healthy blood donors (n = 38) as negative controls, in order to assess the reliability of two saliva tests for the detection of HIV antibodies. These results were confirmed by Western blot. The saliva samples were collected using an oral device (Salivette) maintained in the lateral gingival fold until the individual perceived that it was becoming less rigid due to hydration with saliva. The two tests were a rapid one (Test Pack) and a conventional one (GACELISA). The results for antibody detection in saliva show 99% specificity and 99% sensitivity for the rapid test versus 100% sensitivity and 81% specificity for the conventional test. All results for the saliva samples which were positive in the rapid test were confirmed by Western blot (WHO criteria), and there were no indeterminate Western blot results among the samples which were false-positive in the conventional enzyme immunoassay. No statistically significant differences were observed between the absorbance values of HIV-infected symptomatic and asymptomatic patients. The correlation for the results of the HIV-antibody analysis in the paired sera was 98%. This method of saliva sampling in combination with a rapid and sensitive test for HIV-antibody detection may be applicable in studies conducted with limited technical resources or insufficiently trained health personnel or where blood sample collection is difficult.

Molecular Profile of an Antibody Response to HIV-1 as Probed by Combinatorial Libraries

A large number (33) of human Fab fragments reacting with HIV-1 surface glycoprotein gp120 have been generated by selection from a combinatorial IgGlκ library displayed on the surface of phage. The library was prepared from a long term asymptomatic HIV-seropositive donor. Analysis of the sequences from these Fabs shows the heavy chains can be placed in groups, many of which contain intraclonal variants, almost certainly corresponding to chains used in vivo . Further variants can be accessed via chain shuffling experiments in which a given light chain is recombined with a library of heavy chains. Heavy chain promiscuity, i.e. the ability of heavy chains to pair with different light chains with retention of antigen binding, is dependent on the particular heavy chain considered and probably excludes the identification of in vivo light chain partners. The antibodies examined here are primarily to the CD4 binding site on gp120 and broadly reflect the serum profile of the donor. The antibodies show evidence of extensive somatic modification indicative of an antigen-driven response. The heavy chain CDR3 regions of the antibodies show a remarkably conserved extended length. A number also show strong sequence conservation in CDR3 against a background of considerable diversity in the rest of the VH gene supporting a central role for this region in antigen recognition.

Functional activity of an HIV-1 neutralizing IgG human monoclonal antibody: ADCC and complement-mediated lysis.

The IgG1 kappa, human monoclonal antibody (HMAb), F105, was studied for functional activity in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). F105 reacts with a discontinuous epitope on the CD4 binding site of the HIV-1 envelope glycoprotein, gp120, expressed on the surfaces of infected cells and neutralizes diverse viral strains at antibody concentrations readily achievable in humans. Neither F105 nor serum (diluted 1:50) from HIV seropositive donors mediate CDC against an SF2-infected cell line with rabbit or human sera as a source of complement. F105 and HIV-1 sera mediate ADCC against the SF2 strain. Normal human serum reduced spontaneous lysis of SF2 by peripheral blood monocytes (PBM). Although mixing of F105 with normal human serum reduced the lysis observed (36 +/- 8 vs. 42 +/- 8%), this still was significantly greater than lysis in media (30 +/- 5%) or normal human serum (23 +/- 6%) (p less than .05). A murine antibody to CD16 significantly reduced spontaneous lysis observed with media (30 +/- 5 vs. 18 +/- 3%) while normal mouse serum had no effect (31 +/- 7%). ADCC mediated by F105 is completely abrogated by the anti-CD16 antibody (42 +/- 8 vs. 22 +/- 4%), while only a fraction of ADCC mediated by HIV sera is inhibited by anti-CD16 (60 +/- 9 vs. 46 +/- 6%), suggesting that several populations of effector cells function in ADCC mediated by the polyclonal sera. Thus, F105, as opposed to polyclonal sera, mediates ADCC through a CD16+ PBM population.

Human and murine monoclonal antibodies directed against a conserved sequence from gp41 (aa583–599) of human immunodeficiency virus type 1

Human spleen cells from an HIV-seropositive donor were immunized in vitro with the aa583-599 peptide conjugated to an heptalysyl core. This sequence was derived from the putatively HIV-immunosuppressive region of HIV1 gp41. The same conjugated peptide was used to immunize mice. One human and one mouse IgM monoclonal antibody (mAb) directed against the aa583-599 peptide were obtained. The two mAb had distinct patterns of reactivity against a panel of 42 peptides with modified sequences. Neither of the mAb inhibited the immunosuppressive effect of aa583-599 octopus-lys-conjugated peptide on anti-CD3 Ab-induced lymphoproliferation. In addition, both mAb did not neutralize cell-free virus transmission or enhance HIV infection. However, HmAb inhibited formation of syncytia between HIV1-infected (but not HIV2-infected cells) and non-infected target cells at concentrations above 20 micrograms/ml, whereas MmAb did not have any effect. The degree of conservation of the aa583-599 region makes HmAb a candidate for use as a group-specific reagent in future HIV1 passive immunotherapy protocols.

Immunological characteristics of the putative CD4-binding site of the HIV-1 envelope protein.

As an extension of previous studies demonstrating the immunosuppressive properties of gp120, we have analyzed the immunological characteristics of gp120 peptides, derived principally from its putative CD4-binding site. Our studies indicate that peptides derived from this region do not stimulate proliferation of lymphocytes from HIV-seropositive donors with relatively normal numbers of CD4+ lymphocytes. No significant proliferation was observed in response to various concentrations of peptide, even in the presence of interleukin-2 (IL-2). Significant proliferation of these lymphocytes was observed in response to two recall antigens, cytomegalovirus (CMV) and tetanus toxoid (TT), and these responses were augmented by IL-2. Peripheral blood mononuclear cells from HIV-seronegative donors were cultured in the presence of TT and CMV and the peptides derived from gp120. Proliferation in the presence of these recall antigens was inhibited by these peptides in a dose-dependent manner. These studies demonstrate that at high concentrations, peptides from the putative CD4-binding site can inhibit proliferation of lymphocytes from normal donors in response to a recall antigen. The apparent immunosuppressive properties of this region highlight the pathogenic role played by HIV-1 envelope protein interactions with host cells.

An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

Detection of cytotoxic T lymphocytes specific for synthetic peptides of gp160 in HIV-seropositive individuals.

Four synthetic peptides corresponding to the IIIB sequence of gp160 of HIV were recently reported to stimulate Th cell function by PBL from HIV-infected, asymptomatic patients. In the present report, we used these same peptides to demonstrate CTL activity in a similar patient population. EBV-transformed B-cell lines from asymptomatic, HIV seropositive and seronegative control donors were pre-incubated with the peptides. Fresh PBL from 19 (76%) of 25 HIV seropositive donors lysed autologous targets pulsed with at least one of the four peptides. Autologous targets pulsed with two non-immunogenic peptides were not lysed. PBL from none of the eight HIV seronegative controls lysed peptide-preincubated autologous targets. The CTL activity was mediated by T cells, was predominantly MHC class I restricted, and was increased by in vitro restimulation of PBL with the peptides. HLA A-2 was identified as a restricting element for all four peptides in different patients, and for three of the peptides in the same donor. HLA-A1 or -B8 may also present some of the peptides. Thus, the same peptides can be recognized by human Th cells and class I MHC-restricted CTL.