A comparative immunogenicity study of HIV-1 virus-like particles bearing various forms of envelope proteins, particles bearing no envelope and soluble monomeric gp120.

To assess the potential of native Envelope glycoprotein (Env) trimers as neutralizing antibody vaccines, we immunized guinea pigs with three types of VLPs and soluble gp120. Particles included "SOS-VLPs" (bearing disulfide-shackled functional trimers), "UNC-VLPs" (bearing uncleaved nonfunctional Env) and "naked VLPs" (bearing no Env). The SOS-VLPs were found to have a density of about 27 native trimers per particle, approximately twice that of live inactivated HIV-1 preparations. As immunogens, UNC- and SOS-VLP rapidly elicited anti-gp120 antibodies focused on the V3 loop and the gp120 coreceptor binding site. Reactivity to the gp41 immunodominant domain was absent in SOS-VLP sera, presumably because gp120-gp41 association is stabilized, effectively covering this epitope. Gp120-immune sera reacted with the receptor binding sites of gp120 and were less focused on the V3 loop. Some Env-VLP sera neutralized primary isolates at modest titers. The measurement of neutralization was found to be affected by the cell lines used. Depending on the assay particulars, non-Env specific antibodies in VLP sera could enhance infection, or nonspecifically neutralize. However, a neutralization assay using TZM-BL cells was essentially clear of these effects. We also describe a native trimer binding assay to confirm neutralization activity in a manner that completely eliminates nonspecific effects. Overall, our data suggests that Env-VLP sera were primarily focused on nonfunctional forms of Env on VLP surfaces, possibly gp120/gp41 monomers and not the trimers. Therefore, to make progress toward a more effective VLP-based vaccine, we will need to find ways to refocus the attention of B cells on native trimers.

Redox-triggered infection by disulfide-shackled human immunodeficiency virus type 1 pseudovirions.

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant's infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins.

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.