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 -transierase (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 Ni2+-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 MAb SV5-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, pl5, pl7, 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 proteins. Multivalent SMAA complexes were made that contained His-pl7-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-pl7-Pk or His-pl7-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, pl5, pl7, 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.

Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells

“…Also, the epitope IYKRWIILGLNK is conserved throughout the clades and has been identified as a cross-clade T-cell target in infected individuals ( 45 ). Thus, our direct characterization of peptides from MVA.HIVconsv-infected cells supports the conserved-region strategy for the development of T-cell vaccines ( 21 , 22 , 30 , 33 ).…”

“…Jurkat cells were chosen as a model cell line for analysis of the HLA-associated peptidome, as they are easily expandable in cell culture, which includes the possibility of transformation with labeled amino acids, and they exhibit good levels of HLA class I expression. Before analysis of the immunopeptidome of MVA.HIVconsv-infected Jurkat cells, the levels of the whole-protein HIVconsv expression was assessed in a time course employing a C-terminal Pk tag recognized by monoclonal antibody (MAb) ( 33 ) in a Western blot analysis. This indicated that the HIVconsv protein reached a peak level by 5 h, maintained this level for at least 8 h, and almost disappeared by 24 hpi ( Fig.…”

Preexisting Vaccinia Virus Immunity Decreases SIV-Specific Cellular Immunity but Does Not Diminish Humoral Immunity and Efficacy of a DNA/MVA Vaccine

“…Previous studies have shown that a relatively small amount of antigen is needed for inducing a strong humoral immune response if the antigen is presented in the form of immune complexes (52-54). We speculate that immune complexes formed between preexisting SIV-specific Ab prior to the boost and low levels of SIV antigen present after the boost contributed to the observed strong boost of SIV-specific humoral immunity in Dryvax-immune animals.…”

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens

“…Two vectors pTH.tat and pTH.rev contain the respective re6 genes into the BamHI site without upstream Ub-R. The SIV macJ5 molecular clone was used as the source of these genes as previously described [2,10,12]. Vector pND14-G1 expressed the SIV mac239 envelope gp120 coding sequence under control of the hCMV IE enhancer/promotor.…”