STRUCTURAL CHARACTERIZATION of DNA-ENCODED HIV VACCINES INDUCED NEUTRALIZING ANTIBODY

ABSTRACT

Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.