Immunologic insights into the critical epitopes of HIV-1 and structure-based characterization of cross-reactive antibodies

HIV-1 escape from neutralizing antibodies, even in the presence of strong host immunity, is associated with variations in envelope proteins that drive antigenic diversification. The virus exploits the error-prone nature of reverse transcriptase and the high mutation rate as key survival strategies. However, the rate of emergence of new variations occurs at a relatively slow pace. In this context, while monospecific antibody responses against invading pathogens are well characterized, the functional relevance of multispecific or cross-reactive antibodies in limiting viral escape remains poorly understood. Interestingly, the immune system often produces cross-reactive antibodies, with an anticipated role in neutralizing point mutations in HIV surface proteins by cross-reacting with mutants and tolerating them. In light of this paradox, we investigated immune evasion in the context of observed antibody cross-reactivity by screening single-chain variable fragment (scFv) antibodies against several crucial HIV-1 gp41 epitopes using a phage display library. Selected cross-reactive scFvs were biochemically characterized for binding affinity and their ability to recognize envelope protein and its mutants expressed on cell surface. Here, high-affinity cross-reactive scFvs showed physiologically relevant affinities with peptide epitopes, their analogs, and the native HIV-1 gp41 protein. We determined the crystal structure of a high-affinity, cross-reactive scFv DE94, and gained insights into the molecular interactions of scFv antibodies with peptide epitopes and their natural mutants using molecular docking studies. This analysis of cross-reactive antibodies could contribute to the development of therapeutics against immune-evading pathogens and pave the way for innovative strategies to combat viral infections, including emerging global threats.