Annual and Interim Progress Report Summaries

Principal Investigator: Michael Frank

Project: Studies to Maximize Immunogenicity

Submitted June 11, 2012

In this first year we have made considerable progress in achieving our goals. The grant was based on two fundamental observations that led to testable hypotheses. The first was that HIV envelope gp140 trimer, the envelope spike required for infectivity, had a life span in vivo measured in minutes. Additional experiments suggested that Env was progressively metabolized into smaller fragments. We hypothesized that rapid proteolysis destroyed critical antigens. Our experiments were indirect and we turned to direct experimental observations. The Duke Mass Spectroscopy group finds that gp140 is one of the most rapidly proteolized proteins that they have studied. It is broken down into a series of fragments that are then quite stable. We suggest that the observed non-neutralizing antibody response to Env, recognizes these stable fragments. Importantly we find that the membrane proximal segment of gp140 (MPER) is rapidly cleaved from gp140, contributing to the poor response to the MPER. Most cross-linking reagents have not prevented proteolysis, but low concentrations of gluteraldehyde and formaldehyde stabilize the molecule.

Our second observation was that Env gp140, resists complement binding almost completely. We presented evidence that complement binding contributes to effective antigen processing and hypothesized that this contributed to a poor antibody response, as observed in complement deficient animals and people. We noted that human serum has limited amounts of mannose binding lectin (MBL) and showed that purified MBL would improve C3 binding. We have been attempting to improve complement binding further by coupling carriers that facilitates complement binding to gp140. Thus far, we have studied a broad range of coupling reagents with no clear success. Our reviewers suggested that we study complement binding by an effective viral vaccine Env antigen, influenza H1N1 hemagglutinin. We have studied this protein and, as predicted, it binds C3 strongly.