Hu: Unmasking Conserved Epitopes
A vaccine capable of generating broadly neutralizing antibodies (bNAbs) against primary isolates of human immunodeficiency virus (HIV-1) remains a major but as yet unmet goal. Although recent data suggest that broadly neutralizing antibodies (bNAbs) can be found in some HIV-infected individuals, they are often directed against conserved epitopes on the envelope glycoprotein such as the Env CD4 binding site (CD4bs), conformational epitopes, and other potentially important regions such as the α4β7 bs that are often masked by glycans on the surface of the virus envelope.
The laboratory of Dr. Shiu-Lok Hu previously showed that removal of a specific conserved glycan on envelope glycoprotein gp120 (N197, near the C-terminus of the V2 variable loop) resulted in the enhanced ability of the mutant Env to induce bNAbs in macaques. The mechanism of increased breadth of neutralizing antibodies is believed to be due to increased exposure and stabilization of CD4bs epitopes through the formation of CD4-independent, trimeric protein that assumes a CD4 "triggered" conformation in the absence of CD4.
Dr. Hu plans to generate an optimized Env immunogen that will induce an improved humoral immune response to HIV by: 1) modifying the N-linked glycans on a variety of Envs to expose critical epitopes, 2) using
in vitro cell culture methods to select for Envs with this triggered conformation, and 3) increasing the surface expression of Env on HIV infected cells through targeted changes in cytoplasmic tail region of the protein. Further, by using a replicating viral vector in a prime/ boost vaccination regimen, Dr. Hu expects to elicit a more robust humoral and cellular immune response than those generated by the non-replicating viral vectors currently being evaluated in human clinical trials. If successful, vaccine candidates could rapidly progress into pre-development studies required for clinical trials in humans.
This work builds upon extensive and complementary expertise of five accomplished investigators: Shiu-Lok Hu and Kelly Lee (University of Washington), James Hoxie and Drew Weissman (University of Pennsylvania, subgrantee) and Shan Lu (University of Massachusetts Medical School, subgrantee).
1. To generate and evaluate specific glycan-modified Env from diverse isolates as vaccine immunogens
2. To design modified Env with the ability to mediate CD4-independent (CD4i) infection and increased expression on the infected cell surface
3. To construct recombinant vaccinia viruses expressing WT and modified HIV-1 Env
4. To evaluate novel Env immunogens in small animal models
5. To use heterologous Env for “prime-boost” immunization of macaques to focus responses to conserved epitopes
6. To use novel approaches to map structural determinants of antigenicity in HIV Env
Most of our efforts in the past year have been directed to the generation and characterization of immunogens, and the development of novel methodologies to better define the antigenic properties of HIV-1 envelope proteins and refine criteria to down-select candidate immunogens for in vivo studies.
We have generated an extensive panel of Env proteins from diverse HIV-1 isolates with or without the highly conserved N197 glycan at the base of the V2 loop. Increased neutralization sensitivity to CD4-binding site antibodies is the conserved phenotype for all the N197 glycan mutants examined, indicating the conserved role of this glycan masking the conserved CD4 binding site. Removal of the N197 glycan also affects the antigenicity of the V2 and V3 loops, indicating a role for the N197 glycan modulating the conformation or the dynamic properties of these loops. We conducted the first in vivo study in rabbits primed with recombinant vaccinia virus expressing 89.6 WT Env or the N197 glycan mutant and boosted with the cognate Env gp160 or gp120. Although moderate to high levels of neutralizing activities were detected in most animals against heterologous tier 1 viruses, there was little or no neutralization against tier 2 isolates. Together with our earlier observations, results from this study indicate the potential importance of efficient priming and the conformation of the immunogen used. Studies to address these issues are underway.
We have also generated recombinant vaccinia viruses expressing WT and several mutant Envs from a clade C isolate with or without the glycans proximal to the integrin binding site in the V2 loop. Preliminary data from our collaborator, Dr. J. Arthos (NIH), indicate that these glycans modulate interactions between Env gp120 to the a4b7integrin. Effect of these glycan mutations on Env immunogenicity will be evaluated in the context of prime-boost immunization in rabbits and macaques.