Saye-Francisco, K. | Search bioRxiv

bioRxiv2025

Engineering HIV antibodies with enhanced breadth and potency of neutralization through multistate affinity maturation

Broadly neutralizing antibodies (bnAbs) against HIV hold promise as therapeutic and prophylactic agents, but realizing this potential requires antibodies that function across the antigenic heterogeneity of the HIV envelope glycoprotein (Env). Although numerous bnAbs have been isolated from infected individuals, their breadth and potency may not be sufficient to tackle global viral diversity, motivating efforts to further improve their neutralization capacity. Here, we address this challenge using a multistate antibody engineering approach integrating deep mutational scanning with combinatorial library screening across diverse Env variants. This strategy enables identification of mutation patterns that confer improved binding across antigenically distinct targets. Starting from one of the best-in-class CD4-binding site bnAbs, we performed iterative optimization to increase binding affinity across diverse Env variants. The resulting lead candidate exhibited improved breadth and up to 100-fold higher potency against pseudoviruses from large cross-clade historical and contemporary panels while maintaining biophysical and pharmacokinetic profiles conducive to clinical development. Structural and molecular dynamics analyses revealed a unique tri-tyrosine aromatic triad and reinforced electrostatic contacts that stabilized the bnAb/Env interface. These findings demonstrate that systematic in vitro engineering can generate bnAbs with enhanced breadth and potency, providing a generalizable strategy for developing therapeutic antibodies against highly diverse pathogens.

bioengineering↗

bioRxiv2025

Combining a rhesus cytomegalovirus/SIV vaccine with neutralizing antibody to protect against SIV challenge in rhesus macaques

A vaccine is widely regarded as necessary for the control of the HIV pandemic and eventual eradication of AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have both been shown capable of vaccine protection against the simian counterpart of HIV, SIV, in rhesus macaques. Here we provide preliminary evidence that combining these orthogonal antiviral mechanisms can provide increased protection against SIV challenge such that replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced in the presence of passively administered incompletely protective levels of neutralizing antibody. The report invites studies involving larger cohorts of macaques and alternate routes of providing neutralizing antibody.

immunology↗

bioRxiv2024

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response. Author SummaryThe elicitation of epitope-specific broadly neutralizing antibodies is highly desirable for an HIV vaccine as bnAbs can prevent HIV infection in robust animal challenge models and humans, but to date, cows are the only model shown to reliably produce HIV bnAb responses on Envelope (Env) immunization. These responses involve Abs with ultralong CDRH3s and are all directed to a single site, the CD4 binding site. To determine whether this is a unique phenomenon or whether cow antibodies can target further bnAb sites on Env, we employed an immunization protocol that generated cow bnAbs to a second site, the V2-apex. We conclude that ultralong CDRH3s are well adapted to penetrate the glycan shield of HIV Env and recognize conserved regions and may constitute protein units, either in the context of antibodies or in other engineered proteins, that could be deployed as anti-HIV reagents.

immunology↗