Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ∼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.

Evidence for both Intermittent and Persistent Compartmentalization of HIV-1 in the Female Genital Tract.

HIV-1 has been shown to evolve independently in different anatomical compartments, but studies in the female genital tract have been inconclusive. Here, we examined evidence of compartmentalization using HIV-1 subtype C envelope (Env) glycoprotein genes (gp160) obtained from matched cervicovaginal lavage (CVL) and plasma samples over 2 to 3 years of infection. HIV-1 gp160 amplification from CVL was achieved for only 4 of 18 acutely infected women, and this was associated with the presence of proinflammatory cytokines and/or measurable viremia in the CVL. Maximum likelihood trees and divergence analyses showed that all four individuals had monophyletic compartment-specific clusters of CVL- and/or plasma-derived gp160 sequences at all or some time points. However, two participants (CAP177 and CAP217) had CVL gp160 diversity patterns that differed from those in plasma and showed restricted viral flow from the CVL. Statistical tests of compartmentalization revealed evidence of persistent compartment-specific gp160 evolution in CAP177, while in CAP217 this was intermittent. Lastly, we identified several Env sites that distinguished viruses in these two compartments; for CAP177, amino acid differences arose largely through positive selection, while insertions/deletions were more common in CAP217. In both cases these differences contributed to substantial charge changes spread across the Env. Our data indicate that, in some women, HIV-1 populations within the genital tract can have Env genetic features that differ from those of viruses in plasma, which could impact the sensitivity of viruses in the genital tract to vaginal microbicides and vaccine-elicited antibodies.IMPORTANCE Most HIV-1 infections in sub-Saharan Africa are acquired heterosexually through the genital mucosa. Understanding the properties of viruses replicating in the female genital tract, and whether these properties differ from those of more commonly studied viruses replicating in the blood, is therefore important. Using longitudinal CVL and plasma-derived sequences from four HIV-1 subtype C-infected women, we found fewer viral migrations from the genital tract to plasma than in the opposite direction, suggesting a mucosal sieve effect from the genital tract to the blood compartment. Evidence for both persistent and intermittent compartmentalization between the genital tract and plasma viruses during chronic infection was detected in two of four individuals, perhaps explaining previously conflicting findings. In cases where compartmentalization occurred, comparison of CVL- and plasma-derived HIV sequences indicated that distinct features of viral populations in the CVL may affect the efficacy of microbicides and vaccines designed to provide mucosal immunity.

Significant differences in the intra-host genetic diversity of Helicoverpa armigera nucleopolyhedrovirus dnapol after serial in vivo passages in the same insect population.

A denaturing gradient gel electrophoresis assay was used to assess the genetic diversity within a region of the DNA polymerase gene (dnapol) in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) populations over serial in vivo passages. There was no evidence of movement towards a consensus dnapol variant composition in the different host larvae after multiple per os passages. The study showed that the HearNPV variant structure after in vivo passages in the same host population is not necessarily convergent, and that it may be reasonable to expect significant differences in intra-host HearNPV genetic diversity after inoculation of larvae with a genotypically-diverse HearNPV inoculum.

Evaluating the antibody response elicited by diverse HIV envelope immunogens in the African green monkey (Vervet) model.

African Green (Vervet) monkeys have been extensively studied to understand the pathogenesis of infectious diseases. Using vervet monkeys as pre-clinical models may be an attractive option for low-resourced areas as they are found abundantly and their maintenance is more cost-effective than bigger primates such as rhesus macaques. We assessed the feasibility of using vervet monkeys as animal models to examine the immunogenicity of HIV envelope trimer immunogens in pre-clinical testing. Three groups of vervet monkeys were subcutaneously immunized with either the BG505 SOSIP.664 trimer, a novel subtype C SOSIP.664 trimer, CAP255, or a combination of BG505, CAP255 and CAP256.SU SOSIP.664 trimers. All groups of vervet monkeys developed robust binding antibodies by the second immunization with the peak antibody response occurring after the third immunization. Similar to binding, antibody dependent cellular phagocytosis was also observed in all the monkeys. While all animals developed potent, heterologous Tier 1 neutralizing antibody responses, autologous neutralization was limited with only half of the animals in each group developing responses to their vaccine-matched pseudovirus. These data suggest that the vervet monkey model may yield distinct antibody responses compared to other models. Further study is required to further determine the utility of this model in HIV immunization studies.

Ad26.COV2.S breakthrough infections induce high titers of neutralizing antibodies against Omicron and other SARS-CoV-2 variants of concern.

The Janssen (Johnson & Johnson) Ad26.COV2.S non-replicating viral vector vaccine has been widely deployed for COVID-19 vaccination programs in resource-limited settings. Here we confirm that neutralizing and binding antibody responses to Ad26.COV2.S vaccination are stable for 6 months post-vaccination, when tested against multiple SARS-CoV-2 variants. Secondly, using longitudinal samples from individuals who experienced clinically mild breakthrough infections 4 to 5 months after vaccination, we show dramatically boosted binding antibodies, Fc effector function, and neutralization. These high titer responses are of similar magnitude to humoral immune responses measured in convalescent donors who had been hospitalized with severe illness, and are cross-reactive against diverse SARS-CoV-2 variants, including the neutralization-resistant Omicron (B.1.1.529) variant that currently dominates global infections, as well as SARS-CoV-1. These data have implications for population immunity in areas where the Ad26.COV2.S vaccine has been widely deployed, but where ongoing infections continue to occur at high levels.

Targeting the N332-supersite of the HIV-1 envelope for vaccine design.

Introduction: Broadly neutralizing antibodies (bNAbs) that are able to target diverse global viruses are widely believed to be crucial for an HIV-1 vaccine. Several conserved targets recognized by these antibodies have been identified on the HIV-1 envelope glycoprotein. One such target that shows particular promise for vaccination is the N332-supersite.Areas covered: This review describes the potential of the N332-supersite epitope as an immunogen design platform. We discuss the structure of the epitope and the bNAbs that target it, emphasizing their diverse modes of binding. Furthermore, the successes and limitations of recent N332-supersite immunization studies are discussed.Expert opinion: During HIV-1 infection, some of the broadest and most potent bNAbs target the N332-supersite. Furthermore, some of these antibodies require less affinity maturation than the high levels typical of many bNAbs, making these potentially more achievable vaccine targets. In addition, bNAbs bind this epitope with multiple angles of approach and glycan dependencies, perhaps increasing the probability of eliciting such responses by vaccination. Animal studies have shown that N332-supersite bNAb precursors can be activated by novel immunogens. While follow-up studies must establish whether boosting strategies can drive the maturation of bNAbs from these precursors, the development of targeted N332-supersite immunogens expands our arsenal of potential HIV-1 vaccine candidates.

Antibody Isotype Switching as a Mechanism to Counter HIV Neutralization Escape.

Neutralizing antibodies (nAbs) to highly variable viral pathogens show remarkable diversification during infection, resulting in an "arms race" between virus and host. Studies of nAb lineages have shown how somatic hypermutation (SHM) in immunoglobulin (Ig)-variable regions enables maturing antibodies to neutralize emerging viral escape variants. However, the Ig-constant region (which determines isotype) can also influence epitope recognition. Here, we use longitudinal deep sequencing of an HIV-directed nAb lineage, CAP88-CH06, and identify several co-circulating isotypes (IgG3, IgG1, IgA1, IgG2, and IgA2), some of which share identical variable regions. First, we show that IgG3 and IgA1 isotypes are better able to neutralize longitudinal autologous viruses and epitope mutants than can IgG1. Second, detrimental class-switch recombination (CSR) events that resulted in reduced neutralization can be rescued by further CSR, which we term "switch redemption." Thus, CSR represents an additional immunological mechanism to counter viral escape from HIV-specific antibody responses.

Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies.

The elicitation of broadly neutralizing antibodies (bNAbs) is likely to be essential for a preventative HIV-1 vaccine, but this has not yet been achieved by immunization. In contrast, some HIV-1-infected individuals naturally mount bNAb responses during chronic infection, suggesting that years of maturation may be required for neutralization breadth. Recent studies have shown that viral diversification precedes the emergence of bNAbs, but the significance of this observation is unknown. Here we delineate the key viral events that drove neutralization breadth within the CAP256-VRC26 family of 33 monoclonal antibodies (mAbs) isolated from a superinfected individual. First, we identified minority viral variants, termed bNAb-initiating envelopes, that were distinct from both of the transmitted/founder (T/F) viruses and that efficiently engaged the bNAb precursor. Second, deep sequencing revealed a pool of diverse epitope variants (immunotypes) that were preferentially neutralized by broader members of the antibody lineage. In contrast, a 'dead-end' antibody sublineage unable to neutralize these immunotypes showed limited evolution and failed to develop breadth. Thus, early viral escape at key antibody-virus contact sites selects for antibody sublineages that can tolerate these changes, thereby providing a mechanism for the generation of neutralization breadth within a developing antibody lineage.