title: Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses.
abstract: Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification 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 (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. 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 immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
authors.affiliation: School of Biological and Medical Sciences, Division of Cell and Molecular Biology, University of St. Andrews, Fife, U.K.
authors.forename: T
authors.initials: T
authors.lastname: Hanke
authors.forename: C
authors.initials: C
authors.lastname: Botting
authors.forename: E A
authors.initials: EA
authors.lastname: Green
authors.forename: P W
authors.initials: PW
authors.lastname: Szawlowski
authors.forename: E
authors.initials: E
authors.lastname: Rud
authors.forename: R E
authors.initials: RE
authors.lastname: Randall
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D008251:Macaca; D051379:Mice; D010957:Plasmids; D015302:Simian Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology*; D014760:Viral Fusion Proteins; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000936:Antigen-Antibody Complex; D014760:Viral Fusion Proteins; D014764:Viral Proteins