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The antigenic variation associated with Human Immunodeficiency Virus type-1 (HIV-1) envelope proteins could limit their utility in vaccines if the immune responses induced are specific for immunodominant variable epitopes. We evaluated the ability of experimental subunit vaccines, containing recombinant forms of the envelope glycoprotein (rgp120) from two HIV-1 variants, to induce immune responses capable of recognizing unrelated HIV-1 variants. A vaccine formulation based on HIV-1IIIB/LAI rgp120 and supplemented with saponin adjuvant (QS-21) induced neutralizing antibodies specific for the HIV-1IIIB/LAI variant. This antibody response was presumably specific for the variable principle neutralizing determinant (PND) of the third variable region of gp120, the V-3 region. This formulation induced cytotoxic T-lymphocytes (CTL) specific for the dominant V-3 epitope but also to an additional unidentified epitope outside of this region. The CTL specific for this second epitope also recognized gp120 from the HIV-1MN and HIV-1RF variants in a "cross-reactive" manner. A second vaccine formulation based on HIV-1MN rgp120 and QS-21 adjuvant induced neutralizing antibodies that were again variant-specific but also CTL that recognized all three HIV-1 variants in a cross-reactive manner. These data demonstrate that CTL capable of recognizing different HIV-1 variants, which are presumed to be specific for a conserved HIV-1 gp120 epitope, can be induced using subunit vaccines with the appropriate adjuvant while variant-specific antibody responses are produced. These findings support further evaluation of this vaccine format.

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The in vivo and in vitro accessory cell requirements of class I major histocompatability complex (MHC) antigen-restricted cytotoxic T-lymphocyte (CTL) responses were determined using cell-depletion experiments coupled with active immunizations using ovalbumin (OVA) as the immunogen and saponin adjuvant (QS-21). To paralyze macrophage activity in vivo, C57BL/6 mice were treated with particulate silica or carrageenan. In vivo depletion of helper T-lymphocytes was accomplished by treatment with GK1.5 rat monoclonal antibody, which is specific for the murine CD4 antigen, and by genetic depletion of class II MHC antigens. Following treatments, the mice were immunized with formulations containing OVA alone or mixed with QS-21 saponin adjuvant, which induces MHC class I antigen-restricted CTL responses. In vivo treatment to paralyze macrophages abrogated these CTL responses but not antigen-specific antibody or lymphocyte proliferative responses. Depletion of CD4+ T-lymphocytes had no effect on CTL responses but significantly reduced proliferation and antibody responses. In vitro depletion and reconstitution experiments were done to compare the contributions of different antigen-presenting cells (APC), specifically dendritic cells (DC) and macrophages. Again, the requirement for macrophages was absolute but there was no indication that DC were involved. These data suggest that antigen processing and presentation functions are critical to the induction of CTL and that they are a function of macrophages but that CD4+ helper T-lymphocyte functions are not required.

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Different forms of recombinant HIV-1 gp160 and tetanus toxoid were adsorbed onto latex microspheres and this particulate form of the proteins was used to measure antigen-specific proliferation in vaccinated rhesus macaques. Proliferative responses to proteins bound to microspheres were significantly greater and allowed for the detection of antigen-specific responses that were not detected using soluble proteins. The responses were antigen-specific and required prior immunization of the animals. Additionally, the presence and magnitude of the proliferative responses was associated with antibody responses to the same proteins suggesting the results were representative of in vivo responses and that the assay format did not induce in vitro artifacts.

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The ability of a saponin adjuvant, QS-21, to induce OVA-specific, class I MHC Ag-restricted CTL was investigated using different forms of soluble OVA and OVA adsorbed onto alum as immunogens. C57BL/6 mice were immunized with soluble native or denatured OVA in formulations that contained increasing quantities of QS-21, and CTL responses were measured using EL4 and E.G7-OVA cells as targets and splenic mononuclear cells as effectors. Ag-specific CTL responses were produced but only if the QS-21 adjuvant was used. Similar responses were induced using alum-adsorbed OVA when mixed with the QS-21 adjuvant but not when used alone. The CTL were specific for an epitope present on the OVA258-276 synthetic peptide, which contains the dominant CTL epitope recognized by C57BL/6 mice. The CD8+ subpopulation of lymphocytes in immune mice was not increased in spleens but increased significantly in vitro after culture with soluble OVA. The CTL activity of splenic mononuclear cell preparations was totally destroyed by treatment with mAb specific to the CD8 Ag plus complement. The ability of the QS-21 adjuvant to induce class I MHC Ag-restricted CTL after immunization with soluble proteins is a characteristic unique to saponin adjuvants.

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The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.

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