Due to their close evolutionary relationship to humans, nonhuman primates have provided invaluable animal models for studying a wide variety of human clinical conditions, including HIV infection and AIDS (9
). While the human and monkey genomes are highly similar, the small differences noted between monkey and human protein sequences are often sufficient to elicit neutralizing antibodies when the human xenogeneic reagents are given to monkeys (24
). Circumventing this potential problem, this study reports three biologically active molecular adjuvants that were designed specifically for macaque vaccine studies. To this end, the nucleic acid sequences of the regions coding for macaque GITRL and SP-D were determined. Macaque SP-D was nearly identical to its human ortholog (Fig. ). Similarly, macaque GITRL differed in only five amino acid positions from its human ortholog (Fig. ). The sequence of macaque CD40L has been previously reported and is also nearly identical to its human ortholog (37
). Nevertheless, the construction of fully autologous macaque proteins should avoid potential cross-species differences in biological activity and reduce or eliminate the possibility that these adjuvant molecules might elicit immune responses directed against themselves. For example, in the mouse system, we were unable to detect any antibodies against murine SP-D-CD40L when a plasmid for this protein was used in a DNA vaccine (35
Plasmids for two forms of macaque soluble CD40L were produced (Fig. ). pmacAcrp30-CD40L is anticipated to form a V-shaped molecule with two trimers of CD40L (11
). pmacSP-D-CD40L is anticipated to form a four-armed cruciate molecule that has four trimers of CD40L (10
). When these plasmids were transfected into 293T cells, proteins of the expected immunoreactivity and size were secreted as assessed by Western blotting (Fig. ). To determine whether the 293T cell-produced proteins were active, both a human B-cell proliferation assay (30
) and a macaque B-cell proliferation assay were used. Both the two-trimer and four-trimer forms of macaque soluble CD40L were highly active on human B cells in the presence of IL-4 (Fig. ), and four-trimer macaque CD40L was active on macaque B cells even in the absence of IL-4 (Fig. ). These results support the prediction that these molecules will also be effective as dendritic cell activators and vaccine adjuvants.
It is of note, however, that the maximal proliferation of human B cells induced by macaque CD40L reagents appeared severalfold lower than that of a similar aliquot of B cells stimulated with the human CD40L reagent (Fig. ), suggesting species-specific differences. Conversely, the use of a human CD40L trimer protein in the stimulation of macaque B cells versus human B cells required fivefold-higher levels of trimer to induce maximal proliferation of macaque B cells in vitro, and this proliferative activity was markedly lower than that exhibited by human B cells (data not shown).
A plasmid for a four-trimer form of macaque soluble GITRL was also produced as a fusion protein with SP-D. When this plasmid was transfected into 293T cells, a protein of the expected immunoreactivity and size was secreted as assessed by Western blotting (Fig. ). To determine whether the 293T cell-produced protein was active, two assays were used. The first assay relied on the ability of GITR stimulation to serve as a costimulus for CD4+
T-cell proliferation in response to a T-cell receptor stimulus (14
). With human CD4+
T cells and immobilized anti-CD3 antibody, macSP-D-GITRL was clearly active in this costimulation assay (Fig. ). The second assay measured the ability of GITR stimulation to abrogate the immunosuppressive effects of CD4+
regulatory T cells on CD4+
T-cell proliferation in a mixed leukocyte reaction (21
). With human cells from two subjects in this MLR-based assay, macSP-D-GITRL was found to be active in reversing CD4+
regulatory T-cell immunosuppression (Fig. ).
In conclusion, these plasmids for multimeric soluble CD40L and GITRL provide macaque reagents for use in a variety of experimental vaccine or immunotherapeutic studies. Given our recent findings that the murine forms of these molecules are strong molecular adjuvants for HIV DNA vaccination in mice, it will be interesting to determine whether the macaque forms of these plasmids can be used to augment immune responses to DNA vaccines in the SIV/macaque model as a preliminary step for their future clinical use.