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Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naive progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of broadly active ADCC antibodies may require mutations that enable high-affinity antigen recognition along with mutations that optimize factors contributing to functional ADCC activity., eLife digest Nearly four decades after the human immunodeficiency virus (HIV for short) was first identified, the search for a vaccine still continues. An effective immunisation would require elements that coax the human immune system into making HIV-specific antibodies – the proteins that can recognise, bind to and deactivate the virus. Crucially, antibodies can also help white blood cells to target and destroy cells infected with HIV. This ‘antibody-dependent cellular cytotoxicity’ could be a key element of a successful vaccine, yet it has received less attention than the ability for antibodies to directly neutralize the virus. In particular, it is still unclear how antibodies develop the ability to flag HIV-infected cells for killing. Indeed, over the course of an HIV infection, an immune cell goes through genetic changes that tweak the 3D structure of the antibodies it manufactures. This process can improve the antibodies' ability to fight off the virus, but it was still unclear how it would shape antibody-dependent cellular cytotoxicity. To investigate this question, Doepker et al. retraced how the genes coding for six antibody families changed over time in an HIV-carrying individual. This revealed that antibodies could not initially trigger antibody-dependent cellular cytotoxicity. The property emerged and improved thanks to two types of alterations in the genetic sequences. One set of changes increased how tightly the antibodies could bind to the virus, targeting sections of the antibodies that can often vary. The second set likely altered the 3D structure in others ways, potentially affecting how antibodies bind the virus or how they interact with components of the immune system that help to kill HIV-infected cells. These alterations took place in segments of the antibodies that undergo less change over time. Ultimately, the findings by Doepker et al. suggest that an efficient HIV vaccine may rely on helping antibodies to evolve so they can bind more tightly to the virus and trigger cellular cytotoxicity more strongly.

A reservoir of HIV-infected cells that persists despite suppressive antiretroviral therapy (ART) is the source of viral rebound upon ART cessation and the major barrier to a cure. Understanding reservoir seeding dynamics will help identify the best timing for HIV cure strategies. Here we characterize reservoir seeding using longitudinal samples from before and after ART initiation in individuals who sequentially became infected with genetically distinct HIV variants (superinfected). We previously identified cases of superinfection in a cohort of Kenyan women, and the dates of both initial infection and superinfection were determined. Six women, superinfected 0.2–5.2 years after initial infection, were subsequently treated with ART 5.4–18.0 years after initial infection. We performed next-generation sequencing of HIV gag and env RNA from plasma collected during acute infection as well as every ~2 years thereafter until ART initiation, and of HIV DNA from PBMCs collected 0.9–4.8 years after viral suppression on ART. We assessed phylogenetic relationships between HIV DNA reservoir sequences and longitudinal plasma RNA sequences prior to ART, to determine proportions of initial and superinfecting variants in the reservoir. The proportions of initial and superinfection lineage variants present in the HIV DNA reservoir were most similar to the proportions present in HIV RNA immediately prior to ART initiation. Phylogenetic analysis confirmed that the majority of HIV DNA reservoir sequences had the smallest pairwise distance to RNA sequences from timepoints closest to ART initiation. Our data suggest that while reservoir cells are created throughout pre-ART infection, the majority of HIV-infected cells that persist during ART entered the reservoir near the time of ART initiation. We estimate the half-life of pre-ART DNA reservoir sequences to be ~25 months, which is shorter than estimated reservoir decay rates during suppressive ART, implying continual decay and reseeding of the reservoir up to the point of ART initiation., Author summary During HIV infection, a reservoir of long-lived latently infected cells is established that persists during antiretroviral therapy (ART) and is the source of virus replication after treatment cessation. A better understanding of when viruses enter the HIV reservoir (reservoir seeding) will aid efforts to target these long-lived HIV infected cells during their establishment. We studied women infected at two different times with two genetically distinct HIV strains (called superinfection), and assessed the genetic relationship between sequences of the HIV strains that circulated throughout infection (pre-ART HIV RNA sequences) and the HIV strains that persisted in reservoir cells (HIV DNA sequences during ART). We estimated when HIV DNA sequences entered the reservoir by identifying the time the most genetically related HIV RNA sequence was detected. In most cases we observed that viruses in the reservoir included both the initial and superinfecting lineages, suggesting reservoir seeding occurs throughout HIV infection. However, the majority of HIV sequences entered the reservoir near the time of ART initiation, suggesting that novel strategies that aim to reduce reservoir size should focus on times immediately prior to ART.