Zolla-Pazner: Epitope-Scaffold Immunogens
A vaccine capable of inducing antibodies that neutralize most of the diverse HIV-1 strains circulating in humans would dramatically reduce human suffering. Evidence for the ability of antibodies to protect against HIV-1 infection comes from studies showing that passively administered antibodies can produce sterilizing immunity in animal models. The fact that HIV-1 mutates to escape naturally occurring human antibodies indicates that these antibodies can indeed block or eliminate virus.
The ideal vaccine would target a region on the surface of the virus that is highly conserved across all families of HIV-1 (known as subgroups or clades). One such region is the third variable region (V3) of the HIV-1 envelope glycoprotein gp120. A large body of data now shows that, due to the structural and biological constraints on V3, the variability of the V3 domain is limited, resulting in epitopes that can be recognized by broadly cross-reactive serum anti-V3 Abs from HIV-infected individuals. Antibodies to V3 are able to block virus infectivity because V3 forms part of the gp120 surface required to interact with the virus co-receptor.
The research team lead by Dr. Susa Zolla-Pazner is exploring whether vaccines that target the immune response to the V3 loop of gp120 induce broadly-reactive, cross-neutralizing antibodies that protect against HIV infection. The researchers are designing immunogens that focus the humoral immune response on structural components of V3 that are common to many or all HIV isolates and have the potential to prevent HIV-1 entry into target cells.
- Dissect the diversity of the natural human antibody response to V3 by producing and characterizing broadly neutralizing human anti-V3 monoclonal antibodies (mAbs) from the cells of patients infected with subgroups A, B or C, the three HIV-1 subgroups that cause ~85% of the current pandemic.
- Use immunogen design cycles which consist of (a) crystallographic resolution of rationally selected V3/mAb complexes, (b) molecular modeling to extend these seed structures to apply to diverse isolates, and (c) empirical and computational methods to design an immunogens (and cocktails of immunogens) that will optimally focus the antibody response on V3 and induce broadly-reactive and potent protective antibodies.
- Select the best immunogen(s) to move forward into non-human primate and clinical trials by testing the ability of candidate immunogens to induce broadly neutralizing antibodies in rabbits. Ultimately, the best of these immunogens will be produced for human trials.
The Zolla-Pazner-led research consortium is dissecting the natural human antibody response to V3 by producing and characterizing broadly neutralizing human anti-V3 mAbs derived from cells of patients infected with HIV-1 isolates bearing envelopes of subgroups A, AG, B, C, G, H and CRF_02AG. For this effort the Zolla-Pazner team has collected more than 750 specimens over the past 4.5 years from HIV-infected individuals in Cameroon and India.
Support from the Gates Foundation has contributed to the development of a panel of anti-V3 mAbs which consists of 30 anti-V3 mAbs derived from non-subtype B-infected individuals and 29 subgroup B-derived anti-V3 mAbs.
Studies were designed to examine the neutralizing activity of the 18 most broadly neutralizing mAbs. These studies, performed and analyzed by the Zolla-Pazner consortium, the Ab-VIMC, VISC, and Monogram BioSciences, Inc., demonstrate that 100% of Tier 1 viruses and ~50% of Tier 2 viruses from subgroup A, B, C, and D are neutralized by one or more anti-V3 mAbs tested at <50 ug/ml.
In order to design and develop new immunogens that focus the immune response on the V3 domain, the structures of 25 V3/antibody complexes have been resolved, providing structural analyses of epitopes present in the crown of the V3 loop that are recognized by broadly-reactive and strain-specific mAbs. The data from these studies have identified five “families” of anti-V3 mAbs and the modes of binding that provide broad reactivity.
All LANL V3 sequences have been modeled either by bioinformatics or ab initio structure prediction. Novel integration of crystallographic, virologic, epidemiologic and bioinformatics data has permitted an estimation of worldwide occurrences of V3 epitopes recognized by the five families of anti-mAbs identified to date.
Using the crystallographic, modeling and bioinformatics data, “shared V3 epitopes” were designed and engineered in silico into the cholera toxin B (CTB) subunit scaffold. In total, we have designed and synthesized 14 V3-scaffold immunogens. These have all been tested for antigenicity in vitro and most have been tested individually and in various combinations for immunogenicity in vivo in rabbits (see below).
Immunization, laboratory studies and data analysis of nine rabbit experiments have been completed. Groups of animals were primed with gp120 DNA and boosted with the various newly designed V3-scaffold immunogens. The rabbit immune responses have been assessed using ELISA and four neutralization assays against multiclade panels of 15 primary isolates and 97 pseudoviruses (psVs) , including the 11 standard Tier 1 psVs, 14 standard Tier 2 psVs from subgroups B and C, and 27 psVs from acutely and chronically infected individuals from clades A, B, C and D. The results show the following:
- Ab VIMC data with our current best prime/boost regimen demonstrate that 50% neutralizing titers have been achieved for 10/11 Tier 1 viruses (clades B and C).
- Ab VIMC data with our current best prime/boost regimen demonstrate that 50% neutralizing titers have been achieved for. 2/7 Tier 2 clade B and 4/7 Tier 2 clade C viruses.
- Monogram data demonstrate that 50% neutralizing titers have been achieved for 17/20 Tier 2 viruses from clades A, B, C and D.
VISC, together with the Zolla-Pazner VDC, has developed new methods for statistical analysis and comparison of neutralization data.
The results of the work supported by the CAVD have established the “proof of Principle” sought in the original grant proposal:
- To focus the immune response on V3, a neutralizing epitope
- To induce cross-clade neutralizing Ab responses in rabbits
- To demonstrate the V3 region of gp120 induces cross-clade neutralizing Abs.