Lymphoproliferative response to synthetic V3 loop P18 peptide and HIV-1 envelope glycoprotein among individuals immunized with gp160 candidate vaccines.

Because T-cell responses are critical for defense against viral infections, the synthetic P18 peptide (RIQRGPGRAFVTIGK) and active component of gp160 protein has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cells, responses which are known to play a role in enhancing the immunological response to foreign antigen, we studied the response of individuals immunized with HIV gp160 candidate vaccines. We investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMC) derived from individuals immunized with gp160 antigens in three different protocols. We found a PBMC proliferative response to synthetic P18 peptide in healthy immunized individuals induced by gp160 antigen with or without vaccinia virus. There was correlation between the proliferative response to P18 peptide and other antigens such as HIV-like proteins and gp160 molecule. HLA-DR typing revealed the possible presentation of P18 peptide by several different class II molecules. Since these class II molecules occur frequently in the general population, P18 peptide appears to contain broadly reactive epitopes and thus is presented by multiple HLA class II molecules. Due to its broad reactivity P18 peptide is one of the candidates for inclusion as a subunit vaccine against HIV-1.

HIV-1-induced immune suppression may result from autoimmune disorders including anti-SLWDQ autoantibodies.

We have previously unravelled the striking SLWDQ pentapeptide identity between HIV-1 env gp120 and the CD4 molecule. We show here that this pentapeptide is required for the functioning of the co-stimulatory MHC-CD4 signal in T4-cell activation since it suppresses antigen-induced T-cell proliferation. Moreover, concerning the MHC class II counterpart, the LNGQEETGVVSTN sequence which strongly inhibits T-cell immune activation is likely to be part of the functional site of the molecule. Interestingly the MHC/gp120 homology described by Young overlaps this MHC region. We further report that the gp120 SLWDQ peptide triggers an immune reaction which is both humoral (anti-SLWDQ antibodies) and cellular (CTLs against autologous targets carrying the pentapeptide) in HIV-1 infected individuals. Finally, anti-SLWDQ antibodies from patients sera purified by column chromatography strongly inhibit antigen-induced immune T-cell activation. This result led us to postulate that these antibodies found in high titers in HIV-1 infected individuals could contribute to set up the progressive systemic immune T-cell suppression characterizing AIDS.

Effect of purified IgGs from HIV-1-infected and non infected individuals on immune activation.

The purification and analysis of IgGs from sera of HIV-1-infected and non infected individuals are reported. The effect of antibodies purified from sera of infected individuals on antigen-induced T cell proliferation was investigated in relation to their possible involvement in an autoimmune reaction in AIDS, in view of the previously unravelled striking peptide similarities between HIV-1 gp120 and the immunoregulatory CD4 and Fas molecules. However, our data do not allow definite conclusions to be drawn. The necessity of purifying antibodies against specific peptides to show their direct effect on T-cell activation is further stressed.

Critical sites: a semantic approach to protein sequences. Application to the HIV-1 envelope molecule.

We have designed two software systems allowing the study of proteins through a comparison to those stored in data banks. The first one, "Automat", locates in a systematic manner all identities shared by a given protein and the proteins in a data bank. The second, "Critic" enables the selection of specific segments in a given molecule by comparing them with those gathered in a data bank. These sites were termed "critical" since they mostly correspond to functional sites (active sites) of the well-known proteins which were studied with the aid of this program (somatostatin, insulin, IL2, etc). Automat allowed us to reveal homologies between HIV-1 and the CD4, which have remained unsolved until now. These similitudes proved to be critical sites (according to Critic). The putative involvement of these sites in the physiopathological processes as induced by HIV-1 are worth considering since the results of our experiments are consistent with this assumption.

Removal of gp160 induced bio-hazards for a safe AIDS vaccine candidate.

In the first AIDS vaccine trial, immunizing preparations were based on HIV-1 Env protein (gp160). Immunogenic properties of gp160 which trigger both a humoral and cellular immune response have since justified its use in various vaccine programs, both past and present. Many reports however have underlined deleterious effects on the immune system--anti-HIV-1 enhanced antibodies, anti-CD4 autoantibodies, and inhibition of T cell activation by HIV-1--particularly associated with the Env protein. The present study shows that gp160 presented in a biologically inactivated but immunogenic form, as used in our trial, could avoid these complications. Bio-hazards associated with gp160 which indeed could be removed by appropriate treatment of the native protein, should be taken into consideration in AIDS vaccine programs.

Cell-mediated immunity against HGP-30, a group-specific peptide of HIV p17 in individuals infected with the AIDS virus.

HGP-30, the synthetic peptide analogue and active component in an HIV-1 (human immunodeficiency virus, type 1) p 17 core-based experimental vaccine, has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cell responses which are known to play a role in enhancing the immunological response to foreign antigens, we studied the response of individuals infected with HIV to HGP-30 at various stages of disease progression. We have investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMCs) derived from individuals infected with HIV-1 to HGP-30. We have found a PBMC proliferative response to HGP-30 in 40% of the healthy seroconverted patients, in 35% of the CDC stage III patients and in 18% of the CDC stage IV patients. There was no correlation between the proliferative response to HGP-30 and other antigens such as HIV-like proteins or tetanus toxoid not to CD4 cell count. HLA-DR typing revealed the possible presentation of HGP-30 by several different class II molecules. Since these class II molecules occur frequently in the general population, HGP-30 appears to contain broadly reactive epitopes and thus is not restricted as are many peptide vaccines. Due to its broad reactivity and extreme conservation in many HIV-1 strains. HGP-30 is one of the promising candidates for inclusion as a subunit vaccine against HIV-1.

Envelope protein and p18(IIIB) peptide recognized by cytotoxic T lymphocytes from humans immunized with human immunodeficiency virus envelope.

Cytotoxic T cells are the main antigen-specific effector cells of the cellular immune system and MHC class I restricted cytotoxic T-lymphocyte (CTL) responses in mice, acting against the HIV-1 envelope protein, are known to be predominantly directed against an amino acid sequence in the third hypervariable domain. We have investigated the epitope specificity of anti-HIV-1 CTL in healthy human volunteers inoculated with a recombinant vaccinia expressing the HIV-1 gp160 envelope gene. Their isolated lymphocytes were stimulated in vitro with autologous HIV-1 infected cells. Our results show that immunization with recombinant virus is able to generate virus-specific CTLs to the HIV-1 gp160 envelope protein and to a 15-residue synthetic peptide corresponding to a highly variable region of the envelope p18(IIIB). The CTL response was restricted by class I MHC molecules HLA-A2 and A3 that commonly occur in the human population.

Identification of CD4 and major histocompatibility complex functional peptide sites and their homology with oligopeptides from human immunodeficiency virus type 1 glycoprotein gp120: role in AIDS pathogenesis.

CD4 molecules interact with class II major histocompatibility complex molecules as a critical costimulatory signal in CD4+ cell immune activation. CD4 also recognizes a specific region of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 forming a binding site for early stages of HIV-1 infection. We designed two software packages, AUTOMAT and CRITIC, which allowed us to identify similarities between regions of HIV-1 proteins and immunoregulatory protein sequences stored in data banks. In this report we have characterized (i) a pentapeptide, SLWDQ, found in both CD4 and HIV-1 gp120, which surprisingly had remained undetected in these two well-studied molecules until now, and (ii) an HLA sequence corresponding to the putative functional site of H2 I-A. We found that a region of gp120 (residues 254-263) known to be similar to a sequence in HLA class II beta chain overlaps this functional region. We showed experimentally that these two CD4 and HLA peptide segments inhibit CD4+ cell immune activation. There is strong inhibition (50% up to 80%) of immune activation by SLWDQ-containing gp120 segments and a lesser inhibition by the gp120 HLA-homologous segment. In addition, we found that SLWDQ induced in HIV-1-infected individuals a humoral (antibody) and cellular (cytotoxic T lymphocyte) immune reaction. We propose that these HIV-1 gp120 segments, together with the known CD4-binding region, may contribute to the HIV-1-induced immunosuppression by two mechanisms affecting CD4-HLA interaction during T-cell immune activation: autoimmune reaction toward CD4 and direct interference with the CD4-HLA costimulatory signal inducing CD4+ cell anergy with, as a consequence, generation of immunosuppression.

Automat and BLAST: comparison of two protein sequence similarity search programs.

Since the early 1980s, protein/DNA sequence similarity search has become of major importance to biologists, and the need for fast and efficient tools grows with the size of databanks. Two programs use the strategy of finite state deterministic automatons to accomplish these searches. One of these two is BLAST, which is now widely used, and the other Automat, which has just been published. The differences and similarities in their basic principles, their use and their performances are analysed in this paper in order to allow optimal use of these important softwares.

Absence of clinical, virological, and immunological signs of progression in HIV-1-infected patients receiving active anti-interferon-alpha immunization: a 30-month follow-up report.

Twenty-seven HIV-1-infected patients, 16 at early stage of disease and without concomitant antiretroviral therapy and 11 at more advanced stage of disease receiving antiretroviral therapy, have been followed since their enrollment, November 1992 and July 1993, respectively, in phase I/II studies to evaluate safety and immunogenicity of an anti-interferon-alpha (IFN-alpha) vaccine, aimed at modulating the impaired cytokine network in AIDS patients by counteracting IFN-alpha overproduction. We compared clinical, virological, and immunological markers of disease progression, including circulating IFN-alpha levels in a 24- to 30-month follow-up period with those of 62 patients fulfilling the same enrollment criteria and comparable for sex, risk factor, and age, regularly followed at our center. Anti-IFN-alpha immunization consisted of four-six intramuscular injections 1 month apart of a water-in-oil emulsion of 500 micrograms formalin-inactivated recombinant IFN-alpha-2b (iIFN-alpha) followed by intramuscular injections of 250 micrograms iIFN-alpha adsorbed onto calcium phosphate every 3 months. Neither clinical deterioration nor a CD4+ cell count decrease from pretreatment values was observed in IFN-alpha-immunized patients in the follow-up period, whereas clinical and immunological disease progressions were observed among open-comparison patients. Furthermore, statistical analysis showed a strong association between occurrence of clinical manifestations and high circulating IFN-alpha titers, while nonprogression of IFN-alpha-immunized patients was associated with decreased levels of circulating IFN-alpha.