Induction of bronchus-associated lymphoid tissue is an early life adaptation for promoting human B cell immunity.

Infants and young children are more susceptible to common respiratory pathogens than adults but can fare better against novel pathogens like severe acute respiratory syndrome coronavirus 2. The mechanisms by which infants and young children mount effective immune responses to respiratory pathogens are unknown. Through investigation of lungs and lung-associated lymph nodes from infant and pediatric organ donors aged 0-13 years, we show that bronchus-associated lymphoid tissue (BALT), containing B cell follicles, CD4+ T cells and functionally active germinal centers, develop during infancy. BALT structures are prevalent around lung airways during the first 3 years of life, and their numbers decline through childhood coincident with the accumulation of memory T cells. Single-cell profiling and repertoire analysis reveals that early life lung B cells undergo differentiation, somatic hypermutation and immunoglobulin class switching and exhibit a more activated profile than lymph node B cells. Moreover, B cells in the lung and lung-associated lymph nodes generate biased antibody responses to multiple respiratory pathogens compared to circulating antibodies, which are mostly specific for vaccine antigens in the early years of life. Together, our findings provide evidence for BALT as an early life adaptation for mobilizing localized immune protection to the diverse respiratory challenges during this formative life stage.

The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection with influenza virus.

Highly pathogenic avian influenza viruses pose a continuing global threat. Current vaccines will not protect against newly evolved pandemic viruses. The creation of 'universal' vaccines has been unsuccessful because the immunological mechanisms that promote heterosubtypic immunity are incompletely defined. We found here that rapamycin, an immunosuppressive drug that inhibits the kinase mTOR, promoted cross-strain protection against lethal infection with influenza virus of various subtypes when administered during immunization with influenza virus subtype H3N2. Rapamycin reduced the formation of germinal centers and inhibited class switching in B cells, which yielded a unique repertoire of antibodies that mediated heterosubtypic protection. Our data established a requirement for the mTORC1 complex in B cell class switching and demonstrated that rapamycin skewed the antibody response away from high-affinity variant epitopes and targeted more conserved elements of hemagglutinin. Our findings have implications for the design of a vaccine against influenza virus.

US National Institutes of Health Prioritization of SARS-CoV-2 Variants.

Since late 2020, SARS-CoV-2 variants have regularly emerged with competitive and phenotypic differences from previously circulating strains, sometimes with the potential to escape from immunity produced by prior exposure and infection. The Early Detection group is one of the constituent groups of the US National Institutes of Health National Institute of Allergy and Infectious Diseases SARS-CoV-2 Assessment of Viral Evolution program. The group uses bioinformatic methods to monitor the emergence, spread, and potential phenotypic properties of emerging and circulating strains to identify the most relevant variants for experimental groups within the program to phenotypically characterize. Since April 2021, the group has prioritized variants monthly. Prioritization successes include rapidly identifying most major variants of SARS-CoV-2 and providing experimental groups within the National Institutes of Health program easy access to regularly updated information on the recent evolution and epidemiology of SARS-CoV-2 that can be used to guide phenotypic investigations.

FLU-LISA (fluorescence-linked immunosorbent assay): high-throughput antibody profiling using antigen microarrays.

Vaccination and natural infection both elicit potent humoral responses that provide protection from subsequent infections. The immune history of an individual following such exposures is in part encoded by antibodies. While there are multiple immunoassays for measuring antibody responses, the majority of these methods measure responses to a single antigen. A commonly used method for measuring antibody responses is ELISA-a semiquantitative assay that is simple to perform in research and clinical settings. Here, we present FLU-LISA (fluorescence-linked immunosorbent assay)-a novel antigen microarray-based assay for rapid high-throughput antibody profiling. The assay can be used for profiling immunoglobulin (Ig) G, IgA and IgM responses to multiple antigens simultaneously, requiring minimal amounts of sample and antigens. Using several influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen microarrays, we demonstrated the specificity and sensitivity of our novel assay and compared it with the traditional ELISA, using samples from mice, chickens and humans. We also showed that our assay can be readily used with dried blood spots, which can be collected from humans and wild birds. FLU-LISA can be readily used to profile hundreds of samples against dozens of antigens in a single day, and therefore offers an attractive alternative to the traditional ELISA.

Quantifiable predictive features define epitope-specific T cell receptor repertoires.

T cells are defined by a heterodimeric surface receptor, the T cell receptor (TCR), that mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs are generated by genomic rearrangement of the germline TCR locus, a process termed V(D)J recombination, that has the potential to generate marked diversity of TCRs (estimated to range from 1015 (ref. 1) to as high as 1061 (ref. 2) possible receptors). Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features, suggesting that it may be possible to predictively model epitope specificity. Here we report the in-depth characterization of ten epitope-specific TCR repertoires of CD8+ T cells from mice and humans, representing over 4,600 in-frame single-cell-derived TCRαß sequence pairs from 110 subjects. We developed analytical tools to characterize these epitope-specific repertoires: a distance measure on the space of TCRs that permits clustering and visualization, a robust repertoire diversity metric that accommodates the low number of paired public receptors observed when compared to single-chain analyses, and a distance-based classifier that can assign previously unobserved TCRs to characterized repertoires with robust sensitivity and specificity. Our analyses demonstrate that each epitope-specific repertoire contains a clustered group of receptors that share core sequence similarities, together with a dispersed set of diverse 'outlier' sequences. By identifying shared motifs in core sequences, we were able to highlight key conserved residues driving essential elements of TCR recognition. These analyses provide insights into the generalizable, underlying features of epitope-specific repertoires and adaptive immune recognition.

Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections.

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

Study of Healthcare Personnel with Influenza and other Respiratory Viruses in Israel (SHIRI): study protocol.

BACKGROUND: The Study of Healthcare Personnel with Influenza and other Respiratory Viruses in Israel (SHIRI) prospectively follows a cohort of healthcare personnel (HCP) in two hospitals in Israel. SHIRI will describe the frequency of influenza virus infections among HCP, identify predictors of vaccine acceptance, examine how repeated influenza vaccination may modify immunogenicity, and evaluate influenza vaccine effectiveness in preventing influenza illness and missed work. METHODS: Cohort enrollment began in October, 2016; a second year of the study and a second wave of cohort enrollment began in June 2017. The study will run for at least 3 years and will follow approximately 2000 HCP (who are both employees and members of Clalit Health Services [CHS]) with routine direct patient contact. Eligible HCP are recruited using a stratified sampling strategy. After informed consent, participants complete a brief enrollment survey with questions about occupational responsibilities and knowledge, attitudes, and practices about influenza vaccines. Blood samples are collected at enrollment and at the end of influenza season; HCP who choose to be vaccinated contribute additional blood one month after vaccination. During the influenza season, participants receive twice-weekly short message service (SMS) messages asking them if they have acute respiratory illness or febrile illness (ARFI) symptoms. Ill participants receive follow-up SMS messages to confirm illness symptoms and duration and are asked to self-collect a nasal swab. Information on socio-economic characteristics, current and past medical conditions, medical care utilization and vaccination history is extracted from the CHS database. Information about missed work due to illness is obtained by self-report and from employee records. Respiratory specimens from self-collected nasal swabs are tested for influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, and coronaviruses using validated multiplex quantitative real-time reverse transcription polymerase chain reaction assays. The hemagglutination inhibition assay will be used to detect the presence of neutralizing influenza antibodies in serum. DISCUSSION: SHIRI will expand our knowledge of the burden of respiratory viral infections among HCP and the effectiveness of current and repeated annual influenza vaccination in preventing influenza illness, medical utilization, and missed workdays among HCP who are in direct contact with patients. TRIAL REGISTRATION: NCT03331991 . Registered on November 6, 2017.

Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env V2.

The RV144 trial demonstrated 31% vaccine efficacy at preventing human immunodeficiency virus (HIV)-1 infection. Antibodies against the HIV-1 envelope variable loops 1 and 2 (Env V1 and V2) correlated inversely with infection risk. We proposed that vaccine-induced immune responses against V1/V2 would have a selective effect against, or sieve, HIV-1 breakthrough viruses. A total of 936 HIV-1 genome sequences from 44 vaccine and 66 placebo recipients were examined. We show that vaccine-induced immune responses were associated with two signatures in V2 at amino acid positions 169 and 181. Vaccine efficacy against viruses matching the vaccine at position 169 was 48% (confidence interval 18% to 66%; P = 0.0036), whereas vaccine efficacy against viruses mismatching the vaccine at position 181 was 78% (confidence interval 35% to 93%; P = 0.0028). Residue 169 is in a cationic glycosylated region recognized by broadly neutralizing and RV144-derived antibodies. The predicted distance between the two signature sites (21 ± 7 Å) and their match/mismatch dichotomy indicate that multiple factors may be involved in the protection observed in RV144. Genetic signatures of RV144 vaccination in V2 complement the finding of an association between high V1/V2-binding antibodies and reduced risk of HIV-1 acquisition, and provide evidence that vaccine-induced V2 responses plausibly had a role in the partial protection conferred by the RV144 regimen.

Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083.

BACKGROUND: Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. METHODS: A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. RESULTS: T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6-1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2-5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). CONCLUSIONS: These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. CLINICAL TRIALS REGISTRATION: NCT01095224.

Comprehensive sieve analysis of breakthrough HIV-1 sequences in the RV144 vaccine efficacy trial.

The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or "signatures" and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection.

Experimental and computational evidence suggests that HLAs preferentially bind conserved regions of viral proteins, a concept we term "targeting efficiency," and that this preference may provide improved clearance of infection in several viral systems. To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study performed during the 2009-2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression). A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24, were associated with pH1N1 mortality (r = 0.37, P = 0.031) and are common in certain indigenous populations in which increased pH1N1 morbidity has been reported. HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection. The computational tools used in this study may be useful predictors of potential morbidity and identify immunologic differences of new variant influenza strains more accurately than evolutionary sequence comparisons. Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Analysis of HLA A*02 association with vaccine efficacy in the RV144 HIV-1 vaccine trial.

UNLABELLED: The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02(+)) participants than in A*02(-) participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02(+) participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02(+) participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials. IMPORTANCE: The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.).

HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.

Mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load.

RATIONALE: Children are an at-risk population for developing complications following influenza infection, but immunologic correlates of disease severity are not understood. We hypothesized that innate cellular immune responses at the site of infection would correlate with disease outcome. OBJECTIVES: To test the immunologic basis of severe illness during natural influenza virus infection of children and adults at the site of infection. METHODS: An observational cohort study with longitudinal sampling of peripheral and mucosal sites in 84 naturally influenza-infected individuals, including infants. Cellular responses, viral loads, and cytokines were quantified from nasal lavages and blood, and correlated to clinical severity. MEASUREMENTS AND MAIN RESULTS: We show for the first time that although viral loads in children and adults were similar, innate responses in the airways were stronger in children and varied considerably between plasma and site of infection. Adjusting for age and viral load, an innate immune profile characterized by increased nasal lavage monocyte chemotactic protein-3, IFN-α2, and plasma IL-10 levels at enrollment predicted progression to severe disease. Increased plasma IL-10, monocyte chemotactic protein-3, and IL-6 levels predicted hospitalization. This inflammatory cytokine production correlated significantly with monocyte localization from the blood to the site of infection, with conventional monocytes positively correlating with inflammation. Increased frequencies of CD14(lo) monocytes were in the airways of participants with lower inflammatory cytokine levels. CONCLUSIONS: An innate profile was identified that correlated with disease progression independent of viral dynamics and age. The airways and blood displayed dramatically different immune profiles emphasizing the importance of cellular migration and localized immune phenotypes.

Putative amino acid determinants of the emergence of the 2009 influenza A (H1N1) virus in the human population.

The emergence of the unique H1N1 influenza A virus in 2009 resulted in a pandemic that has spread to over 200 countries. The constellation of molecular factors leading to the emergence of this strain is still unclear. Using a computational approach, we identified molecular determinants that may discriminate the hemagglutinin protein of the 2009 human pandemic H1N1 (pH1N1) strain from that of other H1N1 strains. As expected, positions discriminating the pH1N1 from seasonal human strains were located in or near known H1N1 antigenic sites, thus camouflaging the pH1N1 strain from immune recognition. For example, the alteration S145K (an antigenic position) was found as a characteristic of the pH1N1 strain. We also detected positions in the hemagglutinin protein differentiating classical swine viruses from pH1N1. These positions were mostly located in and around the receptor-binding pocket, possibly influencing binding affinity to the human cell. Such alterations may be liable in part for the virus's efficient infection and adaptation to humans. For instance, 133(A) and 149 were identified as discriminative positions. Significantly, we showed that the substitutions R133(A)K and R149K, predicted to be pH1N1 characteristics, each altered virus binding to erythrocytes and conferred virulence to A/swine/NC/18161/02 in mice, reinforcing the computational findings. Our findings provide a structural explanation for the deficient immunity of humans to the pH1N1 strain. Moreover, our analysis points to unique molecular factors that may have facilitated the emergence of this swine variant in humans, in contrast to other swine variants that failed.

High capacity clinical SARS-CoV-2 molecular testing using combinatorial pooling.

BACKGROUND: The SARS-CoV-2 pandemic led to unprecedented testing demands, causing major testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. However, such traditional pooled testing was used in practice only when positivity rates were below 2%. METHODS: Here we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel. RESULTS: P-BEST is clinically validated using 3636 side-by-side tests and is able to correctly detect all positive samples and accurately estimate their Ct value. Following regulatory approval by the Israeli Ministry of Health, P-BEST was used in 2021 to clinically test 837,138 samples using 270,095 PCR tests - a 3.1fold reduction in the number of tests. This period includes the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available. CONCLUSIONS: Our data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.

Testing samples for SARS-CoV-2 is usually done on one sample at a time. However, the unprecedented demand for testing during the COVID-19 pandemic led to the adoption of pooled testing strategies, where samples are combined before being tested. This strategy requires two rounds: first, each pool of samples is tested, and then a second testing round is performed on individual samples from positive pools. We developed and implemented a pooling method for SARS-CoV-2 that requires a single round of testing, thus enabling the shorter turnaround times required during a pandemic. The method was approved for clinical use in Israel and was used to successfully test 837,138 clinical samples using fewer than a third of the tests usually required. Our study provides a blueprint for rapid implementation of efficient high-throughput testing in future pandemics.

Constrained evolution drives limited influenza diversity.

H3N2 influenza A viruses have been widely circulating in human populations since the pandemic of 1968. A striking feature of the evolutionary development of this strain has been its 'canalized' nature, with narrow evolutionary trees dominated by long trunks with few branching, or bifurcation events and a consequent lack of standing diversity at any single point. This is puzzling, as one might expect that the strong human immune response against the virus would create an environment encouraging more diversity, not less. Previous models have used various assumptions in order to account for this finding. A new analysis published in BMC Biology suggests that this processive evolution down a single path can be recapitulated by a relatively simple model incorporating only two primary parameters - the mutation rate of the virus, and the immunological distance created by each mutation - so long as these parameters are within a particular narrow but biologically plausible range.

Mapping antibody footprints using binding profiles.

The increasing use of monoclonal antibodies (mAbs) in biology and medicine necessitates efficient methods for characterizing their binding epitopes. Here, we developed a high-throughput antibody footprinting method based on binding profiles. We used an antigen microarray to profile 23 human anti-influenza hemagglutinin (HA) mAbs using HA proteins of 43 human influenza strains isolated between 1918 and 2018. We showed that the mAb's binding profile can be used to characterize its influenza subtype specificity, binding region, and binding site. We present mAb-Patch-an epitope prediction method that is based on a mAb's binding profile and the 3D structure of its antigen. mAb-Patch was evaluated using four mAbs with known solved mAb-HA structures. mAb-Patch identifies over 67% of the true epitope when considering only 50-60 positions along the antigen. Our work provides proof of concept for utilizing antibody binding profiles to screen large panels of mAbs and to down-select antibodies for further functional studies.

Obesity Is Associated with an Impaired Baseline Repertoire of Anti-Influenza Virus Antibodies.

Obesity is a risk factor for severe disease and mortality for both influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. While previous studies show that individuals with obesity generate antibody responses following influenza vaccination, infection rates within the obese group were twice as high as those in the healthy-weight group. The repertoire of antibodies raised against influenza viruses following previous vaccinations and/or natural exposures is referred to here as baseline immune history (BIH). To investigate the hypothesis that obesity impacts immune memory to infections and vaccines, we profiled the BIH of obese and healthy-weight adults vaccinated with the 2010-2011 seasonal influenza vaccine in response to conformational and linear antigens. Despite the extensive heterogeneity of the BIH profiles in both groups, there were striking differences between obese and healthy subjects, especially with regard to A/H1N1 strains and the 2009 pandemic virus (Cal09). Individuals with obesity had lower IgG and IgA magnitude and breadth for a panel of A/H1N1 whole viruses and hemagglutinin proteins from 1933 to 2009 but increased IgG magnitude and breadth for linear peptides from the Cal09 H1 and N1 proteins. Age was also associated with A/H1N1 BIH, with young individuals with obesity being more likely to have reduced A/H1N1 BIH. We found that individuals with low IgG BIH had significantly lower neutralizing antibody titers than individuals with high IgG BIH. Taken together, our findings suggest that increased susceptibility of obese participants to influenza infection may be mediated in part by obesity-associated differences in the memory B-cell repertoire, which cannot be ameliorated by current seasonal vaccination regimens. Overall, these data have vital implications for the next generation of influenza virus and SARS-CoV-2 vaccines. IMPORTANCE Obesity is associated with increased morbidity and mortality from influenza and SARS-CoV-2 infection. While vaccination is the most effective strategy for preventing influenza virus infection, our previous studies showed that influenza vaccines fail to provide optimal protection in obese individuals despite reaching canonical correlates of protection. Here, we show that obesity may impair immune history in humans and cannot be overcome by seasonal vaccination, especially in younger individuals with decreased lifetime exposure to infections and seasonal vaccines. Low baseline immune history is associated with decreased protective antibody responses. Obesity potentially handicaps overall responses to vaccination, biasing it toward responses to linear epitopes, which may reduce protective capacity. Taken together, our data suggest that young obese individuals are at an increased risk of reduced protection by vaccination, likely due to altered immune history biased toward nonprotective antibody responses. Given the worldwide obesity epidemic coupled with seasonal respiratory virus infections and the inevitable next pandemic, it is imperative that we understand and improve vaccine efficacy in this high-risk population. The design, development, and usage of vaccines for and in obese individuals may need critical evaluation, and immune history should be considered an alternate correlate of protection in future vaccine clinical trials.

Correlates of protection for booster doses of the SARS-CoV-2 vaccine BNT162b2.

Vaccination, especially with multiple doses, provides substantial population-level protection against COVID-19, but emerging variants of concern (VOC) and waning immunity represent significant risks at the individual level. Here we identify correlates of protection (COP) in a multicenter prospective study following 607 healthy individuals who received three doses of the Pfizer-BNT162b2 vaccine approximately six months prior to enrollment. We compared 242 individuals who received a fourth dose to 365 who did not. Within 90 days of enrollment, 239 individuals contracted COVID-19, 45% of the 3-dose group and 30% of the four-dose group. The fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple VOCs reducing the risk of symptomatic infection by 37% [95%CI, 15%-54%]. However, a group of individuals, characterized by low baseline titers of binding antibodies, remained susceptible to infection despite significantly increased neutralizing antibody titers upon boosting. A combination of reduced IgG levels to RBD mutants and reduced VOC-recognizing IgA antibodies represented the strongest COP in both the 3-dose group (HR = 6.34, p = 0.008) and four-dose group (HR = 8.14, p = 0.018). We validated our findings in an independent second cohort. In summary combination IgA and IgG baseline binding antibody levels may identify individuals most at risk from future infections.