Alpha-2-macroglobulin (a2M) incorporation of proteins enhances in vitro antigen presentation by 100-1000-fold and is equally effective as complete Freud's adjuvant (CFA) for eliciting antibodies in vivo. Long-term objectives of the proposed studies are: (1) to evaluate the use of A2M as a novel adjuvant for antigen processing of subunit vaccines, and (2) to confirm the potential utility of using this novel formulation by showing, as proof of principle, induction of potent humoral immune responses capable of neutralizing the recently-demonstrated, interleukin-10(IL-10)-inducing activity of HIV-1 nef(Nef). These studies may provide a safe, efficient way to enhance antigen processing of a variety of viral or bacterial proteins, thus allowing generation of strong immune responses even in the absence of replicating immunogens. Vaccines developed with this novel formulation may have utility not only in preventing initial infections with various pathogens, but also in boosting immune responsiveness in previously infected individuals. These objectives will be accomplished through three specific aims: (1) generation of vectors for and expression of recombinant Nef and demonstration in murine, guinea pig, and rabbit leukocytes of IL-10 induction by Nef; (2) incorporation of recombinant Nef into murine, guinea pig and rabbit a2M and determination of the in vivo immunogenicity of such formulations compared to """"""""free"""""""" Nef or Nef formulated in other adjuvants; and (3) determination of the ability of sera from animals immunized with Nef formulated in a2M to neutralize the IL-10-inducing activity of Nef, in vitro and/or in vivo.
These specific aims will be accomplished as follows. Recombinant Nef will be obtained by constructing the Nef plasmid pGEX-Nef, encoding the entire mature protein, by insertion of a full-length Nef gene from pUC19 into a glutathione-S-transferase (GST) gene fusion vector, pGEX-3X. GST-Nef fusion protein, expressed in E. coli transformed with pGEX-Nef, will be purified by a combination of glutathione-Sepharose chromatography, digestion with factor Xa to release the GST, and affinity chromatography with high-titered, anti-Nef rabbit IgG coupled to Sepharose. Contaminating endotoxins will be removed using Deloxi-Gel affinity chromatography and final purity determined by gel electrophoresis, mass spectrophotometry, and amino acid sequencing. Induction of IL-10 will be determined using Nef-stimulated peripheral blood leukocytes or elicited peritoneal cells and ELISA measurements of culture supernatants or RT-PCR of cultured cells. a2M for each species will be prepared using standardized procedures established by the investigators, and incorporation of Nef into a2M will be accomplished using previously published techniques. Animals, immunized on a monthly schedule with Nef, Nef incorporated into a2M, or Nef mixed with CFA, will be bled monthly and titers to Nef determined by ELISA and immunoprecipitation. High-titered sera to Nef will be tested for their ability to block Nef induction of IL-10 in leukocyte cultures pre-exposed to dilutions of the sera.
