Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

Abstract

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

Figures

FIG. 1

Groups of 10 guinea pigs were immunized with HIV gp120 DNA in saline, PLG, or PLG plus AlPO4 at doses of 25 or 250 μg of DNA. As positive controls, animals were immunized with 50 μg of recombinant gp120 protein with or without MF59 adjuvant. All animals were immunized at 0 and 4 weeks, and sera were collected at 2 weeks post-second immunization. Data are presented as geometric mean ELISA titer ± standard error of the mean (SEM) for n = 10.

FIG. 2

Groups of 10 guinea pigs were immunized with HIV gp140 DNA in saline or PLG at a dose of 50 μg of DNA. As positive controls, animals were immunized with 25 μg of recombinant gp120 protein with MF59 adjuvant. All animals were immunized at 0, 4, and 8 weeks, and sera were collected at 2 weeks post-second (A) and -third (B) immunization. Some groups were immunized with DNA only (D/D/D) or protein plus MF-59 only (P/P/P) or primed with DNA and boosted with protein plus MF-59 (D/P or D/D/P). Data are presented as geometric mean ELISA titer ± SEM for n = 10.

FIG. 3

Groups of five guinea pigs were immunized with a combination of HIV gp140 DNA and HIV gag DNA in saline or PLG at doses of 1 and 0.5 mg of DNA, respectively. Animals were immunized at 0, 4, and 8 weeks, and sera were collected at 3, 7, 11, and 32 weeks. Data are presented as geometric mean ELISA titer ± SEM (n = 5) for anti-Gag (A) and anti-Env (B) antibodies.

FIG. 4

Groups of five rhesus macaques were immunized with HIV gag DNA in saline (solid triangles) or PLG (open circles) at a DNA dose of 0.5 mg. Animals were immunized at 0 and 4 weeks, and sera were collected at 2, 6, and 11 weeks. Data are presented as geometric mean ELISA titer ± SEM for n = 5. For comparison, anti-Gag antibody titers are shown for unimmunized animals (solid squares) (n = 4).

FIG. 5

Gag-specific cytolytic activity of PBMC from individual rhesus macaques given two doses of PLG/CTAB-formulated pCMV-gag DNA (A) or unformulated pCMV-gag DNA (B). Two weeks after the second DNA immunization, PBMC were stimulated with a pool of overlapping Gag peptides and cultured in the presence of IL-2 for 8 days. Serial dilutions of cultures (1:15, 1:45, 1:135, and 1:405) were added to 51Cr-labeled rVVgagpolSF2-infected autologous B-LCL (●) or rVVgp160envSF162-infected autologous B-LCL (○). 51Cr release was determined 4 h after addition of cultured PBMC to B-LCL.