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

Abstract

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.

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows

“…The Abs neutralized the same viruses, with Moo1 exhibiting a geomean IC 50 of 0.015 µg/ml and Moo2 a geomean IC 50 of 0.011 µg/ml with 42% breadth. The neutralization pattern was similar to that of Bess1 and Bess8 [28], though more potent than Bess8 and lacking neutralization of isolate X1632 >50 µg/ml. They all also displayed incomplete neutralization (<95%) of isolate BJOX2000, which is not uncommon among trimer-specific antibodies and was seen in Bess and ElsE bnAbs [28, 33–37].…”

“…The neutralization pattern was similar to that of Bess1 and Bess8 [28], though more potent than Bess8 and lacking neutralization of isolate X1632 >50 µg/ml. They all also displayed incomplete neutralization (<95%) of isolate BJOX2000, which is not uncommon among trimer-specific antibodies and was seen in Bess and ElsE bnAbs [28, 33–37]. Overall, the Moo antibodies were moderately broad and highly potent but less so than the best of the Bess, ElsE, NC-Cow, and Mel antibodies [27–29].…”

“…Our previous studies had also shown that, with the choice of appropriate trimer immunogens, V2-apex bnAbs could be induced in cows, although this took more immunizations, and the bnAbs were less potent and broad compared to those induced to the CD4bs by BG505 trimer [27, 28]. Here, we wished to see if, by mutation to greatly reduce bnAbs binding to the CD4bs, we could switch the induction of bnAbs to other sites using a mutated BG505 SOSIP trimer as an immunogen.…”

“…Cow PBMCs were sorted as previously described with study specific modifications [28, 50]. PBMCs were thawed until only a small ice piece remained in a 37°C bath and immediately added to 10mL of 37°C prewarmed 50% RPMI (Gibco) and 50% FBS buffer.…”

“…Initially, it was unclear if broadly neutralizing antibody responses could develop to target epitopes beyond the CD4bs, given that the response to this site was so overwhelming. However, we found that using one or multiple V2-apex bnAb-focusing trimer immunogens as well as a cocktail of trimers, we could elicit a cross-clade and somewhat potent bnAb response that targeted the V2-apex in two cohorts of cows [28]. Some questions remained unanswered.…”