Inducing antibodies capable of neutralizing a broad array of HIV isolates is the holy grail for vaccine research. One of the key obstacles is the virus’s outer envelope, which is densely cloaked in sugary molecules that shield the parts that are vulnerable to antibody-mediated attack. When unmodified HIV envelopes are used to make vaccines, the antibody responses that result are largely useless because they target these irrelevant decoys. But Ema Crooks from James Binley’s laboratory at the Torrey Pines Institute for Molecular Studies may have come up with a way to address the problem. The strategy involves using specific enzymes to digest the “junk” envelope, leaving behind the envelope structures (trimers) that are more likely to induce effective antibodies. A similar strategy was described at the recent Keystone HIV vaccines meeting by John Moore’s research group at Cornell University. The next step will be to test these new junk-depleted HIV envelopes as vaccine immunogens.
JVI Accepts, published online ahead of print on 6 April 2011
J. Virol. doi:10.1128/JVI.00154-11
Ema T. Crooks, Tommy Tong, Keiko Osawa, and James M. Binley
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA
HIV-1 viruses and virus-like particles (VLPs) bear non-native "junk" forms of Envelope glycoprotein (Env) that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a co-dependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160, termed "gp160ER" that bypasses protein maturation and trafficks directly into particles. Gp160ER was found to bind two copies of mAb 2G12, consistent with its exclusively high mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer-VLPs" may be useful neutralizing antibody immunogens.
Keystone Symposia: HIV Evolution, Genomics and Pathogenesis (X7), Whistler Conference Centre, Whistler, British Columbia, Canada, March 20–25, 2011
Generation of stable, deglycosylated HIV-1 Env trimers for crystallization and immunogenicity studies
Depetris, R.S.1, Julien, J.P.2, Pejchal, R.2, Rabidou, D.1, Kachare, M.1, Olson, W.3, Wilson, I.2, Sanders, R.1 & Moore, J.P.1
1 Dept. of Microbiology and Immunology, Weill Cornell Medical College, 2 The Scripps Research Institute, 3 Progenics Pharmaceuticals
The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development programs aimed at eliciting humoral responses. The extensive glycosylation of the Env trimer hinders immunogenicity and crystallography studies, because glycans both shield conserved neutralization epitopes and create structural heterogeneity. We have evaluated different ways to obtain stable, properly folded, fully deglycosylated gp140 trimers. This goal can be achieved by inhibiting the generation of complex carbohydrates by expressing the proteins in mutant cell lines, then treating them with appropriate glycosidases under mild conditions compatible with trimer integrity. The deglycosylated Env proteins are trimeric, with stability comparable to wild type. Their antigenic structure is generally conserved, except for glycan-dependent epitopes. The deglycosylated trimers do not aggregate significantly, and have no increased sensitivity to proteolytic digestion. Deglycosylated Env trimers are promising candidates for crystallization studies and are worth evaluating as immunogens.