Development of highly neutralizing nanobodies against HIV-1, SARS-CoV-2 and other pathogens

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

HIV-1 and -2 are lentiviruses that cause acquired immunodeficiency syndrome (AIDS) in humans. To date, no effective vaccine has been developed and according to the World Health Organization nearly 40 million people are infected with HIV across the world. Because preventive measures and treatments are not widely available, AIDS is the leading cause of death in areas such as Sub-Saharan Africa. Therefore, there is an urgent need to develop efficient methods, including vaccines and immunotherapies, to prevent HIV infection in susceptible populations. A near-native trimer mimic, BG505 DS-SOSIP, has been shown to be a nice immunogen for HIV-1 vaccination in various animal models. Here we injected this immunogen in llama to develop heavy chain-only antibodies, aiming to generate broad and potent neutralizing nanobodies (bNNABs) against HIV-1. Two nanobodies that we identified through phage screening, G36 and R27, were further engineered into a trimeric form with the antibody hinge region of llama IgG2a and Fc domain of human IgG1 (G36x3-IgG2a and R27x3-IgG2a). These antibodies demonstrated greater than 90% breadth and substantially higher potency than the previously published best in class, J3. Combination of these antibodies with the potent V2-directed antibody, CAP256V2LS, yielded human-llama bispecifics of even greater neutralization potency. We found the bispecific antibodies to have synergistic neutralization, with improved breadth and potency over both nanobody x3-IgG2a and CAP256V2LS. CAP256L-R27x3LS neutralized 95% of an 80-virus panel at a geometric mean IC50 of 0.003 ug/ml. The cryo-EM structure of CAP256L-R27LS revealed the mechanism of such synergy to be simultaneous binding of R27 and CAP256V2LS to the prefusion-closed Env trimer. We further determined the pharmacokinetics of the bispecific antibody CAP256L-R27x3LS to be comparable to the parent antibody CAP256V2LS, with suitable auto-reactivity. The bispecific CAP256L-R27x3LS may therefore be sufficient to fulfill anti-HIV therapeutic and prophylactic clinical goals. Overall, we described a system whereby we have identified a nanobody through immunization that is 10 times more potent than the prior best in class nanobody and combined it with CAP256V2LS to make an even more potent bispecific; we believe this system can be used to identify even more potent nanobodies and bispecifics. The HMPV surface glycoprotein F mediates the fusion of viral and cellular membrane and is an attractive target for HMPV vaccine and neutralizing antibodies development. Last fiscal year, we immunized llama with the third generation prefusion stabilized F protein developed at the NIHs Vaccine Research Center and isolated dozens of nanobodies targeting different epitopes on F protein. We tested 25 nanobodies representing different CDR families for their neutralizing activities against HMPV. Four nanobodies neutralized both HMPV A strain and B strain at plaque reduction neutralization (PRNT) 60s below 0.1 ug/ml. Structural analysis of both neutralizing and non-neutralizing nanobodies will likely assist further refinement of F immunogen. We expect that engineering with Fc conjugation and multimerization can lead to a potent nanobody/antibody therapeutic for HMPV infection. Indeed, two nanobodies, NB73 and NB42, when combined as a bispecific and fused to the Fc region of human IgG1, neutralize HMPV A strain at PRNT60 of 2.7 ng/ml, rivaling the most potent human antibodies reported to date.