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RTS,S/AS01E has been tested in a phase 3 malaria vaccine study with partial efficacy in African children and infants. In a cohort of 1028 subjects from one low (Bagomoyo) and two high (Nanoro, Kintampo) malaria transmission sites, we analysed IgG plasma/serum concentration and avidity to CSP (NANP-repeat and C-terminal domains) after a 3-dose vaccination against time to clinical malaria events during 12-months. Here we report that RTS,S/AS01E induces substantial increases in IgG levels from pre- to post-vaccination ( p  < 0.001), higher in NANP than C-terminus (2855 vs 1297 proportional change between means), and higher concentrations and avidities in children than infants ( p  < 0.001). Baseline CSP IgG levels are elevated in malaria cases than controls ( p  < 0.001). Both, IgG magnitude to NANP (hazard ratio [95% confidence interval] 0.61 [0.48–0.76]) and avidity to C-terminus (0.07 [0.05–0.90]) post-vaccination are significantly associated with vaccine efficacy. IgG avidity to the C-terminus emerges as a significant contributor to RTS,S/AS01E-mediated protection. RTS,S/AS01E has been tested in a phase 3 malaria vaccine trial and has shown partial efficacy in children and infants. Here, the authors analyze IgG concentration and avidity to CSP in ~1000 participants and show that IgG avidity to the C-terminus of CSP is significantly associated with vaccine-mediated protection.

Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01–12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30–38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development. A longitudinal study of an individual patient developing neutralizing antibodies against HIV-1 (targeting the V1V2 region of gp120) reveals how such neutralizing antibodies develop and evolve over time, providing important insights relevant to vaccine development. HIV-neutralizing antibody formation examined A better understanding of how HIV-1-neutralizing antibodies are generated could be a useful contribution to the design of improved AIDS vaccines. John Mascola and colleagues have now elucidated the immunological pathway of an important category of HIV-1-neutralizing antibody — those that target the variable V1V2 region of the viral envelope. These antibodies are more frequently elicited than CD4-binding site antibodies in the early stages of HIV infection and feature modest affinity maturation, a process that favours mutations in antibody variable domains that enhance antigen binding.

A mutation in VRC01, a broadly neutralizing, HIV-1-specific antibody, confers enhanced binding to the neonatal Fc receptor, increasing the antibody half-life in the serum and localization in mucosal tissues, where it provides superior protection against rectal simian HIV-1 infection in macaques. Enhanced anti-HIV activity in mutant VRC01 antibody The recent discovery of broad and potent anti-HIV-1 antibodies has renewed interest in their use for passive protection against human immunodeficiency virus-1 in humans. This paper describes a mutation in the HIV-specific broadly neutralizing antibody VRC01 that confers enhanced binding to the neonatal Fc receptor and increases the antibody half-life in serum and mucosal tissues. It conferred superior protection in a rectal simian-HIV challenge model in macaques when compared to wild-type VRC01. To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn) 1 , 2 , whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) 3 was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining FcγRIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian–human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (B GC ) cells that last for at least 6 months. A 186-fold increase in B GC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of B GC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding B GC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells 1 , 2 . Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous B GC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses. Using HIV Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B cells lasting at least 6 months, showing promise in regard to difficult vaccine targets.

Human papillomavirus (HPV) vaccination is a major strategy for preventing cervical and other ano-genital cancers. Worldwide HPV vaccination introduction and coverage will be facilitated if a single dose of vaccine is as effective as two or three doses or demonstrates significant protective effect compared to ‘no vaccination’. In a multi-centre cluster randomized trial of two vs three doses of quadrivalent HPV vaccination (Gardasil™) in India, suspension of the vaccination due to events unrelated to the study led to per protocol and partial vaccination of unmarried 10–18 year old girls leading to four study groups, two by design and two by default. They were followed up for the primary outcomes of immunogenicity in terms of L1 genotype-specific binding antibody titres, neutralising antibody titres, and antibody avidity for the vaccine-targeted HPV types and HPV infections. Analysis was per actual number of vaccine doses received. This study is registered with ISRCTN, number ISRCTN98283094; and with ClinicalTrials.gov, number NCT00923702. Of the 17,729 vaccinated girls, 4348 (25%) received three doses on days 1, 60, 180 or later, 4979 (28%) received two doses on days 1 and 180 or later, 3452 (19%) received two doses on days 1 and 60, and 4950 (28%) received one dose. One dose recipients demonstrated a robust and sustained immune response against HPV 16 and 18, albeit inferior to that of 3- or 2-doses and the antibody levels were stable over a 4 year period. The frequencies of cumulative incident and persistent HPV 16 and 18 infections up to 7 years of follow-up were similar and uniformly low in all the vaccinated study groups; the frequency of HPV 16 and 18 infections were significantly higher in unvaccinated age-matched control women than among vaccine recipients. The frequency of vaccine non-targeted HPV types was similar in the vaccinated groups but higher in the unvaccinated control women. Our results indicate that a single dose of quadrivalent HPV vaccine is immunogenic and provides lasting protection against HPV 16 and 18 infections similar to the three- and two-dose vaccine schedules, although the study suffer from some limitations. Data on long term protection beyond 7 years against HPV infection and cervical precancerous lesions are needed before policy guidelines regarding a single dose can be formulated and implemented. Significant and long-lasting protective effect of a single dose can be a strong argument to introduce one dose of the HPV vaccine in many low income countries where the current standard of care for cervical cancer prevention is ‘no intervention’.

Affinity maturation in B cells generates antibodies with increasingly enhanced antigen-binding properties. Imkeller et al. investigated the maturation of human B cells that express protective antibodies against the circumsporozoite protein of the malaria-causing parasite Plasmodium falciparum (PfCSP). The repetitive structure of PfCSP induces mutations in B cells, facilitating direct interactions between two repeat-bound antibodies against PfCSP, which enhance antigen affinity and B cell activation. Such interactions may optimize binding and promote clustering of surface antibodies in general. Science , this issue p. 1358 A repetitive malaria antigen induces clonal selection and affinity maturation of human B cells expressing protective antibodies. Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from experimental alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody–antigen complex structures indicate that aromatic, mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These aromatic side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope–epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochemical features ubiquitously distributed over all protein surfaces.

Immunoglobulin E (IgE) antibodies play a central role in immune responses against helminth and protozoan parasites; however, they also contribute to allergies. IgE antibodies (and the B cells generating them) are rare and thus poorly characterized. Croote et al. performed single-cell RNA sequencing of peripheral blood B cells from patients with peanut allergies and delineated each cell's gene expression, splice variants, and antibody sequences (see the Perspective by Gould and Ramadani). Unlike other isotypes, circulating IgE B cells were mostly immature plasmablasts. Surprisingly, certain IgE antibodies manifested identical gene rearrangements in unrelated individuals. These IgE antibodies showed high affinity and unexpected cross-reactivity to peanut allergens. Science , this issue p. 1306 ; see also p. 1247 Single-cell sequencing of IgE B cells from allergic individuals reveals surprising insights. Immunoglobulin E (IgE) antibodies protect against helminth infections but can also cause life-threatening allergic reactions. Despite their role in human health, the cells that produce these antibodies are rarely observed and remain enigmatic. We isolated single IgE B cells from individuals with food allergies and used single-cell RNA sequencing to elucidate the gene expression and splicing patterns unique to these cells. We identified a surprising example of convergent evolution in which IgE antibodies underwent identical gene rearrangements in unrelated individuals. Through the acquisition of variable region mutations, these IgE antibodies gained high affinity and unexpected cross-reactivity to the clinically important peanut allergens Ara h 2 and Ara h 3. These findings provide insight into IgE B cell transcriptomics and enable biochemical dissection of this antibody class.

There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation. Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709.