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. The laboratory of Dr. Shiu-Lok Hu previously showed that removal of a specific conserved glycan (N197) on envelope glycoprotein gp120, near the C-terminus of the V2 variable loop, resulted in the enhanced ability of the mutant Env to induce cross-reactive neutralizing antibodies in macaques. The mechanism of increased breadth of neutralizing antibody response is unknown, but believed to be due to increased exposure and stabilization of epitopes in the CD4 binding site (CD4bs).
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.
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
The overall goal of our program is to examine how HIV-1 envelope proteins (Env) with specific modifications and biological phenotypes may impact their functional, structural/biophysical, and antigenic/immunogenic properties. In the past year, we focused on three Env-specific modifications: (1) removal of the conserved N197 glycan; (2) truncation and modification of the cytoplasmic tail; and (3) selection of Env that can mediate CD4-independent (CD4i) virus entry. Based on their unique phenotypes and structural and antigenic properties, we downselected a limited number of Env for immunogenicity studies in small animal models and non-human primates.
Results from these studies indicate that the Env modifications we introduced, individually or in combination, can enhance Env antigenicity and immunogenicity. Key observations are as follows:
Cytoplasmic tail modifications in HIV-1 Env/89.6 that increase the surface expression of Env ~8-fold in 293T cells resulted in enhanced immunogencity in mice immunized with a recombinant vaccinia prime followed by gp120 protein boost. Specifically, mice immunized with a mutated Env (termed TM1) induced 7-fold higher Env-specific IgG following two vaccinia primes, and 20-fold higher Tier 1A HIV-1 neutralizing antibodies following two primes and two boosts as measured by the CAVIMC.
Macaques immunized with CD4i Env showed higher and broader HIV-specific response directed to gp70-V1/V2 fusion proteins than those immunized with their CD4-dependent WT Env, similar to our earlier findings from mouse models. Importantly, all but one immunized macaque generated cross-reactive antibody-dependent cellular cytotoxicity (ADCC) activities.
Importantly, we were able to elicit cross-reactive neutralizing antibody (Nab) responses against heterologous Tier 2 viruses in rabbits primed with a replication-competent vaccinia virus and boosted by subunit gp120, in a regimen similar to that used in the RV144 trial. Nab were elicited against ~50% of a panel of global tier 2 primary isolates with IC50 titers up to 1:100 in the standard TZM-bl assay done by the CA-VIMC (Townsley et al. J Virol. 2016 Sep 12;90(19):8644-60. doi: 10.1128/JVI.00853-16. Print 2016 Oct 1. PMID: 27440894). Some of the Nab were directed against the CD4 binding site (CD4bs). These rabbits also generated antibodies that recognized protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. These findings demonstrate that antibody responses that have been correlated with protection against HIV-1 acquisition in humans can be elicited in a preclinical model by a poxvirus prime-gp120 boost strategy and that improvements may be achievable by optimizing the nature of the priming and boosting immunogens. Removal of the N7 glycan modified the epitope specificity of the Ab responses; but did not improve the titer or the breadth of Nab. Structural and biophysical studies of gp120 monomer and SOSIP trimers with or without the N197 glycan indicate that the effect exerted by this glycan on Env antigenicity is only evident in the context of the trimer, but not gp120 (Liang et al., J Virol. 2016 Sep 29;90(20):9224-36. doi: 10.1128/JVI.01116-16. Print 2016 Oct 15. PMID: 27489265). This finding indicates that the use of monomeric gp120 as a boosting immunogen may be suboptimal.
We are currently completing the analysis of the functional antibody responses and the epitope specificity of the cross-reactive tier 2 Nab activities. Together with structural and biophysical studies of the modified Env, we aim to gain further insight on the basis of the enhanced antibody responses observed and to design immunogens that may elicit protective immunity against HIV.