Haynes: Role of IgA in HIV-1 Protection
HIV-1 infection typically occurs across a mucosal surface. Effective mucosal immunity against HIV-1 is likely to be required for the success of candidate vaccines, and it is also expected that the capacity for passively administered broadly neutralizing antibodies (bNabs) to protect against HIV-1 infection will depend on their trafficking to mucosal surfaces. The work in this grant will systematically define the requirements for mucosal protection against HIV-1 with antibodies.
Antibodies of the IgA isotype are a key line of defense against pathogen invasion. IgA1 and IgA2 subclasses can exist as dimeric (dIgA) or multimeric forms comprised of two or more monomeric IgA covalently linked by a joining, or J, chain. Plasma cells produce IgA antibodies at mucosal surfaces, and in some cases it is this locally-produced, rather than systemic, antibody that populates the mucosal compartment. dIgA is transported to the epithelial cell apical surface where it is complexed with a secretory component, resulting in secretory IgA (sIgA). sIgA plays a critical role in protection against a number of mucosal pathogens. In spite of its importance in mucosal host defense, the roles that monomeric, dimeric IgA and sIgA play in prevention of HIV-1 transmission are incompletely defined. In this regard, the recent immune correlates analysis of the RV144 vaccine trial raised the hypothesis that monomeric IgA may mitigate otherwise protective IgG responses.
The consortia led by Dr. Barton Haynes at Duke University will establish cell lines that produce J chain and J chain plus secretory component to enable the production of a series of monomeric, dimeric, and secretory IgA antibodies with the same antigen-binding domains. These forms will be evaluated for their capacity to inhibit HIV-1 infection in a variety of
in vitro assays, tissue explant models, and
in vivo in the non-human primate model of HIV/AIDS. The source materials will come from HIV-infected individuals and the RV144 vaccine trial and related studies, and the IgA antibodies will be isolated through single cell sorting of memory B-cells fluorescently labeled with HIV envelope antigens and via clonal IgA B-cell cultures.
1. Engineer CHO cell lines to produce J-chain, to enable expression of dimeric IgAs
2. Engineer CHO cell lines for production of J-chain and Secretory Piece, for the production of secretory IgAs
3. To develop capacity for cell sorting of mucosal B-cells expressing IgA that binds to the HIV envelope protein gp120
4. Develop clonal cell cultures for mAb isolation from IgA-expressing B-cells
5. To make and evaluate the dimeric IgA forms of bNabs against HIV, and the corresponding IgGs, to enable evaluation of their protective efficacy in model systems
6. To establish a quantitative in vitro model, using surface plasmon resonance, for IgA blocking of HIV binding to receptors on the apical surface of epithelial cells
7. To evaluate IgAs for protection from HIV infection in binding assays and an ex vivo tissue explant model
8. To produce selected dimeric and secretory IgAs under GCLP conditions, in anticipation of cGMP manufacturing of IgAs
9. Conduct passive protection studies in non-human primates using the selected IgAs delivered systemically or mucosally
We have successfully produced a number of IgA2s and IgA1s for study, including CH38 and CH29 from RV144, that bind to the C1 ADCC gp120 site; the CD4 binding site, VRC01-like bnAb CH31; and the ADCC-mediating, virus-capturing gp41 mAb, 7B2. These antibodies have been produced in monomeric and dimeric forms in CHO cells. We have also characterized panels of these monomeric and dimeric IgAs in binding and neutralization assays, virion capture, monocyte/macrophage phagocytosis, NK and PMN ADCC, and galactoceramide blocking. We have been able to phenotype and sort IgA-positive antigen-speci c memory B cells and selectively grow them in vitro. We have selected CH31for further testing in non-human primate studies with localized instillation of antibody as a microbicide as well as systemic passive infusion to evaluate in vivo antibody ef cacy in preventing the transmission of SHIV Bal. Recent ndings include the inability of non-neutralizing antibodies to protect mucosal tissue explants ex vivo while broadly neutralizing antibodies (bnAbs) do protect in this system, the ability of CH31 broad neutralizing antibody to protect against R5 tier 2 SHIV challenge when applied both locally and injected systemically in rhesus macaques, and the superiority of IgG vs. IgA isoforms of CH31 to protect against SHIV challenges systemically and locally.