Macrophage inflammatory protein-1alpha (MIP-1alpha) expression plasmid enhances DNA vaccine-induced immune response against HIV-1

Abstract

CD8+ cell-secreted CC-chemokines, MIP-1alpha, and MIP-beta have recently been identified as factors which suppress HIV. In this study we co-inoculated MIP-1alpha expression plasmid with a DNA vaccine constructed from HIV-1 pCMV160IIIB and pcREV, and evaluated the effect of the adjuvant on HIV-specific immune responses following intramuscular and intranasal immunization. The levels of both cytotoxic T lymphocyte (CTL) activity and DTH showed that HIV-specific cell-mediated immunity (CMI) was significantly enhanced by co-inoculation of the MIP-1alpha expression plasmid with the DNA vaccine compared with inoculation of the DNA vaccine alone. The HIV-specific serum IgG1/IgG2a ratio was significantly lowered when the plasmid was co-inoculated in both intramuscular and intranasal routes, suggesting a strong elicitation of the T helper (Th) 1-type response. When the MIP-1alpha expression plasmid was inoculated intramuscularly with the DNA vaccine, an infiltration of mononuclear cells was observed at the injection site. After intranasal administration, the level of mucosal secretory IgA antibody was markedly enhanced. These findings demonstrate that MIP-1alpha expression plasmid inoculated together with DNA vaccine acts as a strong adjuvant for eliciting Th1-derived immunity.

Fig. 1

Construction of expression plasmid pCAGGSMIP-1α. pCAGGS vector was digested with Xho I restriction enzyme, blunted, and ligated with blunted MIP-1α cDNA.

Fig. 2

Immunoglobulin subclasses of HIV-specific antibody. BALB/c mice were given 2 μg of DNA vaccine alone or vaccine combined with 10 μg of pCAGGSMIP-1α by intramuscular (i.m.) and intranasal (i.n.) immunization. After 4 weeks, anti-HIV-IgG1 (□) and IgG2a titres (▪) were assayed by ELISA. Optical density (OD) data are expressed as means OD ± s.e.m. *A significant difference from that obtained using the DNA vaccine alone (P < 0.05).

Fig. 3

Cytotoxic T lymphocyte (CTL) activity of MIP-1α expression plasmid. (a) By intramuscular administration. (b) By intranasal administration. Two separate groups of mice were immunized with 2 μg of DNA vaccine formulated with the indicated dose of MIP-1α expression plasmid. Splenocytes were isolated after 4 weeks and cultured for 5 days with the V3 peptide. The CTL activity was titrated at E:T ratios of 5, 20 and 80. Data are means ± s.e.m. of three to four mice. *A significant difference from that obtained using the expression vector alone (P < 0.01).

Fig. 4

Histological examination of injected mice. (a) Injected with 2 μg of DNA vaccine alone. (b) Injected with 2 μg of DNA vaccine and 10 μg of MIP-1α expression plasmid. (Mag. × 100.) (c) Injected with 2 μg of DNA vaccine and 10 μg of MIP-1α expression plasmid. (Mag. × 200.) At 1, 3, 5, 7 and 14 days after injection, muscles were resected, fixed with 10% buffered formalin, and embedded in paraffin. Thin sections were then prepared and stained with haematoxylin and eosin for light microscopic observation.