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

DNA vaccinations effectively induce both humoral and cellular immune responses to immunogens from diverse infectious agents. However, DNA vaccines expressing the HIV-1 envelope glycoprotein (Env) are poorly immunogenic when expressed from wild-type (wt) DNA sequences. Two recent approaches used to enhance the immunogenicity of Env expressed from a DNA vaccine are the fusion of the molecular adjuvant, C3d, to a soluble form of Env and the use of codon-optimized (co) env gene inserts. Independently, each approach enhances antibody titer and cellular responses against Env expressed from gene inserts. The goal of this study was to examine if both codon-optimization of env gene inserts and C3d conjugation to Env could function in a synergistic manner to enhance immunogenicity. Mice (BALB/c) were inoculated with decreasing doses (2.0 μg, 0.2 μg or 0.02 μg) of co DNA expressing Env alone or fused to three copies of murine C3d (mC3d3) gene. Mice vaccinated with the highest dose (2.0 μg) of DNA had high anti-Env specific antibody titers regardless of the addition of mC3d3. At lower doses (0.2 μg and 0.02 μg) of DNA, mice vaccinated with Env-mC3d3 had enhanced immune responses compared to mice vaccinated with DNA expressing Env only. In addition, mice vaccinated with Env-mC3d3 at the highest doses of DNA had enhanced interleukin-4 secreting cells, while mice vaccinated with the lowest dose of DNA had enhanced interferon-gamma secreting cells. Therefore, both codon-optimization of env sequences and C3d conjugation to Env appear to enhance anti-Env antibodies in an independent and additive manner.