The murine model was developed to assess the effects of maternally transferred HIV hyperimmune globulin or human intravenous immune globulin on the immunization of the offspring at 18–21 days of age with rgp120SF2‐complete Freunds adjuvant. Either HIV hyperimmune globulin or intravenous immune globulin was administered intraperitoneally to post‐partum BALB/c mice and was transferred via milk to the offspring. Both HIV hyperimmune globulin and intravenous immune globulin inhibited the offspring anti‐rgp120SF2 IgG response to the vaccine. The HIV hyperimmune globulin inhibition persisted for 28 days after immunization while the intravenous immune globulin inhibition was still present at 63 days after immunization. In addition, the intravenous immune globulin had a more generalized immunosuppressive effect, inhibiting the IgG response to both rpg120SF2 and an additional protein antigen, hen egg‐white lysozyme. Effects of maternal or exogenously administered pre‐existing antibody, including control antibodies (intravenous immune globulin), on the newborn response to HIV and other vaccines must be carefully evaluated when vaccine studies proceed in newborns.

Small animals were immunized with plasmid DNA encoding HIV-1 envelope gp120 either intramuscularly by needle injection (mice and guinea pigs) or epidermally with the Accell gene gun (guinea pits). Subsequently, the animals were boosted with a recombinant gp120 protein subunit vaccine in an oil-in-water based adjuvant, MF59. Antibodies and cytotoxic T-lymphocyte (CTL) immune responses to the HIV envelope glycoprotein were observed in animals immunized with gp120 DNA derived from the HIV-1SF2 laboratory strain or from HIV-1 field isolates. Titers of ELISA antibodies and serum neutralizing antibodies against the HIV-1SF2 laboratory isolate were substantially increased in DNA-immunized animals following a single boost with recombinant gp120 protein subunit. This DNA prime/protein subunit boost immunization approach may be important for vaccination against infectious agents such as HIV for which it is difficult to raise strong antiviral humoral responses with DNA vaccination alone.

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.

A large stock preparation of the HIV-1SF2 isolate has been derived after serial passage in human peripheral blood mononuclear cells (PBMCs). This viral stock has a titer of 10(4.9) TCID50 in human PBMCs and 10(4.2) TCID50 in chimpanzee PBMCs. By inoculation into animals the 50% chimpanzee infectious dose titer was found to be about 10(2.3). Virus isolation from animals was achieved on most occasions within 1-4 weeks after inoculation and then became transient. Viral RNA and DNA PCR analyses confirmed the virus infection of the chimpanzees. Anti-HIV antibody levels in the inoculated animals ranged from 1:400 to 1:6400 as measured by ELISA. About 680 vials of this stock preparation, frozen at -190 degrees C, are available for future studies of vaccines and antiviral therapies.

A flow cytometric assay based on expression of the activation antigen CD69 was developed to analyze immunological responses of T cells from human immunodeficiency virus (HIV)-infected (HIV+) or HIV-seronegative (HIV-) donors after in vitro simulation by antigens and polyclonal activators. The levels of CD69 on freshly-isolated or unstimulated, cultured CD3+, CD4+, or CD8+ peripheral blood lymphocyte (PBL) subsets were low and did not differ greatly between HIV+ and HIV- donors. The frequencies of CD3+, CD4+, and CD8+ lymphocytes from HIV+ donors that expressed CD69 after culture with antigenic or mitogenic stimuli were significantly lower than in HIV- donors. Comparison of CD69 expression with [3H]thymidine incorporation revealed that both assays could detect lymphocyte responses to antigenic or mitogenic stimuli. The CD3+ PBL from HIV+ or HIV- donors did not show increased CD69 expression after culture with soluble or cross-linked recombinant envelope glycoprotein, gp120. The gp120, however, significantly inhibited CD69 expression in phytohemagglutinin-stimulated T cells in vitro and may also affect T-cell activation in vivo. These studies demonstrate the usefulness of this CD69 expression assay for the rapid assessment of defects in immune responses of phenotypically defined lymphocyte subsets in HIV+ patients and for testing the effects of agents that modulate immune activation.

Human immunodeficiency virus-1 (HIV-1) infects both humans and chimpanzees, but in the chimpanzee, HIV-1 infection leads only very rarely to loss of CD4 T cells or to AIDS-like disease. The pathogenetic basis for this difference in host range is not understood. In previous studies, using CD4 T cells from HIV-1 seronegative human donors, we demonstrated that crosslinking of CD4-bound gp120, followed by signaling through the T cell receptor for antigen (TCR), resulted in cell death by apoptosis. To determine whether activation-induced apoptosis correlates with progression to AIDS, we studied the chimpanzee. Our data suggest that, although human CD4 T cells respond to CD4 ligation with anergy and apoptosis upon activation, chimpanzee CD4 T cells do not undergo apoptosis after cross-linking of CD4-bound gp120, followed by signaling through the TCR. In addition, proliferation assays show that chimpanzee CD4 T cells do not become anergic after CD4 ligation. Thus, it is possible that, in the chimpanzee, the absence of cellular anergy and apoptotic cell death after CD4 ligation by HIV-1 gp120 protect this primate species from progression to AIDS-like disease.

The effect of maternally transferred monoclonal antibody (MAb) on the offspring antibody response to rgp120s F2 was examined in a murine model. Two MAbs were studied: MAb 83.1, which recognizes a determinant in the V3 loop of gp120 from human immunodeficiency virus-I (HIV-1) SF2, and MAb 26.2D3, which recognizes a conserved N-terminal region of gp120 from HIV-1SF2 Offspring were immunized at 18-21 days of age with 100 µg of rgp120SF2 in complete Freund's adjuvant. Offspring immunized in the presence of preexisting MAb 83.1 but not MAb 26.2D3 demonstrated inhibition of the IgG anti-V3 response. The total IgG anti-rgp120SF2 response was not affected by preexisting MAb. Since newborns at risk for HIV may be immunized in the presence of maternal or administered anti-HIV antibody, alternative strategies may be required to circumvent inhibition of the infant's epitope-specific response to HIV immunization by preexisting antibody.

Objective: To determine whether vaccination with recombinant HIV-1(SF2) gp120 in a novel oil-in-water adjuvant emulsion, MF59, protects chimpanzees against challenge with HIV-1(SF2), the homologous virus isolate.Methods: Two vaccinated chimpanzees and two control animals were challenged with 25-50 animal infectious doses of a stock of HIV-1(SF2) that had been grown in mitogen-activated human peripheral blood mononuclear cells (PBMC). The animals were monitored by a series of serologic [enzyme-linked immunosorbent assay (ELISA), Western blot, and neutralization assays] and virologic [virus culture, RNA and DNA polymerase chain reaction (PCR)] assays for infection.Results: Both control animals showed evidence of seroconversion in ELISA and Western blot assays. In addition, virus was detected in the early, acute phase of infection of both control animals by (1) plasma RNA PCR, (2) virus culture, and (3) PBMC DNA PCR assays. One vaccinated animal showed no serologic or virologic evidence of infection. The other vaccinated animal has not seroconverted, and there was no evidence of plasma viremia. However, virus was detected at early timepoints in this animal's PBMC, and transient lymphoproliferation to HIV-1 proteins not in the vaccine was observed. These observations suggest that the former animal was protected from challenge while the latter may have experienced a transient or curtailed infection.Conclusion: Two types of vaccine-induced protective immune responses were observed when chimpanzees immunized with rgp120(SF2) were challenged with the homologous virus isolate: a response consistent with the 'sterilizing immune response' documented in the chimpanzee model in previous studies, as well as one that did not completely protect from infection, showing curtailment of the acute phase and a failure of the animal to seroconvert.

A vaccine breakthrough occurred in a phase 1 clinical trial of a human immunodeficiency virus (HIV) type 1 candidate subunit vaccine. The vaccine antigen, gp120SF2, is a fully glycosylated protein produced in mammalian cells from the HIVSF2 isolate. After 4 immunizations, the subject developed neutralizing antibodies and lymphoproliferative responses to the gp120 protein. About 18 weeks after the last immunization, the subject became HIV infected. During the acute phase of infection, there was high virus burden, a decline in CD4+ T lymphocytes, increases in rgp120SF2-binding antibodies and HIVSF2- and HIVMN-neutralizing antibodies, and transient lymphoproliferative responses to HIV-1 envelope and core proteins. The nucleotide sequence of the V3 loop from 2 virus isolations displayed close similarity to the V3 sequence of the vaccine antigen. Thus, the immunologic responses induced by the vaccine in this subject did not protect him from HIV-1 infection.

A murine model was developed for assessing the effects of passively transferred polyclonal maternal anti-gp120 antibodies on the subsequent immunization of the offspring with recombinant gp120SF2 in complete Freund's adjuvant (rgp120SF2-CFA). Adult female BALB/c mice were immunized with rgp120SF2-CFA 6 weeks before mating. The 3-week-old offspring were subsequently immunized with the same vaccine and followed for 9 weeks. Both the total IgG anti-rgp120SF2 and the anti-V3 IgG antibody response to vaccine were inhibited in the experimental animals. The total IgG anti-rgp120SF2 response was < 20% of the control response (P < .001) 9 weeks after immunization. Anti-V3 antibody was also decreased. As vaccine studies begin in infants, the effects of preexisting antibody on the infant response to human immunodeficiency virus vaccines must be considered.

El desarrollo de una vacuna preventiva contra el virus de la inmunodeficiencia humana de tipo 1 (VIH-1) es una de las mayores esperanzas para el control de la pandemia en los próximos años. Sin embargo, es claro que una de las mayores dificultades de una vacuna contra el virus es su alta tasa de mutación, lo que le permite evadir la respuesta inmune del húesped. La producción de anticuerpos neutralizantes (AcN) contra la envoltura vírica parece tener un papel importante en el control de la infección y en la ejecución de una protección eficaz luego de la inmunización. Diversos trabajos han mostrado que el dominio V1/V2 de la glucoproteína 120 del VIH-1 se encuentra involucrado en el tropismo vírico durante la infección, en el enmascaramiento de epítopes neutralizantes conservados, en los cambios conformacionales tras la unión a los correceptores y en la inducción de AcN. Sin embargo, hay pocos estudios enfocados sobre este dominio. Por otra parte, por ser uno de los dominios altamente glucosilados, numerosos estudios han determinado la influencia de los hidratos de carbono sobre la producción de AcN. Por tanto, la presente revisión está enfocada en la importancia de los AcN dirigidos contra epítopes de las regiones variables (principalmente V1/V2), su importancia en la protección contra la infección por el VIH-1, el papel que desempeñan esas regiones en la evasión de la respuesta inmune y, finalmente, se discute la importancia de los AcN en la búsqueda de una vacuna eficaz contra el virus.The development of a preventive vaccine against human immunodeficiency virus type-1 (HIV-1) provides hope for control of the pandemic over the coming years. Nevertheless, it is clear that one of the greatest difficulties in achieving this vaccine is the high mutation rate of the virus, which enables it to evade the host's immune response. The production of neutralizing antibodies (NAb) against the HIV-1 envelope proteins is believed to play an important role in controlling the infection and in providing effective protection following vaccination. Several studies have shown that the V1/V2 domain of the HIV-1 gp120 envelope protein is involved in viral tropism during infection, in masking conserved neutralizing epitopes, in the conformational changes occurring after coreceptor binding, and in NAb induction. Nonetheless, this domain has been poorly investigated. However, because the V1/V2 domain is highly glycosylated, numerous studies have determined the influence of carbohydrates on NAb production. The present review focuses on the importance of NAb directed against epitopes of the variable regions, mainly V1/V2, their importance in protecting against HIV-1 infection, and the role these regions play in evading the immune response. Lastly, we will discuss the importance of NAb in the search for an effective vaccine against HIV-1.

We have described previously the generation of seven HIV-SF2 Nef-specific, CD4+ T-cell clones, identification of epitopes within which are recognized by these clones, and the MHC alleles that restrict their responses1. In this study, we have extended this characterization to include evaluation of antigen-processing and presentation requirements and cytotoxic activity. Clones were generated from five HIV-1 uninfected donors by in vitro stimulation of peripheral blood mononuclear cells with purified recombinant Nef1. In experiments with fixed cells, with the exception of two clones, recognition of Nef, but not Nef peptides, required processing. Also, at higher concentrations of antigen, the clones themselves were capable of presenting Nef peptides, but not soluble Nef. All clones had the ability to specifically lyse autologous. Epstein-Barr virus-transformed lines sensitized with Nef synthetic peptides, or, in some cases, soluble Nef. The cytotoxic activity mapped to the same epitopes identified for the proliferative response (a.a. 14–22, 47–53, 68–77, 70–77, 195–203 and 185–192) and was restricted by the same HLA class II molecules (DRw6, DQw7, DRw15(2), DR1 and DP5). Sensitization of the cytolytic clones with specific Nef peptides, but not soluble Nef, resulted in autolysis.

Priming of CD8+ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC-restricted CTL responses is not clear. To address this issue, recombinant HIV-1 gp120 envelope proteins produced in yeast insect, or mammalian cells were compared for the ability to elicit CD8+ CTL activity in mice. Envelope-specific CD8+ T lymphocytes were detected in BALB/c mice immunized with env 2-3, a 55-kDa yeast-derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy-terminus of the molecule. Similar levels of V3-directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope-specific CD8+ CTL activity was elicited when N-linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N-linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.

Human T-cell clones with specificity to the HIV-1 nef protein were generated by the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors with purified nef from the HIV-SF2 isolate produced in genetically engineered yeast. Here the characterization is described of a total of seven discrete clones derived from five different donors. Each clone was CD3+ CD4+ CD8- as determined by FACS analysis. The epitopes recognized by these clones were identified using synthetic overlapping peptides spanning the entire length of nef. Six discrete helper T-cell epitopes located in five distinct regions of nef were identified by this approach. Three of these epitopes are more than 80% conserved among all HIV-1 nef proteins for which sequence data are available. The remaining epitopes are in regions of nef that vary among isolates. Many of the epitopes recognized by our clones overlap T-cell epitopes identified by others examining T-cell responses to nef in HIV-1-infected patients and immunized animals. Using partially class II-matched EBV-transformed B-cell lines, we were able to identify five different HLA class II alleles which encode restricting elements for the in vitro nef-specific proliferative response of these clones (DR1, DRw15(2), DRw6, DQw7, DP5).

A phase 1 study of 42 non-human immunodeficiency virus type 1 (HIV)-infected volunteers was initiated to determine the safety and immunogenicity of an HIV subunit vaccine consisting of recombinant envelope gp120 derived from HIVSF2 (rgp120SF2) combined with a novel adjuvant, MF59, with or without the immunomodulator muramyl tripeptide dipalmitoyl phosphatidylethanolamine (MTP-PE). All injections contained adjuvant MF59, and subjects were grouped according to MTP-PE dose. Injections were given on days 0, 30, 180, and 365. The vaccine was well tolerated with limited local and systemic reactions. These immunizations induced rgp120SF2-specific binding antibodies that persisted > or = 24 weeks. After three immunizations, all subjects receiving the antigen developed neutralizing antibodies to HIVSF2, and serum from 67% of these subjects also cross-neutralized HIVMN. ELISA-reactive antibodies to the HIVSF2 V3 region and strong lymphoproliferative responses to HIVSF2 envelope proteins were detected in all rgp120SF2-immunized subjects.

A phase 1 study of 42 non-human immunodeficiency virus type 1 (HIV)-infected volunteers was initiated to determine the safety and immunogenicity of an HIV subunit vaccine consisting of recombinant envelope gp120 derived from HIVSF2 (rgp120SF2) combined with a novel adjuvant, MF59, with or without the immunomodulator muramyl tripeptide dipalmitoyl phosphatidylethanolamine (MTP-PE). All injections contained adjuvant MF59, and subjects were grouped according to MTP-PE dose. Injections were given on days 0, 30, 180, and 365. The vaccine was well tolerated with limited local and systemic reactions. These immunizations induced rgp120SF2-specific binding antibodies that persisted > or = 24 weeks. After three immunizations, all subjects receiving the antigen developed neutralizing antibodies to HIVSF2, and serum from 67% of these subjects also cross-neutralized HIVMN. ELISA-reactive antibodies to the HIVSF2 V3 region and strong lymphoproliferative responses to HIVSF2 envelope proteins were detected in all rgp120SF2-immunized subjects.