EXPRESSION AND PURIFICATION OF NONGLYCOSYLATED SIV PROTEINS, AND THEIR USE IN INDUCTION AND DETECTION OF SIV-SPECIFIC IMMUNE-RESPONSES

Two commercially available expression vectors were modified to generat e plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9 cPk had a short oligopeptide tag termed Pk at their carboxy termini an d either glutathione S-transferase (GST) or a small histidine (His) ta g, respectively, at their N termini. GST fusion proteins can be purifi ed on immobilized glutathione and proteins coupled to the His tag sele ctively bind to Ni2+-NTA columns. The Pk tag is recognized by monoclon al antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purifie d in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification step s significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAb SV5-P-k in the second purificatio n step, Pk-linked antigens can be assembled directly into solid matrix -antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIVmac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk prot eins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immun izations of mice with this mixture, antibodies could be detected to al l five SIV antigens. When compared to single-protein immunizations, th e immunogenicity of some of the proteins in this cocktail was either e nhanced or decreased. Mice were also immunized with His-p17-Pk or His- p17-Pk-antibody complexes in the presence or absence of alum. The anti body-antigen complexes induced two- to four-fold higher antibody level s than antigen alone but did not appear to be more immunogenic in indu cing lymphoproliferative responses. Sera from SIV-infected macaques we re tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p 17, p27, rt, and vif were readily detected, antibodies against proteas e and vpx were present at much lower levels, but no antibodies were de tected to nef, rev, tat, or vpr. Thus, we have developed a comprehensi ve range of reagents (available on request) that can be used to examin e immune responses to SIV in both mice and monkeys.