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Background:  The effectiveness of HIV DNA vaccines can be improved by the addition of immunostimulants. One of the most powerful endogenous immunostimulatory molecules is CD40 ligand (CD40L, CD154), a trimeric tumor necrosis factor (TNF) superfamily ligand expressed on the surface of activated CD4+ T cells that has been difficult to use as a solubilized protein.

Methods:  Based on in vitro studies, it was hypothesized that multimerization (i.e., many trimers) would be needed to utilize soluble CD40L in a vaccine formulation. Consequently, three kinds of CD40L plasmids were tested in mice: pTr-CD40L, a 1-trimer-soluble CD40L containing an isoleucine zipper; pAcrp30-CD40L, a 2-trimer CD40L produced as a fusion protein with the body of Acrp30, a spontaneously multimerizing collectin; and pSP-D-CD40L, a 4-trimer CD40L produced as a fusion protein with the body of pulmonary surfactant protein D (SP-D), another collectin. Antigen-specific splenic CD8+ immune responses were measured using CTL assays, IFN-γ ELISpot, and major hystocompatibility class I tetramers.

Results:  When combined with a plasmid for secreted, codon-optimized Gag (pScGag) and co-injected into mice intramuscularly three times at two-week intervals, 1-trimer soluble CD40L had almost no adjuvant effects but the 2- and 4-trimer soluble CD40L molecules led to strong CD8+ T-cell responses in direct proportion to their valence (4 > 2 > 1). No toxicity was appreciated for any of the constructs. Similar results were found using an antigen plasmid for Env. However, no version of CD40L significantly augmented antigen-specific proliferative responses by splenocytes or enhanced antibody production. Consequently, glucocorticoid-induced TNF receptor ligand (GITRL), another TNF superfamily ligand that co-stimulates CD4+ T cells and abrogates the immunosuppressive activity of CD4+CD25+ regulatory T cells, was tested as a 4-trimer plasmid, pSP-D-GITRL. This molecule not only stimulated CD8+ T-cell responses but also stimulated antigen-specific proliferative responses and antibody production.

Conclusions:  These results indicate that fusions between immunostimulatory TNF superfamilies and collectins can significantly enhance responses to HIV DNA vaccines and act as a novel vaccine adjuvant.

Keywords: CD40 Ligand; DNA Vaccines; Regulatory T cells