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.     

Human T cells recognize multiple epitopes of an immediate early/tegument protein (IE62) and glycoprotein I of varicella zoster virus.

Infection with varicella zoster virus (VZV) elicits persistent cell-mediated immunity directed against the immediate early (IE62) protein and the glycoprotein I (gp I) in most healthy subjects. In these experiments, synthetic peptides corresponding to residues of the IE62 protein and gp I were used to identify linear amino acid sequences of these immunogenic VZV proteins that were recognized by peripheral blood T lymphocytes from VZV-immune individuals of known major histocompatibility complex (MHC) type. All of 12 VZV-immune donors had T-cell proliferative responses, defined as a stimulation index (SI) greater than or equal to 2.0, to at least two of ten synthetic IE62 peptides; the mean number of IE62 peptides recognized by T cells from VZV-immune donors was seven. Five of the ten IE62 peptides stimulated T cells from 75% to 83% of the VZV-immune donors; the other five IE62 peptides were recognized by T cells from 42% to 67% of the subjects. All VZV-immune donors also had T proliferation responses to at least two of ten synthetic gp I peptides; the mean number of peptides recognized was six. Six of the ten gp I peptides were recognized by T cells from 67% to 92% of the VZV-immune donors; the frequency of donors responding to the other gp I peptides ranged from 42% to 58%. None of five nonimmune donors demonstrated T-cell proliferation to any of the IE62 or gp I peptides. A combination of two IE62 peptides provided epitopes that could be recognized by T cells from all twelve VZV-immune donors, regardless of DR type. Similarly, one gp I peptide in combination with either of two other gp I peptides induced proliferation of T cells from all immune subjects. Memory T cells with specificity for multiple short amino acid sequences of the IE62 protein and gp I were detected in subjects who had had primary VZV infection more than 20 years earlier. These observations indicate that natural VZV infection elicits a diverse cell-mediated immune response to viral proteins that is not restricted to only one or two immunodominant regions. Although the usefulness of peptide vaccines remains to be established, multiple epitopes of the IE62 protein and gp I were identified that could be presented by antigen-presenting cells (APC) and recognized by T cells from most subjects in an "outbred" human population.     

Reversal of the inhibitory effects of HIV-gp120 on CD4+ T cells by stimulation through the CD28 pathway

The effects of exposure to HIV-gp120 on proliferation and cytokine production by T cell lines were investigated. T cell lines were generated by stimulation of peripheral blood mononuclear cells from several healthy donors with cross-linked anti-CD3 antibodies and IL-2. These T cell lines exhibited the characteristics of Th1 cells, producing IL-2 and interferon-gamma (IFN-γ), but not IL-4, on stimulation with anti-CD3 antibodies. In the presence of gp120, stimulation with anti-CD3 antibodies was inhibited in terms of both proliferative responses and the secretion of IL-2 and IFN-γ. Similar effects were observed when a T cell line was stimulated in the presence of a synthetic peptide representing the CD4-binding region of gp120. Neither gp120 nor the CD4-binding region peptide had any effect on IL-4 production by the T cell lines. Stimulation through the CD28 pathway partially restored both the proliferative effect and cytokine production by T cell lines in response to anti-CD3 antibodies in the presence of gp120. Anti-CD28 antibodies also partially restored cytokine production when purified CD4⁺ T cells from a T cell line were stimulated with anti-CD3 antibodies in the presence of gp120. Anti-gp120 antibodies partially or completely reversed the inhibitory effects of gp120 on T cell proliferation. These results indicate that stimulation through the CD28 pathway may restore defective CD4⁺ T cell responses in HIV-infected individuals.     

Comparison of the Number of IL–4 and IFN–7 Secreting Cells in Response to the Malaria Vaccine Candidate Antigen Pf155/RESA in Two Groups of Naturally Primed Individuals Living in a Malaria Endemic Area in Burkina Faso

The enzyme-linked immunospot (ELISPOT) assay was used to enumerate the number of IFN-γ and IL–4 producing cells after in vitro stimulation with a highly purified recombinant malaria vaccine candidate antigen (r-Pf155/RESA) or synthetic peptides corresponding to its major T-cell epitopes. Two groups of naturally primed individuals living in rural areas of Burkina Faso were studied. The donors comprised one group of healthy (non-parasitemic) mainly adult people and one parasitemic mainly younger people. IL–4 producing cells were detected in response to PHA but no such cells were detected in response to the malarial antigens. The most frequent IFN–7 responses were seen with r-Pf155/RESA. Thus, after stimulation with this antigen 52% of the donors responded positively in the ELISPOT assay, while only 17% responded to the synthetic peptides, suggesting that the r-Pf155/RESA contained T-cell epitopes not covered by the peptides used in this study. The number of IFN-γ producing cells in response to the malarial antigens did not differ between the two groups. However, IFN-γ levels found in sera from the parasitemic individuals were significantly higher than in those from healthy donors. This latter finding and the lack of differences seen in the number of IFN-γ producing spots in the two groups indicate that IFN-γ producing cells may have sequestered to other organs in the parasitemic group.     

Immunization with subunit human immunodeficiency virus vaccine generates stronger T helper cell immunity than natural infection.

Healthy, human immunodeficiency virus seronegative (HIV-) volunteers were multiply immunized with a recombinant gp160 (rgp160) candidate acquired immunodeficiency syndrome (AIDS) vaccine. Peripheral blood lymphocytes from volunteers immunized with 40 micrograms or with 80 micrograms (two volunteers per group) of rgp160, as well as from control donors, were tested for T helper (Th) cell function either prior to immunization, 8 to 12 months after the third immunization, or 2 to 5 months after the fourth immunization. The Th cell functional tests included antigen-induced in vitro interleukin 2 (IL 2) production and proliferation in response to influenza A virus (FLU) and to four synthetic peptides of HIV gp120 and gp160, previously demonstrated to be recognized by T cells from HIV naturally infected patients. Our results demonstrate the following: (a) immunization of HIV- individuals with rgp160 results in IL 2 production and T cell proliferation in response to HIV determinants; (b) boosting with rgp160 enhances Th function; (c) HIV-specific Th function is up to 100-fold greater in the multiply immunized volunteers than that observed in asymptomatic, HIV-infected individuals; and (d) multiple immunization with rgp160 does not impair Th function to a non-HIV antigen such as influenza A virus. These results indicate that immunization of uninfected individuals with an HIV subunit vaccine results in much stronger Th cell immunity than does natural infection and suggests that vaccination against HIV may be possible.     

Enhanced proliferative cellular responses to HIV-1 V3 peptide and gp120 following immunization with V3:Ty virus-like particles.

The induction of CD4+ T-helper (Th) cell responses is likely to be an important requirement of vaccine candidates designed to prevent or moderate human immunodeficiency virus-1 (HIV-1) infection. We have investigated the ability of hybrid Ty virus-like particles carrying the V3 loop region of the HIV-1 IIIB envelope gp120 (V3:Ty-VLP) to elicit V3-specific proliferative responses. Significant proliferation in response to stimulation in vitro with homologous IIIB V3 peptide was observed following immunization of mice with V3:Ty-VLP either as an aluminium hydroxide precipitate or without adjuvant. Responses to MN V3 peptide were also observed in certain mouse haplotypes. To assess the effect of presenting the V3 loop in this particulate form, we compared the responses induced by V3:Ty-VLP with those obtained with two non-particulate immunogens, recombinant gp120 (rgp120) and V3 peptide conjugated to albumin. V3-specific responses to V3 peptide in vitro were reproducibly higher following immunization with V3:Ty-VLP than with either rgp120 or V3-albumin coagulate (V3-alb). The data indicate that immunization with the V3 loop as a hybrid Ty-VLP results in enhanced proliferative responses to V3 peptide and recognition of rgp120 in vitro. Some cross-reactivity of Th cells for V3 sequences from different isolates was also observed.     

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.     

Lymphotoxin activates hepatic T cells and simultaneously induces profound thymic atrophy.

The resistance and susceptibility of T cells to human immunodeficiency virus (HIV)-gp120 induced anergy was examined. Antigen-dependent proliferation of polyclonal T cells was markedly inhibited by gp120, whereas from the analysis of monoclonal populations, T cells resistant to the effects of gp120 could be identified. Similarly, exposure of monoclonal T cells to gp120 in the absence of accessory cells, also demonstrated that some T cells could resist the induction of anergy. Loss of antigen recognition was associated with phenotypic modulation of CD3 and CD28, which was not observed in T cells resistant to functional inactivation by gp120. Modulation of CD4 was not related to induction of anergy in the monoclonal T cells examined in this study. Inhibition of T-cell responses by anti-CD4 antibodies was compared to that by gp120. Anti-CD4 antibodies, which cross-compete with gp120 for binding to CD4, inhibited the response to antigen of monoclonal T cells. In contrast, no tolerogenic signals were delivered by pretreating T cells with the anti-CD4 antibodies in the absence of accessory cells, indicating that inhibition was due to abrogation of the interaction of CD4 with major histocompatibility complex (MHC) class II molecules expressed on accessory cells. Although the free CD4-binding region peptide of gp120 could inhibit polyclonal T-cell responses, only the carrier-bound peptide was able to modulate cloned T cells, suggesting a conformational requirement for functional inactivation through engagement of CD4. The results reported here using clonal CD4+ T-cell populations demonstrate that effects of gp120 on antigen-dependent proliferation are not uniform, and that therapeutic intervention might be directed at T-cell populations identified as susceptible to HIV-gp120 induced anergy.     

Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells.

In earlier studies, primary proliferative and cytotoxic T-cell (CTL) responses to influenza virus were produced in vitro by using mouse dendritic cells (DC) pulsed with virus or viral peptide as the stimulus for syngeneic T cells in 20-microliters hanging-drop cultures. We have now adapted this system for producing primary responses with cells from non-immune donors to produce primary proliferative and CTL responses to human immunodeficiency virus I (HIV) and to HIV peptides in vitro using cells from normal human peripheral blood. All donors in this study were laboratory personnel with no history of HIV infection. DC enriched from peripheral blood were exposed to HIV in vitro and small numbers were added to T lymphocytes in 20-microliters hanging drops. Proliferative responses to virus-infected DC were obtained after 3 days in culture. After 6 days, CTL were obtained that killed virus-infected autologous--but not allogeneic--phytohaemagglutinin (PHA)-stimulated blast cells. Proliferative and CTL responses were obtained using cells from 14 random donors expressing a spectrum of major histocompatibility complex (MHC) types but the CTL, once produced, showed killing restricted by the MHC class I type. Treatment of cultures with monoclonal antibody (mAb) to CD4-positive cells at the beginning of culture blocked the development of both proliferative and CTL responses, but treatment after 5 days had no effect on the CTL activity. Treatment with MCA to CD8-positive cells at the beginning of culture did not block proliferation significantly, but treatment either before or after the 5-day culture period blocked CTL responses. Collaboration between proliferating CD4-positive cells and CD8-positive cells may thus be required to produce CTL of the CD8 phenotype. DC exposed to HIV also produced CTL that killed autologous blast cells pulsed with gp120 envelope glycoprotein. However, DC infected with whole virus did not produce CTL that lysed target cells pulsed with a synthetic peptide, which included a known T-cell epitope of gp120 (representing amino acids 111-126). DC pulsed with gp120 were a poor stimulus for the development of CTL. In contrast, DC pulsed with the peptide (111-126) stimulated both proliferative and CTL responses. The latter killed not only target cells pulsed with the peptide itself or with gp120 but also killed virus-infected autologous blast cells. CTL were again obtained reproducibly with this peptide using donors expressing a spectrum of MHC types.(ABSTRACT TRUNCATED AT 400 WORDS)     

A gp120 HIV peptide with high similarity to HLA class II beta chains enhances PPD-specific and autoreactive T cell activation.

The recent report that anti-gp120 antibodies can be induced by allogeneic stimuli in experimental animals in the absence of HIV, has focused attention on the structural similarities between gp120 and MHC. Here we report that some HIV+ individuals develop antibodies which similarly react with the gp120 HIV sequence (aa 254-263) and with the HLA-DR beta chains (aa 142-151). As these two peptides share a high level of similarity, we have investigated the role of this gp120 region on HLA class II mediated T cell recognition. The synthetic peptide corresponding to the gp120 HIV sequence aa 254-263 has been tested on T cell line (TCL) activation. Both the PPD-specific and the self-HLA reactive TCL proliferation increased in the presence of this peptide. Prepulsing experiments indicate that this enhancing effect carried out by HIV peptide is exerted at the level of antigen presentation. Moreover, the specificity of this interaction is supported by the fact that a MoAb specific for this HIV peptide blocked the autoreactive TCL proliferation, similarly to the inhibition carried out by anticlass II antibody. These data support the hypothesis that the functional homology between the HIV peptide and the HLA beta chain described may be involved in the pathogenesis of AIDS.     

Antibody-dependent cellular cytotoxicity (ADCC) is directed against immunodominant epitopes of the envelope proteins of human immunodeficiency virus 1 (HIV-1).

In this study, epitopes of HIV envelope proteins that are involved in ADCC were identified. Peripheral blood mononuclear cells (PBMC) were obtained from adults with asymptomatic HIV infection or early symptoms of AIDS. These PBMC, which were reported to be "armed" in vivo with HIV-specific antibodies, were used as effector cells in 51Cr release assays. Target cells consisted of CD4 lymphocytes from healthy seronegative donors, coated with the IIIB strain of HIV-1 or with one of seven synthetic peptides. Cytotoxicity was detected against CD4 lymphocytes coated with HIV-1 IIIB or with the peptides env aa 507-518, corresponding to the carboxy-terminus of gp120, and env aa 597-611, corresponding to the region of the cysteine loop of gp41. The magnitude of target cell lysis was directly related to the quantity of peptide used. In contrast, target cells coated with the peptide gag aa 129-135, corresponding to the p17/p24 cleavage region of the gag precursor, were not killed. The same immunodominant regions which were involved in ADCC were recognized in enzyme-linked immunoabsorbent assays (ELISA) by the majority of 107 sera from HIV-infected adults. We conclude that the immunodominant epitopes located at the carboxy-terminus of gp120 and the cysteine loop of gp41 serve as recognition structure for antibodies, capable of mediating ADCC against HIV-infected cells.     

Synthetic peptides identify promiscuous human Th1 cell epitopes of the secreted mycobacterial antigen MPB70

MPB70 is a secreted protein of Mycobacterium bovis and Mycobacterium tuberculosis which stimulates both cellular and humoral immune responses during infection with bovine and human tubercle bacilli. In addition, vaccination with MPB70 has been shown to induce Th1 cell responses and protection in animal models of tuberculosis. The present study was carried out to map the dominant human Th1 cell epitopes of MPB70 in relation to major histocompatibility complex (MHC) class II restriction in healthy subjects showing strong T-cell responses to complex mycobacterial antigens. Peripheral blood mononuclear cells (PBMC) from HLA-DR-typed donors were tested with complex mycobacterial antigens (whole-cell M. tuberculosis and M. tuberculosis culture filtrates), with MPB70 purified from the culture filtrate of M. bovis BCG Tokyo, and with 13 synthetic peptides (25-mers overlapping by 10 residues) covering the sequence of MPB70. The donors that responded to the complex antigens and MPB70 also responded to the cocktail of synthetic MPB70 peptides. Testing of PBMC with individual peptides showed that peptides p5 (amino acids [aa] 61 to 85), p6 (aa 76 to 100), p8 (aa 106 to 130), p12 (aa 166 to 190), and p13 (aa 181 to 193) were most frequently recognized in proliferation and gamma interferon (IFN-gamma) assays. Testing of antigen-specific CD4(+) T-cell lines with the individual peptides of MPB70 confirmed that peptides p8, p12, and p13 contain immunodominant Th1 cell epitopes of MPB70. MHC restriction analysis with HLA-typed donors showed that MPB70 and its immunodominant peptides were presented to T cells promiscuously. The T-cell lines responding to MPB70 and peptides p8, p12, and p13 in IFN-gamma assays mediated antigen-peptide-specific cytotoxic activity against monocytes/macrophages pulsed with the whole-protein antigen or the peptides. In conclusion, the promiscuous recognition of MPB70 and its immunodominant peptide defined epitopes (aa 106 to 130 and 166 to 193) by IFN-gamma-producing Th1 cells supports possible application of this secreted antigen to subunit vaccine design.     

Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors.

The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T-cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287-467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significantly frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes, mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.     

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/mm³, 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.     

Immunogenicity of the ALLAVGATK (gp10017 – 25) peptide in HLA-A3.1 melanoma patients

A T cell line recognizing autologous and allogeneic HLA-A3.1 melanomas was obtained from a disease-free melanoma patient (patient 15392). By transfection of a tumor cDNA library and in vitro sensitization experiments, the ALLAVGATK gp100/Mel17-derived peptide was found to be the epitope recognized by this melanoma-specific T cell line. The role of the ALLAVGATK peptide in the systemic immune response to melanoma of this patient was evaluated. When pulsed on the autologous peripheral blood mononuclear cells, the ALLAVGATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single restimulation in vitro was sufficient to raise gp100-specific T lymphocytes, indicating a high frequency of epitope-specific T cells. gp100-specific T cells were also induced from T lymphocytes purified from tumor-invaded lymph nodes (tumor-associated lymphocytes, TAL). TAL-derived effectors displayed lower peptide affinity and lower tumor recognition than effectors elicited from peripheral blood lymphocytes (PBL). To further evaluate its immunogenicity, ALLAVGATK was used to stimulate PBL derived from six additional HLA-A3.1 melanoma patients and seven healthy donors. After 7 weeks of peptide stimulation in vitro the generation of anti-gp100 and tumor-specific T cell lines was achieved in one out of the six patients analyzed. Taken together these data indicate that an in vivo priming leading to a systemic immunity against gp100 in HLA-A3 melanoma patients may occasionally occur and that the immunogenicity of ALLAVGATK peptide in melanoma patients is comparable to that of other HLA-A2-restricted epitopes derived from gp100/Mel 17 protein.     

The effect of a single amino acid substitution within the V3 loop of HIV-1 gp120 on HLA-DR1-restricted CD4 T-cell recognition.

Viral variation has been proposed to play a role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection, and is an important consideration in vaccine design. During the course of an infection, isolates with sequence changes in CD8 T-cell and B-cell epitopes arise. To determine whether sequence variation within the V3 loop of HIV-1 gp120 affects HLA-DR beta 1*0101-restricted CD4 T-cell recognition, we have generated CD4 T-cell clones (TLC) specific to gp120 V3 loop peptides. Four HLA-DR beta 1*0101-restricted groups of TLC were defined by distinct patterns of responses to a panel of peptides, consistent with a highly diverse T-cell repertoire recognizing the 30 amino acid stretch (296-326) of the gp120 V3 loop. Nevertheless, a single residue change at position 311 was found to abolish the recognition of two of the four groups of TLC. This was not due to an effect of the residue at 311 on binding to major histocompatibility complex (MHC), because: (1) irrespective of the residue at 311, peptides competed well with the influenza haemagglutinin peptide 307-319 for binding to cell-bound DR1; and (2) R311-specific TLC were also HLA DR beta 1*0101 restricted. Instead, the substitution of arginine for serine at position 311 blocked the interaction of the peptide with the T-cell receptor. Thus, despite the diversity of the T-cell response to the V3 loop of HIV-1, a single amino acid change can have a considerable influence on the responding T-cell population. As residue 311 is one of the most variable of the V3 loop residues, these results suggest that CD4 recognition can also exert pressure on viral variation consistent with a role for these cells in antiviral immunity.     

HIV vaccine therapy

The immunoregulatory mechanisms responsible for affective the in vivo post-infection control of HIV are unknown. In the setting of natural HIV infection, host-directed immune responses directed against gp120 are minimal. This includes poor gp120 epitope specific antibody responses to C1, C2 and C3; lack of T-cell recognition and proliferation to gp120; and minimal envelope cytotoxic T-cell responses. The historical important of anti-envelope responses in the control of other viral pathogens, coupled with the paucity of anti-envelope responses elicited as a consequence of HIV infection, formed the basis for our rationale to pursued a research program which focused on post-infection vaccination utilizing HIV enveloped derived products. Recently we have demonstrated the scientific feasibility of post-infection vaccination with an HIV envelope based vaccine to broaden host-directed HIV-specific immune responses to include seroconversion to gp120 C1, C2 and C3; the induction of T-cell recognition to gp160; and cytotoxic T-cell responses to envelope peptides. The in vivo safety and immunogenicity of the product in adult patients with early HIV infection has been demonstrated. An expanded phase II trial with rgp160 (MGS) is ongoing to examine the potential role of HIV-specific vaccine therapy to alter the natural history of HIV infection. Finally, we are exploring the value of post-infection immunization in defining specific immune responses and their in vivo HIV immunoregulatory relevance.     

T cell epitopes of the major fraction of rye grass Lolium perenne (Lol p I) defined using overlapping peptides in vitro and in vivo. I. Isoallergen clone1A.

One hundred and fifteen overlapping synthetic peptides spanning the entire sequence of the iso-allergen clone1A of Lol p I from rye grass Lolium perenne were synthesized by the multi-pin technique. The peptides were overlapping 12mers, offset by two residues and overlapping by 10 residues. Sets of six adjacent overlapping peptides (except pool-1, 15, 20) were pooled and were used in vitro and in vivo to map the T cell epitopes on Lol p I. Six atopics who were skin test and RAST positive to rye grass showed T cell responses to L. perenne extract (LPE) and its major fraction (Lol p I). Five out of six showed T cell responses in vitro to peptide pool-17, while five non-atopics did not respond to any of the peptide pools. By testing the individual peptides of pool-17, we have located the T cell epitope on Lol p I. Interestingly, when we tested pool-17 and its single peptides in vivo by intradermal skin testing we found in one patient a typical DTH after 24-48 h to pool-17 and its peptides (peptides 3 and 4) which exactly matched the in vitro responses. By defining the T cell epitopes in this way a greater understanding of the allergic response to pollen will be obtained, and a more effective and less dangerous vaccine may be possible for treating patients with hay fever.     

gp120- and TNF-alpha-induced modulation of human B cell function: proliferation, cyclic AMP generation, Ig production, and B-cell receptor expression

BACKGROUND: It is well known that HIV-1 infection induces profound alterations in the immune system, including hyperactivation of B cells. TNF-alpha induces HIV-1 replication and immunodysregulation, including polyclonal B-cell activation. OBJECTIVE: We sought to determine the effects of surface-binding HIV-1 envelope glycoprotein (gp120) and TNF-alpha on human B-cell function. METHODS: HIV-1 seronegative peripheral blood human B cells were purified and activated by CD40 mAb and IL-4. In vitro studies of B-cell proliferation, cyclic AMP (cAMP) generation, receptor expression, and Ig production were performed. RESULTS: gp120, an Ig superantigen, stimulated HIV-1 seronegative and HIV-1 seropositive human B-cell cAMP generation, proliferation, and Ig production. These gp120-induced B-cell responses were demonstrated to be specific as evidenced by the abrogation of the stimulatory response in the presence of anti-gp120 mAb, blocking of CD4 resulting in no change on gp120-induced B-cell responses, and the binding of gp120 in these B cells. TNF-alpha also stimulated cAMP generation, proliferation, and Ig production in B cells, and the binding of gp120 to these B cells stimulated by TNF-alpha further enhanced cell proliferation, cAMP generation, and Ig production. Antigenic expression of the B-cell receptor CD79b was down-regulated by gp120 but was not altered by the addition of TNF-alpha. CONCLUSION: gp120 modulation of TNF-alpha-induced B-cell receptor- and cAMP-mediated signal transduction events may be involved in the B-cell abnormalities observed in HIV-1 infection.