Biology subjects

Kuraoka, M.

Publications and source records attributed to Kuraoka, M..

13 recordsLinked to original sources

bioRxiv2026

Genetically restricted human antibodies recognize a conserved β-hairpin in postfusion influenza hemagglutinin

A public class of broadly protective, human antibodies (Abs) binds a conserved {beta}-hairpin exposed in the postfusion conformation of influenza virus hemagglutinin (HA). We here describe use of in vitro immunoassays and high-resolution cryogenic electron microscopy analysis of Ab:HA complexes to explain the apparent genetic restriction of these Abs and the wide range of influenza A and B subtypes they recognize. We show that recognition of the {beta}-hairpin epitope requires 1) germline-encoded residues unique to certain alleles of the human immunoglobulin heavy chain gene IGHV1-2, 2) a short (<14-residue) HCDR3 to avoid steric clash with HA, and 3) several Ab light chain residues that are germline-encoded by various IGLV2-family genes or IGLV7-46. Despite the genetic restrictions, the capacity to make {beta}-hairpin Abs is widespread among humans. We recovered examples of these Abs from 14 genetically unrelated donors, and a previously reported analysis of the IGHV genotypes of ~2,500 individuals sampled from major ethnic populations estimates that >75% of people have the necessary IGHV1-2 alleles to make these Abs. We conclude that an immunogen that displays the {beta}-hairpin epitope might reliably elicit broadly protective influenza antibodies in most humans.

immunology↗

bioRxiv2026

Apoptotic Quality Control of Cellular Competence Limits Cryptic Germinal Center B Cell States

Germinal center (GC) B cells undergo extensive apoptosis, but how this process shapes evolving GC populations remains unclear. Using mice lacking intrinsic apoptotic effectors BAK and BAX in B cells, we found that mitochondrial apoptosis was dispensable for affinity maturation but critical for controlling GC size and cellular composition, showing that GC population dynamics and affinity maturation can be uncoupled. BAK/BAX-deficient mice mounted enlarged and prolonged GC responses, largely due to the accumulation of non-proliferating Ki-67loCD71lo dark zone (DZ) B cells. CD71hi and CD71lo DZ cells exhibited similar B-cell receptor (BCR) avidity, retained potential for proliferation and plasmacytic differentiation, and shared clonal lineages, indicating recurrent emergence of non-proliferating DZ populations. A subset also carried Somatic-hypermutation-derived defects that abolished antibody expression despite apparently productive V(D)J sequences; reversion of a single amino acid substitution often restored expression. Mitochondrial apoptosis thus enforces cellular quality control in the GC by limiting the persistence of non-proliferating and expression-defective states while remaining dispensable for affinity maturation.

immunology↗

bioRxiv2026

Antibody feedback limits specific B-cell entry and alters germinal center repertoires by antibody-antigen complexes

Antibody (Ab) feedback impacts germinal center (GC) responses. Mice given IgG1 or IgG2c recombinant Ab (rAb) specific for the receptor binding site (RBS) of H3 hemagglutinin (HA) and later immunized with the H3 HA trimer exhibited altered GC repertoires. Passive RBS rAb had no effect on the magnitude of ensuing primary GC responses but reduced the numbers of RBS-specific GC B cells. These losses were matched by increases in "unspecific" B cells which did not bind the HA immunogen, with no changes in B cells specific for distal epitopes. These effects were independent of IgG subclass. Higher doses of passive rAb resulted in reduced epitope-specific affinity maturation and clonal proliferation in GCs. Passive rAb generated Ab:HA complexes and favored recruitment of rare HA-specific GC B cells with enhanced avidity for the rAb:HA immune complex (IC). We show that some no- and low-affinity GC B cells represent responses to local ICs.

immunology↗

bioRxiv2026

Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

Germline-targeting or lineage-design vaccine strategies are being used to induce HIV broadly neutralizing antibody (bnAb) responses. These strategies assume that genetically diverse individuals respond similarly to the same immunogen by mobilizing comparable germline precursors, although outcomes vary across bnAb epitopes. Here we explored this premise using a simian-HIV infection model in rhesus macaques. Antigen-unbiased Env- reactive B-cell populations were profiled, followed by systematic analysis of antibody function and B-cell receptor (BCR) genetics. We find that while global Env-reactive B-cell response profiles and antibody functional properties are similar across individuals, underlying BCR genetics are diverse, particularly among non-bnAbs. These results indicate that functional convergence of Env-reactive antibody responses does not necessarily require genetic convergence and suggest that specific germline-targeting may not be an absolute prerequisite for successful vaccine design. These findings support an epitope-focused framework in which bnAb epitopes are engineered to enhance population-level immunogenicity, with potential applicability to other challenging pathogens.

immunology↗

bioRxiv2025

Iterative immunogen optimization to focus elicited responses on a conserved, subdominant influenza hemagglutinin epitope

Designing effective vaccination strategies against genetically diverse viruses, such as HIV or influenza, is hindered by the ability of these pathogens to mutate and readily evade immune responses. While these viruses contain conserved regions that cannot easily mutate without affecting viral fitness, these sites are subdominant, meaning that they are not the primary target of antibodies elicited by natural infection or current vaccines. Here, we integrated recent advances in machine learning methods for protein structure prediction and design to engineer protein immunogens that focus immune responses on a conserved, subdominant HA epitope targeted by broadly cross-reactive influenza antibodies. Iterating between computation-guided optimization and in vivo analyses generated immunogens that faithfully displayed the target epitope and redirected humoral responses toward it upon vaccination. These results provide a blueprint for applying recent protein engineering approaches to immunogen design and may inform the development of broadly protective influenza vaccines.

immunology↗

bioRxiv2025

Fluorescence-barcoded cell lines stably expressing membrane-anchored influenza neuraminidases

The discovery of broadly protective antibodies to the influenza virus neuraminidase (NA) has raised interest in NA as a vaccine target. However, recombinant, solubilized tetrameric NA ectodomains are often challenging to express and isolate, hindering the study of anti-NA humoral responses. To address this obstacle, we established a panel of 22 non-adherent cell lines stably expressing native, historical N1, N2, N3, N9, and NB NAs anchored on the cell surface. The cell lines are barcoded with fluorescent proteins, enabling high-throughput, 16-plex analyses of antibody binding with commonly available flow cytometers. The cell lines were at least as efficient as a Luminex multiplex binding assay at identifying NA antibodies from a library of unselected clonal IgGs derived from human memory B cells. The cell lines were also useful for measuring the magnitude and breadth of the serum antibody response elicited by experimental infection of rhesus macaques with influenza virus. The membrane-anchored NAs are catalytically active and are compatible with established sialidase activity assays. NA-expressing K530 cell lines therefore represent a useful tool for studying NA immunity and evaluating influenza vaccine efficacy.

immunology↗

bioRxiv2023

A broad antibody class engages the influenza virus hemagglutinin head at its stem interface

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

microbiology↗

bioRxiv2023

A class of antibodies that overcome a steric barrier to cross-group neutralization of influenza viruses

Antibody titers that inhibit the influenza virus hemagglutinin (HA) from engaging its receptor are the accepted correlate of protection from infection. Many potent antibodies with broad, intra-subtype specificity bind HA at the receptor binding site (RBS). One barrier to broad H1-H3 cross-subtype neutralization is an insertion (133a) between positions 133 and 134 on the rim of the H1 HA RBS. We describe here a class of antibodies that overcome this barrier. These genetically unrestricted antibodies are abundant in the human B-cell memory compartment. Analysis of the affinities of selected members of this class for historical H1 and H3 isolates suggest that they were elicited by H3 exposure and broadened or diverted by later exposure(s) to H1 HA. RBS mutations in egg-adapted vaccine strains cause the new H1 specificity of these antibodies to depend on the egg adaptation. The results suggest that suitable immunogens might elicit 133a-independent, H1-H3 cross neutralization by RBS-directed antibodies.

microbiology↗

bioRxiv2022

An epitope-enriched immunogen increases site targeting in germinal centers

Antibody immunodominance is the asymmetric elicitation of responses against protein antigens. For influenza hemagglutinin (HA), antibody responses often target variable regions on HA and do not provide lasting protection. Next-generation influenza vaccines should elicit antibodies targeting conserved regions such as the receptor binding site (RBS). Understanding how presenting an epitope on a rationally-designed immunogen influences immune responses could help achieve this goal. Here, we compared an engineered RBS-enriched immunogen and its non-enriched counterparts to characterize RBS-directed responses. We found that enriching the RBS-epitope on a single immunogen preferentially expands RBS-directed responses relative to a cocktail of the non-epitope-enriched immunogens. Single B cell analyses showed a genetically diverse RBS-directed population that structural characterization showed engagement of the RBS with canonical features shared with both its receptor and human broadly neutralizing antibodies. These data show how epitope-enriched immunogens can expand responses to a conserved viral site, while maintaining genetic and structural diversity.

immunology↗

bioRxiv2022

Infant antibody repertoires during the first two years of influenza vaccination

The first encounter with influenza virus biases later immune responses. This "immune imprinting", formerly from infection within a few years of birth, is in the U.S. now largely from immunization with a quadrivalent, split vaccine (IIV4). In a pilot study of IIV4 imprinting, we characterized, by single-B-cell cultures, NextGen sequencing, and plasma antibody proteomics, the primary antibody responses to influenza in two infants during their first two years of seasonal influenza vaccination. One infant, who received only a single vaccination in Year 1, contracted an influenza B (IBV) infection between the two years, allowing us to compare imprinting by infection and vaccination. That infant had a shift in hemagglutinin (HA)-reactive B-cell specificity from largely influenza A (IAV)-specific in Year 1 to IBV-specific in Year 2, both before and after vaccination. HA-reactive B cells from the other infant maintained a more evenly distributed specificity. In Year 2, class-switched HA-specific B cell IGHV somatic hypermutation (SHM) levels reached average levels seen in adults. The HA-reactive plasma antibody repertoires of both infants comprised a relatively small number of antibody clonotypes, with one or two very abundant clonotypes. Thus, after the Year 2 boost, both infants had overall B cell profiles that resembled those of adult controls. ImportanceInfluenza virus is a moving target for the immune system. Variants emerge that escape protection from antibodies elicited by a previously circulating variant ("antigenic drift"). The immune system usually responds to a drifted influenza virus by mutating existing antibodies rather than by producting entirely new ones. Thus, immune memory of the earliest influenza exposure has a major influence on later responses to infection or vaccination ("immune imprinting"). In the many studies of influenza immunity in adult subjects, imprinting has been from an early infection, since only in the past two decades have infants received influenza immunizations. The work reported in this paper is a pilot study of imprinting in two infants by the flu vaccine, which they received before experiencing an influenza infection. The results suggest that a quadrivalent (four-subtype) vaccine may provide an immune imprint less dominated by one subtype than does a monovalent infection.

immunology↗

bioRxiv2021

Recall of B cell memory depends on relative locations of prime and boost

Re-entry of memory B cells to recall germinal centers (GCs) is essential for updating their B-cell antigen receptors (BCRs). Using single B-cell culture and fate-mapping, we have characterized BCR repertoires in recall GCs following boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit to recall GCs progeny of primary GC B cells more efficiently than do distal boosts. Recall GCs following local boosts contain significantly more B cells with elevated levels of Ig mutations and higher avidity BCRs. This local preference is unaffected by blockade of CD40:CD154 interaction that terminate active, primary GC responses. Local boosts with heterologous antigens elicit secondary GCs with B-cell populations enriched for cross-reactivity to the priming and boosting antigens; in contrast, cross-reactive GC B cells are rare following distal boosts. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses. One Sentence SummaryThe participation of memory B cells in recall germinal centers depends on whether the boost is local or distal to the priming site.

immunology↗

bioRxiv2020

A prevalent focused human antibody response to the influenza hemagglutinin head interface

Novel animal influenza viruses emerge, initiate pandemics and become endemic seasonal variants that have evolved to escape from prevalent herd immunity. These processes often outpace vaccine-elicited protection. Focusing immune responses on conserved epitopes may impart durable immunity. We describe a focused, protective antibody response, abundant in memory and serum repertoires, to a conserved region at the influenza hemagglutinin head interface. Structures of eleven examples, eight reported here, from seven human donors demonstrate the convergence of responses on a single epitope. The eleven are genetically diverse, with one class having a common, IG{kappa}V1-39, light chain. All of the antibodies bind HAs from multiple serotypes. The lack of apparent genetic restriction and potential for elicitation by more than one serotype may explain their abundance. We define the head interface as a major target of broadly protective antibodies with the potential to influence the outcomes of influenza infection.

immunology↗

bioRxiv2019

Structure-guided molecular grafting of a complex broadly neutralizing viral epitope

Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a "universal" influenza vaccine will come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use non-circulating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These "resurfaced" HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can enrich for RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.

immunology↗