Search

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1(DH12)) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1(DH12), nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1-V2 and V4 regions of HIV-1(DH12) could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.

A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol. This activity was mediated by both CD4+ and CD8+ lymphocytes. On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.

A safety and immunogenicity trial was conducted in vaccinia-immune and vaccinia-naive human immunodeficiency virus (HIV)-uninfected adults who were randomized to receive 10(6) or 10(7) TCID50 of canarypox (ALVAC) vector expressing HIV-1MN gp160 or 10(5.5) TCID50 of ALVAC-rabies virus glycoprotein control at 0 and 1 or 2 months and ALVAC-gp160 or 50 microg of HIV-1SF2 recombinant (r) gp120 in microfluidized emulsion at 9 and 12 months; others received rgp120 at 0, 1, 6, and 12 months. All vaccines were well-tolerated. Neither vaccinia-immune status before vaccination nor ALVAC dose affected HIV immune responses. HIV-1MN and HIV-1SF2 neutralizing antibodies were detected more often (100%) in ALVAC-gp160/rgp120 recipients than in recipients of ALVAC-gp160 (<65%) or rgp120 (89%) alone. ALVAC-gp160/rgp120 also elicited more frequent HIV V3-specific and fusion-inhibition antibodies, antibody-dependent cellular cytotoxicity, lymphoproliferation, and cytotoxic CD8+ T cell activity than did either vaccine alone. Trials with ALVAC expressing additional HIV components and rgp120 are underway.

Effective human immunodeficiency virus (HIV) vaccination may require induction of neutralizing antibodies (NAs) and CD8+ cytotoxic T lymphocytes (CTL) to prevent transmission and control early infection. Recombinant envelope proteins induce NAs but rarely CD8+ CTL responses, and vaccinia vectors containing HIV-1 envelope elicit CD8+ cytotoxicity but few NAs. To benefit from both approaches, 56 vaccinia-naive subjects were randomized to a regimen of priming with recombinant vaccinia gp160LAI and boosting with recombinant gp120SF-2, gp120LAI, gp120MN, or gp160MN. Of 51 persons for whom assays were done, 26 demonstrated envelope-specific CTL. Boosting with gp120, compared with gp160, elicited significantly more NAs and CD4-blocking antibodies. Neutralization of the homologous and heterologous HIV-1 laboratory strains occurred in all subjects receiving vac/env and gp120 and was detectable in 91% of the subjects for >6 months. Thus, vaccine regimens in which one component elicits primarily CTL and the other NAs offer promise for the development of an effective HIV-1 vaccine strategy.

Objective: To evaluate the safety and immunogenicity of recombinant glycoprotein (rgp) 120, a candidate vaccine for the human immunodeficiency virus (HIV), formulated with a novel adjuvant, MF59, with or without a biological response modifier, MTP-PE., Design: Multicenter, double-blind, randomized trial., Setting: University medical centers., Participants: 49 healthy, HIV-seronegative volunteers 18 to 60 years of age who were at low risk for HIV type 1 (HIV-1) infection., Interventions: In part A of the study, 32 participants were randomly assigned to receive either 15 micrograms of rgp 120 in MF59, 15 micrograms of rgp 120 in MF59 plus 50 micrograms of MTP-PE, 50 micrograms of rgp 120 in MF59, or 50 micrograms of rgp 120 in MF59 plus 50 micrograms of MTP-PE. Participants were vaccinated at 0, 1, 6, and 12 to 18 months. In part B, 17 participants were randomly assigned to receive five monthly injections of either 50 micrograms of rgp 120 in MF59 or MF59 alone followed by a booster injection at 12 to 18 months., Main Outcome Measures: Local and systemic reactions; laboratory measures of hepatic, renal, immunologic, and bone marrow toxicity; and HIV-specific serologic and cell-mediated immune responses., Results: 13 patients in part A received 50-micrograms doses of rgp 120; type-specific neutralizing antibody responses against the SF-2 strain of HIV-1 (HIV-1/SF-2) were induced in all 13. Nine of the 13 had crossreactive neutralizing activity against the MN strain of HIV-1 (HIV-1/MN), and 2 had crossreactive neutralizing activity against the IIIB strain of HIV-1 (HIV-1/IIIB). Twelve patients had typespecific fusion inhibition activity; only 1 had crossreactive fusion inhibition activity against HIV-1/MN. The monthly vaccination schedule used in part B resulted in decreased antibody titers, indicating that a rest period in the schedule is necessary for maximal immunogenicity. Lymphoproliferative responses against gp120 were induced in all vaccine recipients. The stimulation index to gp120 was persistently greater than 15 for 6 months after the last booster vaccination was given. CD8+ cytotoxic T-lymphocyte activity was detected in 1 of the 11 participants tested. Vaccine that contained MTP-PE caused a greater number of moderate or severe local and systemic reactions (of 16 participants, 4 had local reactions and 13 had systemic reactions) than did vaccine formulated with MF59 alone (of 16 participants, 7 had local reactions [P < 0.01] and 0 had systemic reactions [P < 0.001])., Conclusions: The SF-2 rgp120 vaccine is safe and immunogenic. Three vaccinations with rgp120 in MF59 can induce type-specific and crossreactive neutralizing antibody against B-subtype laboratory strains of HIV-1. Human immunodeficiency virus-specific lymphoproliferative responses were induced in all vaccinated participants, and CD8+ cytotoxic T-lymphocyte activity was shown in one participant. A trend toward the augmentation of lymphoproliferative and humoral responses by MTP-PE was seen in the participants receiving 15 micrograms of rgp120. However, MTP-PE caused a statistically significant increase in the incidence of local and systemic side effects, which was felt to outweigh the small increase in immunogenicity provided by this biological response modifier in an otherwise well-tolerated vaccine.

The ability of antibody induced by MN and IIIB recombinant gp120 (rgp120) human immunodeficiency virus type 1 (HIV-1) vaccines the bind to oligomeric native and monomeric recombinant HIV-1 envelope glycoproteins (rgp 120) was measured in 25 uninfected, healthy adult volunteers. A major focus was to evaluate the effect of simultaneous and sequential immunization with vaccines representing different strains of HIV-1 on the ability to broaden cross-reactivity of antibodies against these and other HIV-1 strains. A flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to envelope glycoprotein expressed by infected and rgp120-coated target cells was used, MN rgp120 HIV-1 vaccine given alone and coadministered with IIIB rgp120 HIV-1 vaccine elicited antibody which bound to cells infected with HIV-1MN, HIV-IIIB, HIV-1RF, and HIV-1-SF2. The presence of envelope glycoprotein-binding antibody detected by FIFA correlated to a moderate degree with functional antibody against HIV-1MN and HIV-IIIB. Priming immunization with IIIB rgp120 HIV-1 vaccine followed by booster injections of MN rgp120 HIV-1 vaccine resulted in increased cross-reactive antibody binding to these and heterologous clade B HIV-1 strains infecting cells. MN rgp120 HIV-1 vaccine given alone was better able to induce cross-reactive antibody to cells infected with heterologous HIV-1 laboratory strains than was IIIB rgp120 HIV-1 vaccine given alone. The vaccines induced binding antibody to rgp120 possessing the amino acid sequence of a clade E HIV-1 strain as measured by enzyme-linked immunosorbent assay. Levels of antibody binding to cells infected with clade B HIV-1 and cells coated with monomeric rgp120 were greater than that induced by HIV-1IIIB-based gp160 vaccines in previous studies.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

A phase I double-blind trial was done to examine the safety and immunogenicity of a prototype synthetic human immunodeficiency virus type 1 MN strain (HIV-1MN) third variable region domain (V3) branched peptide vaccine in HIV-1-uninfected healthy adult volunteers. Subjects were randomly assigned to receive 20, 100, or 500 micrograms of vaccine or alum adjuvant control on days 0, 28, and 168. The vaccine was well-tolerated and appeared safe. Induction of binding antibody to V3 MN branched peptide was vaccine dose-related and was detectable in 9 of 10 subjects in the highest-vaccine-dose group. HIV-1MN-neutralizing antibody was detected after the third 500-micrograms dose in 8 of 10 subjects at the 90% neutralization end point. V3 MN peptide stimulated lymphocyte proliferation in 15 (75%) of 20 subjects after vaccination. In conclusion, this prototype vaccine was safe and it induced humoral and cell-mediated immune responses.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein. In one experiment, two chimpanzees were immunized multiple times; one animal received antigens derived only from HIV-1LAI, and the second animal received antigens from both HIV-1LAI and HIV-1MN. In another experiment, four chimpanzees were immunized in parallel a total of five times over 18 months; two animals received purified gp160 and V3-MN peptides, whereas the other two animals received the recombinant canarypox virus and gp160. At 3 months after the final booster, all immunized and naive control chimpanzees were challenged by intravenous inoculation of HIV-1SF2; therefore, the study represented an intrasubtype B heterologous virus challenge. Virologic and serologic follow-up showed that the controls and the two chimpanzees immunized with the live recombinant canarypox virus became infected, whereas the other animals that were immunized with gp160 and V3-MN peptides were protected from infection. Evaluation of both cellular and humoral HIV-specific immune responses at the time of infectious HIV-1 challenge identified the following as possible correlates of protection: antibody titers to the V3 loop of MN and neutralizing antibody titers to HIV-1MN or HIV-1LAI, but not to HIV-1SF2. The results of this study indicate that vaccine-mediated protection against intravenous infection with heterologous HIV-1 strains of the same subtype is possible with some immunogens.

Genetically engineered, noninfectious HIV-1-like particles containing processed envelope glycoproteins represent potential candidate immunogens for a vaccine against HIV-1. However, since the gp120 glycoprotein is known to be rapidly lost from the surface of infected cells and purified virions as a result of its low-affinity interaction with gp41, shedding of this extracellular subunit could compromise the immunogenic potential of particle-based HIV-1 vaccine candidates. In this study, we demonstrate for the first time the feasibility of producing fully assembled HIV-1-like particles containing only unprocessed gp160 glycoproteins. Monkey kidney Vero cells were transfected with an inducible, human metallothionein-based expression vector containing most of the HIV-1LAI coding sequences that were genetically modified to introduce safety mutations and destroy the major cleavage site of the HIV-1 envelope glycoprotein. A stably-transfected cell line was isolated and shown to secrete HIV-1-like particles containing unprocessed gp160. Immunization with these particles induced HIV-1 cross-neutralizing, syncytium-inhibiting and env-CD4 blocking antibodies. Thus, these novel HIV-1-like particles represent alternative candidate immunogens for the development of a particle-based AIDS vaccine.

The safety and the immunogenicity of a recombinant canarypox live vector expressing the human immunodeficiency virus type 1 (HIV-1) gp160 gene from the MN isolate, ALVAC-HIV (vCP125), followed by booster injections of a soluble recombinant hybrid envelope glycoprotein MN/LAI (rgp160), were evaluated in vaccinia-immune, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 20) received vCP125 (10(6) TCID50) at 0 and 1 month, followed randomly by rgp160 formulated in alum or in Freund's incomplete adjuvant (FIA) at 3 and 6 months. Local and systemic reactions were mild or moderate and resolved within the first 72 hr after immunization. No significant biological changes in routine tests were observed in any volunteer. Two injections of vCP125 did not elicit antibodies. Neutralizing antibodies (NA) against the HIV-1 MN isolate were detected in 65 and 90% of the subjects after the first and the second rgp 160 booster injections, respectively. Six months after the last boost, only 55% were still positive. Seven of 14 sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp 160 was detected in 25% of the subjects after vCP125 and in all subjects after the first booster injection and 12 months after the first injection. An envelope-specific cytotoxic lymphocyte activity was found in 39% of the volunteers and characterized for some of them as CD3+, CD8+, MHC class I restricted. The adjuvant formulation did not influence significantly the immune responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: To evaluate the safety and immunogenicity of the MN strain of recombinant gp120 (MN rgp120) as a vaccine prototype to prevent human immunodeficiency virus (HIV)., Design: Double-blind, randomized, placebo-controlled study with subjects vaccinated at 0, 4, 24, and 48 weeks and followed up through 64 weeks., Setting: The AIDS Vaccine Evaluation Units in St Louis, Mo, Nashville, Tenn, and Rochester, NY, conducted the clinical study. Laboratory studies were conducted at Duke University, Raleigh, NC; data analysis was done by the Data Coordinating and Analysis Center at the EMMES Corporation, Potomac, Md., Participants: Fifty-seven persons seronegative for HIV, at low risk for acquiring HIV infection, and 18 to 60 years of age., Interventions: The MN rgp120 vaccine was administered to 12 volunteers each in doses of 100 micrograms, 300 micrograms, or 600 micrograms, and 12 volunteers received a combination of 300 micrograms of MN rgp120 vaccine and 300 micrograms of vaccine from rgp120 of strain IIIB. Nine volunteers received alum adjuvant alone (control)., Main Outcome Measures: Safety was assessed by monitoring lymphocyte subsets, serum creatinine, and liver enzymes. Immunogenicity was measured by enzyme-linked immunosorbent assay using the immunogen and synthetic peptide corresponding to the variable region 3 domain of gp120. Functional antibody assays included CD4 binding blocking; antibody-dependent, cell-mediated cytotoxicity; and neutralization of homologous and heterologous HIV strains., Results: No severe adverse reactions occurred. In 33 of 48 volunteers, two doses of vaccine induced antibodies that neutralized the homologous strain HIV-1/MN. Three doses of vaccine induced antibodies that neutralized MN (in 46 of 48 volunteers), SF-2 (in 45 of 48 volunteers), or IIIB strains of HIV-1 (in 30 of 48 volunteers)., Conclusion: The vaccines were safe and immunogenic. Multiple injections of vaccine broadened and increased the neutralizing antibody response.

The third variable domain (V3 domain) of HIV-1 gp120 is involved in virus neutralization by antibody, in determination of cell tropism, and in syncytium-inducing/non-syncytium-inducing capacity. Antibodies are highly specific tools to delineate the role of different V3 amino acid sequences in these processes, and to dissect events occurring during synthesis of gp120/160, gp120-CD4 interaction, cellular infection, and syncytium formation. We describe here an IgG1 murine monoclonal antibody (MAb), coded IIIB-V3-01, that was raised with a synthetic peptide (FVTIGKIGNMRQAHC) derived from the carboxy-terminal flank of the HIV-1 IIIB V3 domain. The binding site of this antibody was mapped to the sequence IGKIGNMRQ, using Pepscan analysis. In ELISA, this antibody binds to E. coli-derived gp120 from HIV-1 IIIB, which is denatured and not glycosylated. The antibody showed no neutralizing activity against HIV-1 IIIB, MN, SF2, or RF in a virus neutralization assay and in a syncytium formation inhibition assay. In addition, this antibody did not react with gp120 expressed on the surface of IIIB-infected MOLT-3 cells in FACS analysis. To assess whether the epitope defined by MAb IIIB-V3-01 is hidden on native gp120, reactivity of the antibody with SDS-DTT-denatured or DTT-denatured glycosylated gp120 (CHO cell produced) was tested. Both these treatments exposed the epitope for binding. From these data we conclude that the epitope defined by MAB IIIB-V3-01 is hidden on glycosylated recombinant gp120, and is not accessible on gp120 expressed on the membrane of HIV-1, IIIB-infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Twelve vaccinia-naive volunteers were inoculated with recombinant vaccinia virus expressing the human immunodeficiency virus type 1 (HIV-1) strain IIIB (HIV-1IIIB) envelope glycoprotein gp160 and subsequently immunized with 640 micrograms of recombinant (r) gp160 protein produced in baculovirus. After booster immunization with rgp160, the sera of all vaccinees showed strong antibody responses detected by Western blot and ELISA; 8 had neutralizing activity and 5 had fusion inhibition activity against the homologous strain; 5 blocked binding of CD4 cells to gp120. Cross-reactive neutralization of HIV-1MN was detected in 3 of 8 sera that neutralized HIV-1IIIB. The combination of live recombinant vaccinia followed by subunit booster immunization was more immunogenic than either product alone and represents a promising approach for HIV-1 immunoprophylaxis. Further definition of recombinant vaccinia safety and augmentation of immune responses to geographically prevalent HIV-1 strains will be necessary before expanding clinical trials to high-risk groups.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.

The influence of human anti-human immunodeficiency virus type 1 (HIV-1) antibody on HIV-1 infection of freshly isolated normal human peritoneal macrophages and blood monocytes was examined. Each of 14 HIV antibody-positive human serum samples was found to block the infection of four virus isolates (human T-cell lymphotropic virus type IIIBa-L [HTLV-IIIBa-L], HTLV-IIIB, D.U. 6587-7, and D.U. 7887-8) at serum dilutions ranging from 10(-1) to 10(-2). Three of these isolates (HTLV-IIIBa-L, D.U. 6587-7, and D.U. 7887-8) infected cultures of monocytes and macrophages rapidly and produced high levels of virus reverse transcriptase and p24 antigen. A fourth virus isolate (HTLV-IIIB) infected the monocytes and macrophages more slowly and produced low levels of viral protein. More dilute HIV antibody-positive sera had no significant effect on the overall level or rate of virus infection or expression. Complement did not appear to influence the course of infection by any combination of antisera or virus examined. Successful HIV-1 infection of the peritoneal macrophages and blood monocytes under the conditions tested showed strict dependence on CD4 since a recombinant CD4 polypeptide and an anti-CD4 monoclonal antibody effectively blocked the process.

An intravenous solution of 99% pure globulin (hyperimmune IgG, HIVIG) was obtained from pooled plasma of selected human immunodeficiency virus (HIV-1)-seropositive asymptomatic donors with greater than 400 CD4+/microliters cells per microliter and a high titer of antibody to HIV-1 p24 protein. HIVIG had high titers of antibody to p24, glycoprotein 41 (gp41), and gp120, group-specific neutralizing activity, and binding to the gp120 hypervariable loop region. It inhibited syncytia formation. At low concentration, it enhanced viral production of HIV-1 in infected peripheral blood monocytes but was inhibitory at higher concentration. HIVIG directed group-specific antibody-dependent cellular cytotoxicity against HIV-infected targets. For a period of 6 to 28 months, plasma donors kept stable antibody titers and had a 1.0% decrease in CD4+ cells per month. One gram per kilogram HIVIG injected in two juvenile chimpanzees was well tolerated and did not transmit HIV, as measured by negative cell culture, IgM immune response to HIV proteins, and polymerase chain reaction. The mean half-life of HIV-1 p24 antibody was 15 days. These preliminary data suggest that HIVIG is a safe product suitable for clinical trial in HIV-1-infected individuals.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.

The immunoreactivity of HTLV-III-infected individuals and virus-inoculated chimpanzees with gp120 synthetic peptides of the HTLV-III gp120 envelope principle neutralizing domain (amino acid 301-324 sequences), derived from the HTLV-III isolates 3B, RF, MN, WMJ2, and SC were determined. Sequential bleeds from an infected lab worker and chimpanzees, both infected with the HTLV-IIIB, were immunoreactive only with the 3B peptide. In contrast, 33 HTLV-III-infected individuals were immunoreactive with the HTLV-III(MN) peptide. Of these 33 individuals, 23 were also immunoreactive with the HTLV-III(SC) peptide, and 18 with the HTLV-III(WMJ2) peptide. The data suggest that HTLV-III strains related to MN are most prevalent among HTLV-III-infected individuals. The binding specificities of goat sera generated against either of these synthetic peptides or the C-terminal fragment of gp120 (PB-1, amino acid 287-467, derived from the HTLV-III isolates 3B, RF, MN, WMJ2, and SC) were also determined. Four different ELISA formats (peptide sera/peptide antigens, peptide sera/PB-1 antigens, PB-1 sera/PB-1 antigens, and PB-1 sera/peptide antigens) were utilized to determine the cross-reactivity patterns of goat sera with the antigens. Goat sera generated against MN and SC sequences (PB-1 proteins, as well as synthetic peptides) were highly cross reactive. Thus, patient sera cross reactivity to multiple strains of the principal neutralizing domain may reflect the antigenic relatedness of the virus isolates rather than multiple infection events or strains generated during disease progression.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.

A total of 18 overlapping peptides were synthesized that covered envelope amino acid sequences (amino acids 288-472 of the III-B isolate) of the human immunodeficiency virus type 1 (HIV-1). Antibodies from human immunodeficiency virus-1-infected individuals bound to three of the peptides tested. Guinea pigs were immunized with each of the overlapping peptides and the resultant sera analyzed for biologic activity. One of the peptides elicited antibodies that had both neutralizing and fusion blocking activities that were type specific. This peptide, designated 1-69, was also one of the peptides reactive with the human immunodeficiency virus type 1-positive human sera.

The immune responses produced by four macaques inoculated intradermally with a recombinant vaccinia virus that expresses the envelope gene of human immunodeficiency virus (HIV) were analyzed. Antibody capable of immunoprecipitating the glycosylated envelope protein precursor gp160 was detected in all animals within 3 weeks after the primary intradermal vaccination. The level of antibody was increased following a booster inoculation, and the sera from three of the four animals were then capable of immunoprecipitating gp120. Sera from two of the macaques with anti-gp120 antibody were able to prevent syncytium formation mediated by the parent HIV but not that induced by an unrelated HIV isolate. The fusion-inhibiting antibody was directed toward the highly variable central portion of the gp120 molecule since the effect was abrogated by incubation with PB1. The latter is a recombinant protein produced in Escherichia coli containing amino acids 295-474, a major neutralizing epitope. Sera from the three macaques with anti-gp120 neutralized HIV infectivity in an isolate-specific manner. The serum with highest neutralizing activity was also best at inhibiting syncytium formation. In contrast to the isolate-specific nature of the neutralizing antibody, T lymphocytes from immunized animals proliferated in response to divergent HIV isolates as well as to purified gp120. These studies provide baseline immunologic information for further development of recombinant vaccinia virus vectors.

Fresh circulating PBMC from HIV-1 seropositive individuals have been found to mediate specific, non-MHC restricted lysis of targets expressing the major envelope glycoprotein of HIV-1, gp120, in 6-h 51Cr release assays. This gp120 specific cell-mediated cytotoxicity (CMC) is broadly reactive against target cells infected with a wide range of viral isolates, is IL-2 augmentable, and is mediated by a CD16+, Leu-7+, CD15-, CD3- population of NK/K cells. The presence of FcR (CD16) on these cells suggested that the lytic specificity for gp120 might be directed by cytophilic antibody bound to the cell surface. Affinity purified F(ab')2 antibody fragments specific for the Fc and F(ab')2 portions of human IgG were used in attempts to block gp120 specific lysis. A 1/50 dilution of these antibodies inhibited gp120 specific cytolytic activity by more than 90% while exhibiting a minimal effect on NK/K cell lysis of K562 targets. The blocking activity of these fragments demonstrates the direct involvement of cytophilic antibody in CMC. In attempts to isolate this cytophilic anti-HIV-1 antibody, short 56 degrees C incubations were used to dissociate antibodies from the surface of PBMC of seropositive individuals. The supernatants generated in this manner exhibited specific gp120 activity in antibody-dependent cellular cytotoxicity assays. The ability of Staphylococcal protein A to remove this activity confirms the presence of cytophilic antibody on freshly isolated PBMC. Selective enrichment of specific cell subpopulations revealed the origin of the cytophilic antibody to be CD16+ NK/K cells and not B cells, T cells, or monocytes/macrophages. These studies show that the gp120-specific CMC seen in HIV-1 seropositive individuals is directed by cytophilic antibody bound to circulating CD16+ NK/K cells and represents a form of direct antibody-dependent cellular cytotoxicity which may provide a primary cytotoxic host defense.