Background:  One of the major goals in developing a protective HIV-1 vaccine is to induce neutralizing antibodies that are broadly reactive. Presently, only a few of them have been identified, all of which were isolated from patients. Of these antibodies, 2F5 and 4E10 target the membrane-proximal region (MPR) of gp41 ectodomain. The objective of this study is to generate antigens that can elicit antibodies against the gp41 MPR.

Methods:  Rather than using short peptides that contain 2F5 or 4E10 epitopes, our approach was to use larger protein fragments that contain gp41 MPR. We generated 3 GST-fusion proteins, which consist of C-terminal 30, 64, or 100 amino acids of gp41 ectodomain from an M group consensus sequence (MCON6). Proteins were expressed in E. coli and purified. Antigenic properties of the proteins were evaluated by immunoprecipitation and/or by ELISA using 2F5, 4E10, HIV-Ig, as well as antisera from individual HIV-infected patients. To characterize their immunogenic properties, Balb/c or HLA-DR transgenic mice were immunized. Immune responses were evaluated by peptide ELISA, ELISpot, and neutralization assays.

Results:  A protocol was developed to express large amounts of GST-gp41 fusion proteins in E. coli, to solubilize, to renature, and to purify them (>45 mg/L). Both GST-gp41-100 and -64 were efficiently recognized by HIV-Ig. In contrast, GST-gp41-30 was recognized weakly, indicating poor immunogenicity of gp41 MPR. Unlike HIV-Ig, 2F5 recognized all three proteins equally demonstrating that they all contain conformationally correct 2F5 epitopes. 4E10, on the other hand, recognized GST-gp41-64 and -100 better than GST-gp41-30, suggesting that the conformation of 4E10 epitope is partly influenced by upstream sequences. Evaluation of antisera from individual patients indicated that there is a significant variation amongst patients in antibody responses against the 3 gp41 fragments. Although there was no strict correlation, antisera having higher antibodies against GST-gp41-30, in general, exhibited broader neutralizing activity. One HLA-DR4-restricted T cell epitope and 5 B-cell epitopes have been identified, including ones that overlap caveolin-1- and 2F5-binding sites. Antisera exhibited neutralizing activity, the breadth of which is presently being evaluated.

Conclusions:  MCON6 gp41 antigens we generated are antigenically correct and have great potential to elicit broadly reactive neutralizing antibodies.