Purification and Characterization of Oligomeric Envelope Glycoprotein from a Primary R5 Subtype B Human Immunodeficiency Virus

Abstract

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1 US4 . We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R h ) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R h of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

Human Immunodeficiency Virus Type 1 Subtype B Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope

“…We hypothesize that the gp140 and gp160 DNA vaccines presented native Env conformations in vivo that were boosted poorly or not at all by these adjuvanted, and probably at least partially denatured, gp120 monomer proteins. Although native gp120 can bind conformation-dependent broad neutralizing antibodies (61), elicit neutralizing antibodies (26), and boost DNA priming (3), there is evidence that oligomeric envelope proteins are more effective in boosting broader responses against primary HIV-1 isolates (14,60).…”

“…Production of gp120 was monitored by enzyme-linked immunosorbent assay (ELISA) at all stages of production, and purity was verified by SDS-PAGE. The SF162 protein was prepared as previously described (60).…”

Role of Neutralizing Antibodies in Protective Immunity Against HIV

“…158 Recently, we reported the purification and characterization of stable trimeric Env protein from US4 (Subtype B R5 isolates). 159 We also purified and characterized trimeric protein from SF162 and demonstrated that it elicited strong antibody responses in rabbits which neutralized two out of six heterologous subtype B primary isolates. 168 To further enhance the immunogenicity of trimeric Env, we have introduced a partial deletion of the V2 loop and expressed, purified and characterized this novel Env immunogen.…”

“…Several approaches have been tried with reasonable success. 88,[155][156][157][158][159][160][161][162][163][164][165][166] Yang and colleagues used GCN4-stabilized oligomers to demonstrate that antibodies induced by oligomeric gp140 were more effective at neutralizing heterologous primary isolates, compared to antibodies elicited by the corresponding monomeric gp120 protein. 167 Earl and colleagues made similar observations in rhesus macaques using a trimeric Env protein from SIV.…”

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

“…SF162 envelope glycoproteins were purified as described previously (6,7). Note that all the experiments carried out with the socalled gp140 in this study were in fact done with the V2 deleted version of gp140 (gp140⌬V2SF162).…”