T cell epitope "hotspots" on the HIV Type 1 gp120 envelope protein overlap with tryptic fragments displayed by mass spectrometry

Our previous work has shown that immunodominant T-helper cell epitopes cluster within distinct fragments on a single face of the HIV envelope gp120 protein. We show in this report that the general positions of immunodominant epitopes are shared by T cells derived from BALB/c, C57BL/6, and CB6F1 mice, yet the precise peptides recognized by the responding T cell populations may differ. In addition, we find that gp120 peptides displayed by tryptic digestion and mass spectrometry of a purified HIV envelope protein share location with peptides defined as immunodominant T cell targets. Results are consistent with the suggestion that gp120 peptide location influences antigen processing, which, in turn, influences the specificity of immunodominant T cells.     

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.     

Immunogenicity comparison between codon optimized HIV-1 CRF BC_07 gp140 and gp145 vaccines

To develop an effective vaccine against the most prevalent HIV strain "B'/C recombinant" in China, we compared the immunogenicity of B'/C-derived gp140 and gp145. The codon optimized gp140 and gp145 env gene derived from CN54, an ancestor-like B'/C recombinant strain, were synthesized and cloned into a plasmid as DNA vaccines, designated as pDRVISV140 and pDRVISV145, respectively. BALB/c mice were inoculated three times at week 0, 2, and 4 and sacrificed at week 7. Both T cell immunity and humoral immunity were determined. The mock vector pDRVISV1.0 carrying no HIV immunogen was included as control. Our data showed that B'/C recombinant-derived gp145 mounted stronger T cell and broader linear antibody but less binding antibody immune responses than gp140 did. Though both gp145 and gp140 raised neutralization antibodies against laboratory-adapted strain SF33, both failed to neutralize B' or B'/C clade primary strains. Overall, this is the first time the immunogenicity of B'/C recombinant-derived gp140 and gp145 was examined and compared; our data prefer B'/C-derived gp145 to gp140 as an HIV vaccine immunogen. The failure to induce neutralization antibodies against primary isolates indicates that it is insufficient to enhance the immunogenicity of conserved epitopes by simply employing gp145 or gp140; strategies to enhance antibody responses against conserved epitopes should be explored further.     

An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes

Because cytotoxic T lymphocytes (CTL) may be important for preventing direct cell-to-cell transmission of human immunodeficiency virus (HIV), the agent responsible for acquired immunodeficiency syndrome, we have begun to investigate the epitope specificity and immune response (Ir) gene control of anti-HIV CTL responses in experimental animals. Mice were infected with a recombinant vaccinia virus expressing the HIV gp160 envelope gene, and the primed lymphocytes were restimulated in vitro with a transfected histocompatible cell line expressing the same gene. Our results show that H-2d mice are CTL high responders and H-2k mice are low responders to the HIV gp160 envelope protein under these conditions. Moreover, the H-2d mice respond predominantly to a single immunodominant site represented by a 15-residue synthetic peptide conforming to the amphipathic alpha-helix model of T-cell epitopes and seen by CD4- CD8+ CTL in association with the Dd class I major histocompatibility complex (MHC) molecules. The facts that CTL responses were detected in the context of only one of four class I MHC molecules tested and that the response was limited predominantly to a single epitope indicate that the CTL repertoire elicited by the HIV envelope protein in association with murine class I MHC molecules may be very limited. In addition, this epitope occurs in a highly variable segment of the envelope protein. This puts constraints on the use of a single peptide sequence from this region in a vaccine, as such a vaccine would have to be polyvalent. Nevertheless, this same variability suggests that this region may be under selective pressure from human CTL, and therefore that this site may be immunodominant in humans as well as mice and so of clinical importance in vaccine development.     

Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.     

Presence of maternal antibodies to human immunodeficiency virus 1 envelope glycoprotein gp120 epitopes correlates with the uninfected status of children born to seropositive mothers.

The present study demonstrates that maternal antibodies to certain epitopes of human immunodeficiency virus 1 (HIV-1) proteins are associated with a defined outcome for at-risk pregnancies of HIV-infected women. An initial retrospective analysis of antibodies to synthetic peptides and recombinant proteins representing env, pol, and gag regions of HIV-1 was carried out. Sera studied were from 33 children who were born to HIV-infected mothers and whose clinical outcome was known at the time of analysis. Sera, collected within the first 6 months of life, of uninfected at-risk children were found to selectively contain maternal antibodies to certain peptides containing epitopes of the HIV envelope glycoprotein gp120. To confirm the predictive role of maternal antibodies to defined HIV-1 epitopes, a prospective analysis was then performed on sera from 21 HIV-seropositive mothers and their infants, whose clinical and immunological status was then followed up for a period of at least 15 months. As expected, antibodies to the same envelope protein peptides were detected almost exclusively in sera from mothers of uninfected children. Our data suggest that antibodies against select epitopes of HIV envelope protein gp120 might play an important role in preventing mother-to-child transmission of HIV-1 infection. Accordingly, site-directed serology might be used to predict the outcome of an at-risk pregnancy of an HIV-infected woman.     

Immunogenicity of a vaccine preparation representing the variable regions of the HIV type 1 envelope glycoprotein

Variability of the major antigenic sites of the envelope glycoprotein of HIV-1 constitutes a major problem in the formulation of effective vaccines. We have prepared a synthetic peptide vaccine that represents the major hypervariable epitopes (V1 through V5) of the clade B HIV-1 envelope glycoprotein (gp120). We refer to this preparation as variable epitope immunogen or VEI vaccine. This construct takes into consideration the type and frequency of amino acid substitutions found at each epitope during the evolution of the virus in individual patients and in the target population. Immunization of mice, rabbits, and rhesus macaques with the VEI vaccine resulted in the induction of long-lasting, high-titered HIV-1 antibodies, including antibodies that neutralize primary isolates. We also documented lymphocyte proliferative responses to the VEI vaccine, its individual components, analogs, and subtype-specific peptides representing the major hypervariable regions of HIV-1 gp120. Delayed-type hypersensitivity responses to these antigens were also demonstrated in mice. Our results show that this vaccine is highly immunogenic and safe in animals. Our data suggest that this formulation could become an important component of combination vaccine approaches against HIV-1 and other antigenically variable pathogens.     

HIV-1 envelope T cell epitope "hotspots " among mice and humans and among CD4+ and CD8+ T cell subpopulations

HIV-1-specific T cell responses correlate with control of infection and disease, thus encouraging a full understanding of the peptides and antigen-processing mechanisms that govern T cell activation. We have previously demonstrated that CD4(+) T cell epitopes cluster nonrandomly within envelope protein "hotspot" regions. The current study was initiated to determine whether envelope-specific CD8(+) T cells might share epitope "hotspots" with the CD4(+) T cell population. Identification of CD8(+) T cell determinants by ELISPOT assays with peripheral blood mononuclear cells from four HIV-1-infected individuals, in conjunction with a survey of determinants in the Los Alamos database, revealed similarities among "hotspot" positions for CD4(+) and CD8T(+) cells within mice and humans. These results emphasized the important influence that envelope peptide position may have on antigen processing, and the consequent impact such processing may have on HIV-1-specific CD4(+) and CD8(+) T-cell activities.     

Cross-clade conservation of HIV type 1 Nef immunodominant regions recognized by CD8+ T cells of HIV type 1 CRF02_AG-infected Ivorian (West Africa)

Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.     

Serum IgA of HIV-exposed uninfected individuals inhibit HIV through recognition of a region within the alpha-helix of gp41

BACKGROUND: HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro. OBJECTIVES: To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals. METHODS: Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication. RESULTS: IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the alpha-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5. CONCLUSION: These results have important implications for the development of vaccines and therapeutical strategies against HIV infection.     

Identification of T-cell epitopes without B-cell activity in the first and second conserved regions of the HIV Env protein.

We have previously hypothesized that an effective vaccine against HIV should elicit cell-mediated immunity without antiviral antibody production. As a first step towards this goal we have identified potential T-cell epitopes, without B-cell activity against the native protein, from the first and second conserved sequences, and from three functionally important regions of the HIV-1 envelope protein gp160. For this approach, short peptide sequences selected by established computer programs were synthesized and chemically modified to generate either polymers with disulfide bonds, or micelles with two palmitic acid residues attached to the amino-terminal lysine. In both configurations several peptides were immunogenic without the need for coupling to carrier molecules. Of the 19 peptides we tested in our present studies, seven induced good T-cell proliferative response in mice representing four major histocompatibility complex haplotypes. None of these seven peptides produced antibodies that could recognize the envelope protein gp160.     

Identification of CD4+ T cell epitopes of Taenia solium paramyosin

T cell mediated response is involved in a protective immune response against experimental cysticercosis conferred by immunization with Taenia solium paramyosin (TPmy) to BALB/c mice. In this study, we analysed the TPmy amino acid sequence for predicted CD4+ T cells epitopes. Five different regions of this protein showed that the residues anchor to bind the I-Ad molecule, synthetic peptides containing these epitopes were evaluated for their ability to induce lymphoproliferative responses of spleen cells from TPmy immunized mice. Among them, Tp176 (amino acids 176-192 sequence DDLQRQMADANSAKSRL) was the immunodominant T cell epitope of TPmy. Delineation of this epitope should facilitate analysis of the role of CD4+ T cell response in experimental cysticercosis.     

Generation and structural analysis of soluble oligomeric gp140 envelope proteins derived from neutralization-resistant and neutralization-susceptible primary HIV type 1 isolates

We generated DNA constructs expressing soluble truncated forms of the envelope of SF162, a neutralization-resistant primary human immunodeficiency virus type 1 isolate, and SF162AV2, a neutralization-susceptible virus derived from SF162 after the deletion of 30 amino acids from the V2 loop. The constructs express the entire gp120 subunit and the extracellular region of the gp41 subunit, with either the presence ("cleaved" forms, designated gp140C) or the absence ("fused" forms, designated gp140F) of the gp120-gp41 cleavage site. Both gp140 forms derived from SF162 and SF162deltaV2 are secreted in the cell medium and are recognized by the oligomer-specific anti-gp41 MAb T4. As is the case for the corresponding virion-associated envelope molecules, the CD4-binding region is occluded within both gp140F and gp140C forms. However, structural differences exist between these two forms. The gp140F proteins are less efficiently recognized than the gp140C proteins by antibodies present in the sera of HIV-infected patients with neutralizing activities against SF162 and SF162AV2. Also, the V3 loop is more exposed on gp140F than gp140C. As is the case for intact virions, on CD4 binding both the gp140F and gp140C proteins undergo conformational changes that result in the exposure of the epitope recognized by MAb 17b, which has been implicated in coreceptor binding. In contrast, during these structural changes the exposure of specific V3 loop epitopes is not increased on either gp140C or gp140F. Taken together, our data indicate that although these gp140 forms differ structurally from the native envelope, their similarities, in particular that of gp140C, outweigh their differences.     

Impact of intrapeptide epitope location on CD8 T cell recognition: implications for design of overlapping peptide panels

BACKGROUND: Antigen-specific CD8 T cells following infection or immunization are typically assessed by measuring interferon-gamma production after stimulation with overlapping peptides spanning the region of interest. The effect of epitope location within such peptides is not known but may influence recognition. OBJECTIVE: To examine if peptides containing the appropriate C-terminal anchor amino acid residue would provide more sensitive detection of T cell responses. The impact was examined of epitope location within overlapping peptides on recognition of epitope-specific CD8 T cell responses. METHODS: C-terminal amino acid residues were analyzed in well-defined optimal epitopes for HIV, Epstein-Barr virus, cytomegalovirus and influenza and in peptide-binding motifs. Recognition of known epitopes within longer synthesized peptides by peripheral blood mononuclear cells or CD8 T cell lines was tested using interferon-gamma Elispot at various peptide concentrations. RESULTS: Only 9 of 20 amino acids served as the C-terminal anchor position in 96% of described optimal epitopes and in 95% of peptide-binding motifs. A CD8 T cell response to an epitope within a longer peptide is best detected when the epitope is situated at the C-terminal end of the longer peptide, both when using peptides designed to include the optimal epitope at every possible position and when comparing responses towards optimal epitopes and corresponding overlapping peptides in a larger group of subjects. CONCLUSION: When using overlapping peptides to screen for CD8 T cell responses, more sensitive detection will be achieved using known C-terminal anchor amino acid residues at the C-terminus.     

Development of anti-HIV agents targeting dynamic supramolecular mechanism: entry and fusion inhibitors based on CXCR4/CCR5 antagonists and gp41-C34-remodeling peptides

A molecular mechanism involved both in HIV-entry and -fusion steps has been disclosed in detail: The interaction of an HIV envelope protein, gp120, with chemokine receptors, CXCR4 and CCR5, which were identified as major co-receptors in association with CD4, triggers conformational changes in the gp120-gp41 (another envelope protein) complex, and subsequently forms the trimer-of-hairpins structure of gp41 followed by virus-cell membrane fusion. The elucidation of the above dynamic supramolecular mechanism in HIV-entry and -fusion has provided insights into new type of drugs that can block HIV infection. Based on this, we have developed not only coreceptor antagonists (1) but also fusion inhibitors (2). (1) Potent CXCR4 antagonists, T22 and T140, have been developed through the structure-activity relationship studies on tachyplesins and polyphemusins that are horseshoe crabs' antimicrobial peptides. T22, which was initially found to bind gp120 and CD4, and T140 selectively suppress T-cell line-tropic HIV-1 (X4-HIV-1) entry based on their specific binding to CXCR4. Furthermore, molecular-size reduction of T140 using cyclic pentapeptide templates brought us to find low molecular weight CXCR4 antagonists, such as FC131. (2) Artificial remodeling of a gp41 fragment, C34, has led to development of strong inhibitors of HIV-fusion into cells. These fusion inhibitors effectively block the formation of the trimer-of-hairpins structure of gp41. HIV-entry/fusion inhibitors such as CXCR4 antagonists and C34 analogs would improve the clinical chemotherapy of AIDS and HIV-infected patients. This review article focuses on our recent research on the development of the above two types of inhibitors, including comparative studies with several CXCR4 antagonists besides T22/T140-related compounds and other fusion inhibitors such as Fuzeon (T-20).     

Epitope mapping of human monoclonal antibodies recognizing conformational epitopes within HTLV type 1 gp46, employing HTLV type 1/2 envelope chimeras.

The majority of the antibody response to HTLV-1 surface glycoprotein, gp46, is directed at conformational epitopes. However, the regions of HTLV-1 gp46 that contain conformational epitopes are poorly defined. We previously reported on human monoclonal antibodies (hMAbs) to conformational epitopes within the HTLV-1 surface glycoprotein (gp46) that inhibit HTLV-1-mediated syncytium formation (Hadlock KG, Rowe J, Perkins S, et al.: J Virol 1997;71:5828-5840). To localize the conformational epitopes recognized by these antibodies, chimeric envelope proteins were constructed in which selected regions of the HTLV-1 envelope were replaced with the corresponding sequences from other members of the HTLV family of retroviruses. The chimeras were tested for reactivity with three hMAbs to conformational epitopes in HTLV-1 gp46, PRH-7A, PRH-3, and PRH-4, and one hMAb to a linear epitope, 0.5alpha. hMAb PRH-3 was specifically nonreactive with a chimera that replaced amino acids 32-36 of HTLV-1 gp46 and exhibited sharply reduced reactivity with a chimera that replaced amino acids 224-251 of HTLV-1 with the corresponding HTLV-2 sequence. hMAb PRH-4 was specifically nonreactive with a construct replacing amino acids 1-162 of HTLV-1 gp46 with the corresponding HTLV-2 sequence. Thus, HTLV-1 gp46 contains multiple conformational epitopes located in the amino-terminal portion of the protein.     

An HLA-directed molecular and bioinformatics approach identifies new HLA-A11 HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected Thais.

Only limited cytotoxic T lymphocyte (CTL) epitope mapping has been done in nonsubtype B HIV-infected persons. We used molecular immunogenetic tools to determine HIV-specific CTL responses in HIV-1 Env subtype E-infected female sex workers (FSWs) from northern Thailand, where more than 50% of the population is HLA-A11 positive. EpiMatrix, a computer-based T cell epitope prediction algorithm, and a manual editing approach were used to predict 77 possible HLA-A11 CTL epitopes in HIV-1, some of which were conserved between subtypes B and E. MHC binding of these peptides was determined in an HLA-A11 stabilization assay, and binding peptides were tested for CTL recognition in eight HLA-A11-positive FSWs. Subtype E versions of known HLA-A2 subtype B HIV epitopes were also tested in four HLA-A2 positive FSWs. CTL responses were detected in all HLA-A11-positive and in three of four HLA-A2-positive persons. Among the 12 FSWs responses to peptides were found to Pol in 9 (75%), Env in 7 (58%), Nef in 5 (42%), and Gag in 5 (42%), and to conserved epitopes in 8 (67%). To identify HLA-A11 CTL epitopes in the absence of prediction tools, it would have been necessary to test almost 3000 10-mer peptides. EpiMatrix and manual predictions reduced this number to 77, of which 26 were MHC binding and 12 were CTL epitopes. Six of these HLA-A11 CTL epitopes have not been previously reported and are located in RT, gp120, and gp41. This report of CTL responses in subtype E-infected individuals defines epitopes that may be useful in HIV pathogenesis or vaccine studies.     

Propagation of CD4+ T cells specific for HIV type 1 envelope gp120 from chronically HIV type 1-infected subjects

HIV-specific CD4+ T cell responses, in particular to the HIV envelope antigen gp120, are often undetectable in the peripheral blood of HIV-infected individuals. The failure to detect these cells poses a significant impediment to studying the T cell populations that are considered to be essential for controlling HIV infection and has led to speculation that these cells are entirely depleted during HIV infection. This study was designed to test whether gp120-specific CD4+ T cells exist in HIV-infected subjects and can be expanded from peripheral blood mononuclear cells by in vitro stimulation with the gp120 antigen, allowing better characterization of these cells. Although gp120-specific T cell responses were barely observed in patient cells ex vivo before antigenic stimulation, CD4+ T cells specific for gp120 were successfully propagated from the blood of each asymptomatic chronically HIV-infected subject studied. The dominant epitopes recognized by gp120-specific CD4+ T cells from these HIV-infected subjects were mapped to well-conserved sites in the C1 and C2 domains of gp120. Two CD4+ T cell lines recognizing these two regions were subsequently established. The CD4+ T cell lines proliferated and produced interferon gamma in response to the specific epitopes, and the responses were MHC class II restricted. These T cell lines also exhibited cross-reactivity with gp120 from T cell line-adapted HIV-1 strains IIIB and MN, as well as with gp120 from primary isolates SF33 (subtype B), CA1 (subtype A), and CA10 (subtype A/E). The data demonstrate that CD4+ T cells specific for gp120 are not entirely depleted from the peripheral blood of chronically HIV-infected subjects; these cells are present in low numbers but can be expanded after antigenic stimulation in vitro.     

Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O

An effective vaccine against AIDS should induce both cellular and humoral immune responses. Here we report that immunization of mice with a DNA plasmid encoding a chimeric protein consisting of HIV89.6 Env gp140 and the listeriolysin O (LLO) C-terminal segment (59 amino acids) significantly enhanced both humoral and cellular immune responses against the HIV89.6 Env protein. Plasmid DNA expression vectors with genes codon-optimized for mammalian expression were synthesized for HIV89.6 gp140 as well as for chimeric protein gp140-LLO, in which the coding sequence for the C-terminal 59 amino acids of LLO were fused in frame to the 3' end of the codon-optimized gene for gp140. All plasmid vectors produced high levels of protein expression, and the gp140-LLO chimeric protein was cleaved and secreted as efficiently as gp140. Analysis of humoral immune responses by ELISA showed that the chimeric gp140-LLO construct induced higher antibody responses than the gp140 construct in immunized mice, more notably in the IgG2a antibody subtype. Intracellular cytokine staining and flow cytometry analysis showed that the gp140-LLO construct induced significantly higher levels of cytotoxic T lymphocyte immune responses against the HIV 89.6 Env protein than those observed with the gp140 construct. Our results thus demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses against the HIV89.6 Env antigen in the context of a DNA vaccine.     

Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins.

Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.