Mapping of linear B-cell epitopes on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1).

Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.     

Association between HIV Type 1-specific T cell responses and CD4+ T cell counts or CD4+:CD8+ T cell ratios in HIV Type 1 subtype B infection in China

CD4+ T cell counts and CD4+:CD8+ T cell ratios represent key determinants of HIV disease progression and infectivity. However, the relationship between the HIV-1-specific cytotoxic T lymphocyte (CTL) response and these determinants has not been elucidated for all HIV-1B and HIV-1C proteins. In the present study, virusspecific T cell responses to HIV-1B and HIV-1C proteins were analyzed with interferon gamma (IFN-gamma) enzyme- linked immunospot (ELISpot) assays using synthetic overlapping peptides corresponding to naturally occurring HIV-1B and HIV-1C consensus sequences. For Gag/Gag p24/Gag p17, a correlation between T cell responses and CD4+ T cell count in HIV-1 clade B and clade C was seen: elevated T cell response resulted in higher CD4+ T cell production. A statistically significant correlation between the Pol-specific T cell response and CD4+ T cell counts was also found in HIV-1 subtype C. For all HIV-1B and HIV-1C proteins, a correlation between the HIV-1-specific T cell response and CD4+:CD8+ T cell ratios was found for Tat and Pol proteins. CD4+ T cell counts in patients with Tat and/or Rev T cell response were higher than in patients without Tat and/or Rev T cell response. We suggest that this correlation within HIV-1B and HIV-1C Gag p24/Gag p17 responses makes the Gag p24/Gag p17 region a potential vaccine candidate and that HIV-1-specific CTL epitopes toward Pol are important in controlling HIV-1 infection; we emphasize that future vaccination strategies should include these early antigens, Tat and Rev.     

Identification and characterization of HIV-1 CD8+ T cell escape variants with impaired fitness

In this study, amino acid sequence variation in human immunodeficiency virus (HIV)-1 Gag CD8(+) T cell epitopes was examined in untreated mother-infant pairs. Several HIV-1 CD8(+) T cell escape variants were identified within maternal plasma viral p17 and p24 sequences that were either not detected or did not persist in the plasma of their non-HLA-matched HIV-1-infected infants. Viruses constructed with each of these mutations demonstrated reduced viral replication in vitro and reduced expression of p17 and p24 proteins compared with wild type. Reduced recognition of the variant sequences compared with wild-type sequence was also demonstrated by enzyme-linked immunospot assays. Nontransmission or reversion after transmission was thus associated with reduced viral fitness cost in vivo. Better understanding of the balance between CD8(+) T cell selective pressures and viral fitness cost may facilitate the identification of optimal viral sequences for inclusion in HIV-1 vaccines.     

HIV-1 p24 vaccine protects cats against feline immunodeficiency virus infection

BACKGROUND: Based on previous analysis of feline immunodeficiency virus (FIV)-specific cross-reactive antibodies to HIV-1 p24, cats vaccinated with HIV-1 p24 were evaluated for cross-reactive immunity to FIV. OBJECTIVE:: To determine the level of cross-reactivity that exists between HIV-1 and FIV p24 and its implications for vaccine prophylaxis. METHODS: Specific-pathogen-free cats were immunized three times with HIV-1 p24 in Ribi adjuvant, with (n = 18) or without cytokine (n = 6). Control cats were immunized three times with adjuvant (n = 10) or phosphate-buffered saline (PBS; n = 5). All immunized cats were challenged with either subtypes B or A/B FIV, and monitored by virus isolation, proviral PCR, FIV-specific antibodies, and feline interferon-gamma ELISpot for T-cell activities. RESULTS: Of 18 cats vaccinated with subtype B HIV-1 (HIV-1LAI/LAV, HIV-1UCD1) p24 in Ribi/cytokine adjuvant 14 (78%) were protected against FIV challenges (subtype Agag and Bgag) that infected all 15 adjuvant- or PBS-immunized cats. Furthermore, only three of six (50%) cats vaccinated with FIV p24 in Ribi/cytokine adjuvant were protected against similar FIV challenge. HIV-1 p24 vaccination induced weak cross-reactive antibodies to FIV p24, which did not correlate with vaccine efficacy. However, the peripheral blood mononuclear cells from HIV-1 p24-vaccinated/protected cats at 33-34 weeks post-FIV challenge responded to three T-cell responsive peptides at the carboxyl-terminus of the FIV p24, whereas those cells from the infected control cats had minimal to no responses to the same peptides. CONCLUSIONS: These results suggest the importance of including lentiviral p24 as vaccine immunogen for human AIDS vaccine. Moreover, these results suggest the potential importance of evolutionarily conserved, cross-protective epitopes in vaccine protection.     

Decline in CTL and antibody responses to HIV-1 p17 and p24 antigens in HIV-1-infected hemophiliacs irrespective of disease progression. A 5-year follow-up study.

CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.     

Cytolytic T lymphocytes (CTLs) from HIV-1 subtype C-infected Indian patients recognize CTL epitopes from a conserved immunodominant region of HIV-1 Gag and Nef

Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.     

Reactivities of HIV-1 gag-derived peptides with antibodies of HIV-1-infected and uninfected humans.

A group of 41 peptides, each 24 amino acids long and overlapping with each other by 12 residues spanning the total gag open reading frame (orf) of HIV-1 (HTLV-IIIBH 10 isolate) were synthesized using Fmoc chemistry. The purified compounds were used in ELISA assays and tested for antibody reactivities in sera of human HIV-1-infected and noninfected individuals. Sera of HIV- humans showed reactivity against four defined regions, two in p17, one in p24, and one in p15. The values of these reactivities were elevated especially in serum samples of HIV- individuals showing cross-reaction with gag proteins on Western blot. Amino acid sequence comparison of HIV-1 gag proteins with those of human endogenous retroviruses (ERV K10, ERV 3) revealed significant similarities predominantly in the domains showing elevated antibody cross-reactions. The majority of sera from HIV-1+ individuals showed strong reactivities to the cross-reactive regions and to various other peptide sequences, a sequential epitope recognized by all HIV-1+ sera could, however, not be identified. The results suggest that human individuals may have immune reactions to endogenous retroviral protein sequences, which are enhanced by infections with HIV-1. Specific antibodies to HIV-1 gag proteins are probably mainly directed to tertiary structure defined epitopes formed by particle formation of the p24 monomers to the nucleocapsid.     

Predicting progression to AIDS: combined usefulness of CD4 lymphocyte counts and p24 antigenemia

PURPOSE: To investigate the combined usefulness of CD4 lymphocyte counts and human immunodeficiency virus type 1 (HIV-1) p24 antigen in predicting progression to the acquired immunodeficiency syndrome (AIDS). PATIENTS AND METHODS: CD4 lymphocyte counts and HIV-1 p24 antigen status were evaluated over a 4-year period in 518 HIV-1-seropositive men enrolled in the Multicenter AIDS Cohort Study in Chicago. RESULTS: Twenty-six percent (134 of 518) of the HIV-1-seropositive cohort had detectable p24 antigen during the study period. Men with p24 antigenemia experienced a more rapid decline in CD4 lymphocyte counts than men who were persistently p24 antigen-negative (p less than 0.01). Mean CD4 lymphocyte counts at first detection of p24 antigen were 406 and 455 cells/microL for men with incident and prevalent antigenemia, respectively. Antigen was detected in 61% (63 of 103) of the men who progressed to AIDS and in only 17% (71 of 415) of the men who did not (p less than 0.0001). The 4-year estimated cumulative AIDS incidence was 86%, 63%, and 21% for men with entry CD4 counts less than 200, 200 to 399, and 400 or more cells/microL, respectively. Presence of p24 antigenemia was strongly associated with more rapid disease progression within each of these CD4 groupings (p less than 0.0001). CONCLUSION: Our data indicate that p24 antigenemia can first be detected with moderate CD4 cell depletion, is associated with a more rapid decline in the CD4 lymphocyte population, and combined with CD4 lymphocyte counts is useful in identifying individuals at significantly greater risk of disease progression. Our findings provide important information for assessing HIV-1 disease prognosis over a 4-year period.     

Prevalence of HIV-1 p24 antigenemia in African and North American populations and correlation with clinical status

Sera from 622 individuals and culture supernatants from three HIV-1 viral isolates were assayed for HIV-1 p24 antigen to investigate the frequency of p24 antigenemia in African and North American populations using three commercial HIV-1 p24 antigen assays (Coulter, Du Pont, and Abbott). The prevalence of p24 antigenemia in 89 hospitalized Zairian AIDS patients was significantly lower than in 47 clinically comparable AIDS patients in the USA (17 versus 48%, P less than 0.0001). Prevalence of p24 antigenemia in sera from 200 asymptomatic HIV-1-infected individuals was also lower in individuals from Zaire compared with 83 individuals in the USA (3.5 versus 7%). In African individuals, antigenemia prevalence increased with advanced clinical status: 8% in ambulatory AIDS patients, 17% in hospitalized AIDS patients and 18% in postmortem AIDS patients. Acid hydrolysis treatment of sera from 63 Zairian AIDS patients initially negative for p24 antigen showed an 11% positivity rate confirmed by neutralization, suggesting that immune complexing of p24 antigen may play a role in the observed lower p24 antigenemia rates reported for African individuals.     

Full-length gag sequences of HIV type 1 subtype C recent seroconverters from Pune, India

Although HIV-1 subtype C is the most prevalent subtype worldwide, data on subtype C viruses are rather limited. Very little information is available on the complete HIV-1 subtype C gag sequences from India. We report full-length gag (p55) sequences from six Indian early seroconverters. The samples were collected within few weeks of seroconversion and may represent immunologically naive viruses. The comparison of p55 sequences with other Indian and non-Indian subtype C sequences as well as with nonsubtype C sequences obtained from the Los Alamos database revealed gag as a well-conserved region of the HIV genome (range: 84-95%). The phylogenetic tree indicated that the sequences compared here cluster together within clade C. Two epitopes in the p24 region of the gag gene were subtype C specific while many epitopes in the same region were also present in other clades. The data on HIV-1 subtype C full-length gag sequences would be useful in the design and evaluation of effective subtype C-based HIV vaccines.     

Identification of regions of HIV-1 p24 reactive with sera which give "indeterminate" results in electrophoretic immunoblots with the help of long synthetic peptides

We analyzed nine sera from persons unlikely to be HIV infected which had an IgG reactivity directed against HIV-1 p24, and in two cases also to its precursor p55, but to no other HIV proteins, nor to proteins of the H9 host cell, in electrophoretic immunoblots (EIB). These sera are also referred to as having an indeterminate HIV EIB pattern or as HIV antibody false positive sera. Seven of nine sera reacted with longer (61-77 amino acids) and none with shorter (17-25 amino acids) p24-derived peptides in enzyme immunoassays (EIAs). This is compatible with a conformational (discontinuous) nature of the epitopes involved in many false positive HIV-1 p24 antibody reactions. Four sera reacted with an N-terminal, one with an internal, and two with a C-terminal fragment. Each of the seven sera thus only reacted with one of the long p24 peptides. The specificity and singularity of the reaction was further demonstrated by competition and/or absorption experiments with synthetic peptides. In contrast, 18 of 20 confirmed HIV-1+ sera with p24 reactivity in EIB reacted with at least one and often several of the longer peptides, most frequently the C-terminal one. Thus, the distribution of peptide reactivity of true HIV-1 antibody-positive sera was different from that of the falsely reactive sera. According to two of several explanations, these antibodies may have arisen because of (1) molecular mimicry by chance or by functional selection, (2) immunization by activation, noninfectious exposure, or infection involving non-HIV endogenous or exogenous retroviral antigens. The latter gains some support from our finding of antibody reactions with capsid proteins of the simian viruses, simian sarcoma-associated virus (SSAV), and Mason-Pfizer monkey retrovirus in some of the p24 +/- p55 reactive sera.     

Multiple antigenic epitopes expressed on gag proteins, p26 and p15, of a human immunodeficiency virus (HIV) type 2 as defined with a library of monoclonal antibodies.

Thirty-two hybridoma clones producing monoclonal antibody (MAb) against HIV-2[GH-1] were established from mice immunized with NP-40-disrupted purified whole virus of a Ghanaian isolate of human immunodeficiency virus type 2 (HIV-2), strain HIV-2[GH-1]. Of these 32 MAbs, 20 reacted with p26 and the other MAbs recognized p15 of the HIV-2[GH-1] isolate. From the results of serological characterization by these MAbs, p26 and p15 were identified as capsid proteins and matrix protein, respectively, of HIV-2[GH-1] gag products. In addition to two gag proteins, a 55-kD protein corresponding to the primary translational product of gag gene and 39-kD protein corresponding to an intermediate product of cleavage of p55 were recognized by these MAbs in the lysate of HIV-2[GH-1]-infected cells. Moreover, these MAbs were used to analyze the number of antigenic epitopes on p26 and p15 of HIV-2[GH-1] isolate. The results of cross-reactivity with different HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates and competitive binding assay suggest that there are at least four and five antigenic epitopes in p26 and p15, respectively, of the HIV-2[GH-1] isolate. The biological activity of MAbs was studied by performing syncytium inhibition assay and infection inhibition assays. However, our MAbs could not inhibit syncytium formation and infection by cell-free virus.     

Characterization of mother-infant HIV type 1 gag p17 sequences associated with perinatal transmission.

The gag p17 matrix sequences of human immunodeficiency virus type 1 (HIV-1) from seven infected mother-infant pairs were analyzed after perinatal transmission. The p17 matrix open reading frame was maintained in 143 of the 166 clones analyzed (86.2% frequency of intact p17 open reading frames). The functional domains essential for p17 matrix function in HIV-1 replication, including targeting of Gag to the plasma membrane, virus assembly and release, envelope glycoprotein incorporation into virus particle, virus entry, and localization of the virus preintegration complex to the nucleus of nondividing cells, were highly conserved in most of the sequences. In addition, examination of the three-dimensional structure of the p17 matrix protein in mother-infant isolates showed a high degree of conservation of amino acids required for correct folding and biological activity. Several amino acid motifs common to most of the mother-infant pairs sequences, including pair-specific signature sequences, were observed. There was a low degree of heterogeneity of gag p17 sequences within mothers, within infants, and between mother-infant pairs, but the distances were greater between epidemiologically unlinked individuals. Phylogenetic analyses of 166 mother-infant pairs and 181 other p17 sequences available from HIV-1 databases revealed distinct clusters for each mother-infant pair and for other p17 sequences. In conclusion, these findings indicate that an intact and functional gag p17 matrix is maintained during maternal-fetal transmission and that several motifs in p17 may be associated with perinatal transmission.     

Investigation of a new HIV-1 p24 antigen detection kit based on the enzyme-linked fluorescent immunoassay

We investigated the performance of the new p24 antigen detection kit (VIDAS HIV p24) with the conventional antigen kit (HIV-1 Ag monoclonal; Abbott). The new kit is an enzyme-linked fluorescent immunoassay (ELFA) and all of the assay steps are performed automatically by the VIDAS instrument within 100 minutes. With the seven HIV-1 seroconversion panels, three seroconversions were detected on an average of 6.8 days earlier with ELFA than the conventional EIA kit. ELFA showed negative results for all of the 11 false positive samples by the combined (p24, anti-HIV) detection kit (VIDAS HIV DUO). The results obtained suggest that ELFA are very useful for an earlier diagnosis of HIV infection and re-test for false positive samples by other HIV diagnosis kits.     

Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques

Despite advances in the clinical management of HIV infection, using combinations of antiretroviral pharmaceuticals, a safe and efficacious vaccine is needed to limit the AIDS pandemic. It is now thought that an effective HIV-1 vaccine should prime both cross-neutralizing antibodies and long-lasting cytotoxic CD8+ T lymphocytes (CTLs) recognizing multiple codominant HIV-1 epitopes. To that end, many novel vaccine strategies have been tested. However, only a few of these strategies, beside those relying on live-attenuated viruses, are able to prime strong CTL responses in nonhuman primates and humans. In this study, three rhesus macaques were immunized with HIV-1 p55gag virus-like particles (VLPs) in the absence of adjuvant to assess the potential of such a vaccine to prime CTL responses. After intramuscular injection of p55gag VLP, all three animals mounted CTL responses against HIV-1 p55gag. Notably, these CTLs primed by vaccination recognized naturally processed peptides and were long lived (>8.5 months) both in the peripheral blood and draining lymph node. Furthermore, these CTLs were directed against multiple HIV-1 p55gag epitopes. This indicated that immunization with p55gag VLP primes strong MHC class I-restricted, CD8+ cell-mediated immune responses and suggested that HIV-1 p55gag VLPs should be a reasonable vaccine candidate, when combined with strategies priming cross-neutralizing antibodies.     

Immune response to HIV p24 core protein during the early phases of human immunodeficiency virus infection

The immune response to the p24 core antigen of human immunodeficiency virus type 1 (HIV-1) was studied in serial samples collected prospectively from 52 homosexual males in two separate cohorts from Amsterdam and San Francisco. p24 antibody levels were quantified with an antigen sandwich immunoassay using p24 recombinant antigen as capture and probe. Titers and slopes of dilution curves reflecting antibody affinity were analyzed. Only 45 of 52 men developed a measurable primary immune response to p24. In 17 (33%) patients there was a low response with maximum antibody titer below 66, shallow (low affinity) dilution curve, and 10 of the 17 became HIV antigen positive over a 2 year period. In 24 (46%) of the 52 patients titers ranged from 100-4000, steeper dilution curves were noted, and none became HIV antigen positive. Four (8%) men developed a strong immune response with high titers (greater than 12,000) and high affinity type dilution curve. Over time, after the peak immune response, antibody titer declined in some individuals related in part to the formation of immune complexes between HIV-1 p24 antigen and antibody which were dissociable. In vitro, the addition of increasing amounts of purified p24 antigen corresponded to decreasing antibody titer and a shallower dilution curve suggesting a preferential consumption of high affinity antibodies for complex formation. The magnitude of immune response to HIV-1 p24 antigen varies widely in infected homosexual men. Both the intrinsic ability to mount an immune response and immune complex formation contribute to the measurable antibody level.     

HIV／AIDS病人肝脏HIV-1 RNA和p24抗原的检测

目的了解艾滋病病毒I型（HIV-1）在肝组织的表达和分布情况,探讨HIV／艾滋病（AIDS）病人发生肝损害的可能机制。方法先后采用免疫组织化学方法和核酸原位杂交方法,检测HIV／AIDS病人肝组织病理切片中的HIV-p24抗原和HIV-1核酸。结果14例HIV／AIDS病人肝切片组织的Kupffer细胞、淋巴细胞、血管内皮细胞和肝细胞,均检测到HIV—p24抗原。所有切片组织的Kupffer细胞、淋巴细胞有HIVRNA阳性表达,其中4例标本的肝细胞浆内有HIVRNA表达。结论HIV-1可能在肝脏实质细胞和间质细胞内复制表达,并引起肝脏损害。     

Lymphoproliferative response to HIV type 1 p24 in long-term survivors of HIV type 1 infection is predictive of persistent AIDS-free infection.

To establish immunologic correlates of progression to AIDS in long-term survivors of HIV-1 infection, HIV-1-specific T cell-mediated responses, together with T cell reactivity to recall antigens, were studied in frozen samples collected after 5 and 8 years of documented HIV-1 infection. Eight of 21 homosexual men, who remained asymptomatic and maintained CD4+ T cell numbers >400 cells/microl for 9 years of HIV-1 infection, progressed to AIDS (CDC 1993 definition) within 12.5 years of infection (late progressors, LPs). The remainders showed minimal deterioration of immune parameters (long-term nonprogressors, LTNPs). CD4+ T cell numbers and T cell function measured at years 5 and 8 of follow-up were comparable in the two groups. At both time points responses to recall antigens did not significantly differ between the two groups, although a significant decline of lymphoproliferative responses to Candida and tetanus toxoid was observed in LPs. Circulating HIV-1-specific cytotoxic T lymphocyte precursors were found in broad frequency ranges in both LPs and LTNPs and, similarly, no significant differences were found in comparing the breadth of serum neutralizing activity against heterologous HIV-1 primary isolates. In contrast, lymphoproliferative responses to p24gag, but not p17gag or gp160env, were detected only in LTNPs and were totally absent in LPs at both time points (p < 0.01). Our data suggest that the presence of circulating p24-specific CD4+ T cells may reflect effective viral control and be predictive of subsequent favorable clinical course in long-term asymptomatic individuals.     

Evidence for Gag p24-specific CD4 T cells with reduced susceptibility to R5 HIV-1 infection in a UK cohort of HIV-exposed-seronegative subjects

AIM: To characterize HIV-1 Gag p24-specific CD4 cell responses in HIV-exposed-seronegative (ES) individuals. METHODOLOGY: Twelve ES individuals, of diverse ethnicity and wild type for the CCR5 Delta-32 mutation, were identified. Controls were HIV-negative blood donors. Gag p24-specific and total Vbeta+ CD4 cells that expressed MIP-1beta, IFN-gamma and IL-2 were enumerated by intracytoplasmic cytokine staining. beta-Chemokine expression was correlated with susceptibility to R5 HIV-1 infection, as measured by polymerase chain reaction for integrated HIV-1 and by p24 enzyme-linked immunosorbent assay. RESULTS: Similar numbers of mitogen-stimulated and Vbeta+ MIP-1beta+, IFN-gamma+ and IL-2+ T cells were found in ES and HIV-negative control subjects. However, all ES subjects tested had an HIV Gag p24-specific MIP-1beta+, IFN-gamma+ and IL-2+ CD4 T-cell response that was rare in controls. p24-Specific cells of all ES but no control subjects could be expanded by in-vitro Ag/IL-2 stimulation, and when re-stimulated with an overlapping peptide series showed evidence of a broad CD4 cell memory response directed against multiple regions of Gag p24. Mitogen-stimulated ES CD4 cells were as susceptible to HIV infection as those from control subjects, but p24-specific IFN-gamma+ CD4 cells of six out of seven ES subjects tested were less susceptible to R5 HIV-1 infection than the counterpart fraction depleted of p24-specific IFN-gamma+ cells. The addition of blocking anti-beta-chemokine antibodies did not promote R5 HIV-1 infection of p24-specific IFN-gamma+ cells. CONCLUSION: Specific CD4 cell immunity, characterized by a broadly directed memory Gag-p24 CD4 cell response and reduced susceptibility of specific CD4 cells to R5 HIV-1 infection, is a likely correlate of non-transmission.