Enhancement of Antibodies to the Human Immunodeficiency Virus Type 1 Envelope by Using the Molecular Adjuvant C3d

Abstract

DNA vaccines expressing the envelope (Env) protein of the human immunodeficiency virus have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, the murine and human homologues of the complement component, C3d, were used in a DNA vaccine to enhance the titers of antibody to Env. Initially, plasmids expressing a secreted form of Env (sgp120) fused to one, two, or three copies of the murine homologue of C3d (mC3d) were constructed. Mice were inoculated with four vaccinations of DNA or two DNA vaccinations, followed by two boosts of affinitypurified gp120 protein. Analyses of titers demonstrated that multiple copies of mC3d coupled to sgp120 induced long-lasting, high-titer anti-Env antibody. Priming mice with sgp120-mC3d-DNA, followed by inoculation of purified gp120 protein, elicited the strongest antibody titers; however, the avidity maturation of the antibody was accelerated in the mice inoculated with sgp120-mC3d 3 -DNA. In addition, DNAs expressing sgp120 fused to three copies of the human homologue of C3d (hC3d 3 ) efficiently enhanced the anti-Env antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d 3 elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody responses to Env compared to non-C3d fused forms of Env, and this approach may be one way to overcome the poor ability of DNA vaccines to generate antibodies to Env.

C3d Enhances Immune Responses Using Low Doses of DNA Expressing the HIV-1 Envelope from Codon-Optimized Gene Sequences

“…Plasmids were constructed to express a soluble form of HIV-1 gp120 using wild type or synthetic genes optimized for expression in mammalian cells [2,18,37] from the HIV-1 isolates, YU-2, JR-FL, ADA, and R2 [5,16,21,32]. Each DNA vaccine was designated for the gene insert that the plasmid expressed.…”

“…Mice were anesthetized with 0.03-0.04 ml of a mixture of 5 ml ketamine HCl (100 mg/ml) and 1ml xylazine (20 mg/ml). Gene gun immunizations were performed on shaved abdominal skin using the hand held gene delivery system (Bio-Rad, Hercules, CA, USA) as described previously [5,16,17,24]. Mice were immunized with two gene gun doses containing 1.0 μg per 0.5 mg of approximately 1 μm gold beads (Bio-Rad, Hercules, CA, USA) at a helium pressure setting of 400 psi.…”

“…Independently, each approach enhances antibody titer and cellular responses against Env compared to DNA plasmidsexpressing wild-type (wt) env gene inserts only. The fusion of C3d to an antigen results in enhanced immunogenicity to the fused antigen [5,16,17,19,21,24,31,32,35,36]. Rodents inoculated with DNA plasmids expressing HIV-1 Env fused to multiple copies of human or murine C3d (mC3d) had enhanced anti-Env specific IgG titers and accelerated affinity maturation of antibody [16].…”

“…Rodents inoculated with DNA plasmids expressing HIV-1 Env fused to multiple copies of human or murine C3d (mC3d) had enhanced anti-Env specific IgG titers and accelerated affinity maturation of antibody [16]. In addition, higher titers of neutralizing antibodies were elicited in rodents vaccinated with gp120-mC3d 3 compared to gp120 alone [16]. The precise mechanism of C3d enhancement is unclear; however both CR2-dependent and CR2-independent pathways play a role in C3d immune enhancement [19].…”

“…Two recent approaches used to enhance the immunogenicity of Env expressed from a DNA vaccine are 1) the fusion of the molecular adjuvant, C3d, to a soluble form of Env [5,16,21,32,36] and 2) the use of codon-optimized (co) env gene inserts [5,11,18,21,37]. Independently, each approach enhances antibody titer and cellular responses against Env compared to DNA plasmidsexpressing wild-type (wt) env gene inserts only.…”