Increased immunogenicity for gp120. We have previously mapped the group-specific neutralizing epitope of HIV-1 to the conformation-dependent CD4 binding site on gp120. Unfortunately, this site is not very immunogenic, as found on recombinant gp120 monomers In this project, we have attempted to increase the overall immunogenicity of gp120, while preserving the conformation of this important neutralizing site. We have tried two strategies: conjugate or particulate vaccines. For the first strategy, we have found a way to enzymatically oxidize the oligosaccharide sidechains of gp120, while retaining the high affinity CD4 binding site. Using affinity purified galactose oxidase (after neuraminidase to remove sialic acid), we were able to introduce over 20 aldehydes per molecule without disturbing CD4 binding. We have also developed a novel chemical crosslinker, which can couple these sites to free SH groups introduced into virtually any carrier protein. In this way, we expect to increase the immunogenicity of gp120 by means of a carrier effect, as observed for other antigens. The first example will be HBsAg, which is highly immunogenic in humans at a dose of 20 ug, at least 50X less than is required for gp120 alone. Another effect of these conjugates may be to crosslink multiple copies of gp120 onto the same particle. This may increase immunogenicity by enabling more antigen to enter a given antigen presenting cell. Since these cells have a threshold of about 200 copies before they can stimulate T cells, this may explain the 1000-fold enhanced immunogenicity of HBsAg particles vs. monomers. Hopefully, we may achieve a similar increase in vaccine potency by coupling gp120 into larger aggregates. Our second strategy is to make particulate antigens by adsorbing gp120 onto the surface of virus-sized latex beads. So far, we have succeeded in adsorbing about 200 molecules per bead, and by doubling the radius of the beads, we should be able to increase this four-fold. Each particulate antigen will be analyzed in an in vitro assay, and the most promising ones will be tested for immunogenicity in rabbits, both for total antibodies and for neutralizing antibodies against the group-specific neutralizing site. The cellular assay consists of a T cell line specific for gp120 and an MHC matched antigen presenting cell. The T cells have shown a good response to gp120 and to the appropriate peptide fragment of gp120, and they will now be tested for the response to particulate gp120, to determine the best constructs for testing in rabbits.
