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We previously reported that repeated inoculation of VC1, a macromolecular multicomponent peptide vaccine emulsified with Freund's adjuvant (VC1-F), induced high cytotoxic T lymphocyte (CTL) levels and a substantial level of multivalent antibodies which neutralized various human immunodeficiency virus type 1 (HIV-1) isolates. In the present study, we report that inoculation of VC1-F plus interleukin (IL)-12 expression plasmid can induce a higher antigen-specific CTL response compared to that with VC1-F alone. VC1-F plus IL-12 expression plasmid or VC1-F alone were inoculated to BALB/c mice twice at interval of 2 weeks. Two weeks after the second inoculation, spleen effector cells from these mice were examined. Stronger CTL responses against target cells were observed from the inoculation of VC1-F plus IL-12 plasmid than from that with VC-1F alone, but there was no difference in antibody induction. The inoculation of VC1 plus IL-12 plasmid also produced higher CTL activity than the inoculation of VC1 alone. These augmented CTL activities were not observed using target cells pulsed with non-HIV-specific peptides and different class I haplotype cells. These data demonstrate that co-inoculation of cell-mediated immune potent antigen and IL-12 plasmids can enhance the antigen-specific CTL response. This may be a potential approach for the induction of cellular immunization against HIV-1 and other diseases.

We report on the genetic effect on in vivo production of tumor necrosis factor (TNF)-alpha induced by lipopolysaccharides (LPS) using various congenic mouse strains. B10.A, Bl0.A(3R), B10.AQR, B10.A(5R), and B10.S(7R) produced significantly high TNF-alpha compared with B10.BR, B10.S, C57BL/10, B10.A(2R), B10.A(4R), B10.G, B10.DA(80NS), and B10.RIII(71NS). This suggests that LPS-induced TNF-alpha production is genetically controlled by H-2. Mice with the same alleles on K, A, E, or S loci produced various (high or low) levels of TNF-alpha, thus indicating that regulatory genes are located outside these loci. All strains with H-2Dd produced significantly high levels of TNF-alpha, but strains with other alleles in the H-2D locus produced low levels. Thus, TNF-alpha production appears to be genetically linked to H-2D itself or H-2D linked genes and the allele d is linked to a high responder gene. This was the case with the A background. C3H/HeN (H-2k), however, showed a high TNF-alpha production, suggesting the presence of another controlling gene outside H-2. In addition, high TNF-alpha productivity was transmitted into F1 mice (B10.A X B10.BR) in a dominant fashion. Both LPS-stimulated and unstimulated TNF-alpha mRNA expression in splenic macrophages were enhanced in high responder strains. Thus, we conclude that TNF-alpha production is closely related to genes within or linked to the H-2D locus as well as others outside H-2.

The immunogenicity of a newly constructed macromolecular multicomponent peptide vaccine candidate against human immunodeficiency virus type 1 (HIV-1) was compared with that of previously reported vaccine candidates. This vaccine candidate is composed of a macromolecular multicomponent peptide complex consisting of three V3 region peptides, one Gag region peptide, and a CD4-binding site peptide and was constructed using the multiple-antigen peptide and glutaraldehyde methods. Sera from rabbits immunized with this newly constructed vaccine showed strong antibody titers against each constituent peptide antigen. Furthermore, these antibodies exhibited strong neutralizing and antifusion activity toward HIV-1IIIB, HIV-1MN, and fresh isolates from Japanese HIV-seropositive individuals. These results show that this new vaccine candidate has the capacity to induce strong, polyvalent immunogenicity and therefore may prove to be a powerful peptide vaccine against HIV-1 infection.

We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1) IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51 Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n.-immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-γ production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.