Background:  Eliciting neutralizing antibodies that are broadly reactive remains a critical roadblock to developing a protective HIV-1 vaccine. Presently, only a few of such neutralizing antibodies have been identified, all of which were isolated from patients. Of these antibodies, 3 target the outer domain of gp120 (gp120OD), namely b12, 2G12, and 447-52D. The outer domain is largely immunosilent due to extensive glycosylation. Although the inner domain is highly immunogenic, antibodies against this region fail to neutralize the virus. We hypothesize that the removal of the inner domain will divert antibody responses toward the outer domain of the protein, which would increase the likelihood of eliciting broadly reactive neutralizing antibodies. The objective of this study is to generate a structurally intact gp120OD that is antigenically correct.

Methods:  For constructing gp120OD, we used codon-optimized M group consensus envelope (MCON6). We used known crystal structures of gp120 to select suitable cleavage sites for separating the inner and outer domains. 6xHis tag was attached to the C-terminal end of the protein to facilitate protein purification. The protein was expressed by transient transfection of a plasmid encoding the protein into HeLa or 293T cells. Antigenic properties of the proteins were evaluated by immunoprecipitation and/or by ELISA using a panel of monoclonal antibodies, including b12, 2G12 and 447-52D, as well as HIV-Ig and antisera from individual HIV-infected patients.

Results:  MCON6 gp120OD was efficiently expressed and secreted into cell culture medium. It was quite homogeneous in size with an apparent mass of about 50 kD. The protein is glycosylated; deglycosylation with PNGaseF resulted in an expected size of about 26 kD. The protein could be immunoprecipitated by b12, 2G12, 447-52D, and 654-30D, but not by 48d or 17b. More importantly, gp120OD could co-immunoprecipitate CD4 demonstrating that our protein was able to bind CD4. Together, these results indicated that our gp120OD is structurally intact and antigenically correct. HIV-Ig was able to immunoprecipitate full-length gp120, but not gp120OD, indicating that the OD is indeed poorly immunogenic. However, evaluation of antisera from individual patients indicated that there is a significant variation amongst patients in antibody responses against gp120OD.

Conclusions:  MCON6 gp120OD we generated is antigenically correct and has great potential to elicit broadly reactive neutralizing antibodies.