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.

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.

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.

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.

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.

Obesity is associated with an altered baseline and post-vaccination influenza antibody repertoire.

As highlighted by the ongoing COVID-19 pandemic, vaccination is critical for infectious disease prevention and control. Obesity is associated with increased morbidity and mortality from respiratory virus infections. While obese individuals respond to influenza vaccination, what is considered a seroprotective response may not fully protect the global obese population. In a cohort vaccinated with the 2010-2011 trivalent inactivated influenza vaccine, baseline immune history and vaccination responses were found to significantly differ in obese individuals compared to healthy controls, especially towards the 2009 pandemic strain of A/H1N1 influenza virus. Young, obese individuals displayed responses skewed towards linear peptides versus conformational antigens, suggesting aberrant obese immune response. Overall, these data have vital implications for the next generation of influenza vaccines, and towards the current SARS-CoV-2 vaccination campaign.

Efficient high-throughput SARS-CoV-2 testing to detect asymptomatic carriers.

Recent reports suggest that 10 to 30% of severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) infected patients are asymptomatic and that viral shedding may occur before symptom onset. Therefore, there is an urgent need to increase diagnostic testing capabilities to prevent disease spread. We developed P-BEST, a method for Pooling-Based Efficient SARS-CoV-2 Testing, which identifies all positive subjects within a set of samples using a single round of testing. Each sample is assigned into multiple pools using a combinatorial pooling strategy based on compressed sensing. We pooled sets of 384 samples into 48 pools, providing both an eightfold increase in testing efficiency and an eightfold reduction in test costs, while identifying up to five positive carriers. We then used P-BEST to screen 1115 health care workers using 144 tests. P- BEST provides an efficient and easy-to-implement solution for increasing testing capacity that can be easily integrated into diagnostic laboratories.

Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways.

BACKGROUND: The mammalian inner ear contains sensory organs, the organ of Corti in the cochlea and cristae and maculae in the vestibule, with each comprised of patterned sensory epithelia that are responsible for hearing and balance. The development, cell fate, patterning, and innervation of both the sensory and nonsensory regions of the inner ear are governed by tight regulation involving, among others, transcription factors and microRNAs (miRNAs). In humans, mutations in specific miRNA genes are associated with hearing loss. In mice, experimental reduction or mutations of miRNAs in the inner ear leads to severe developmental and structural abnormalities. A comprehensive identification of miRNAs in the sensory epithelia and their gene targets will enable pathways of auditory and vestibular function to be defined. RESULTS: In this study, we used Next-Generation Sequencing (NGS) to identify the most prominent miRNAs in the inner ear and to define miRNA-target pairs that form pathways crucial for the function of the sensory epithelial cells. NGS of RNA from inner ear sensory epithelial cells led to the identification of 455 miRNAs in both cochlear and vestibular sensory epithelium, with 30 and 44 miRNAs found in only cochlea or vestibule, respectively. miR-6715-3p and miR-6715-5p were defined for the first time in the inner ear. Gene targets were identified for each of these miRNAs, including Arhgap12, a GTPase activating protein, for miR-6715-3p, implicating this miRNA in sensory hair cell bundle development, actin reorganization, cell adhesion and inner ear morphogenesis. CONCLUSIONS: This study provides a comprehensive atlas of miRNAs in the inner ear sensory epithelia. The results provide further support of the essential regulatory role of miRNAs in inner ear sensory epithelia and in regulating pathways that define development and growth of these cells.

The LINC complex is essential for hearing.

Hereditary hearing loss is the most common sensory deficit. We determined that progressive high-frequency hearing loss in 2 families of Iraqi Jewish ancestry was due to homozygosity for the protein truncating mutation SYNE4 c.228delAT. SYNE4, a gene not previously associated with hearing loss, encodes nesprin-4 (NESP4), an outer nuclear membrane (ONM) protein expressed in the hair cells of the inner ear. The truncated NESP4 encoded by the families' mutation did not localize to the ONM. NESP4 and SUN domain-containing protein 1 (SUN1), which localizes to the inner nuclear membrane (INM), are part of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the nuclear envelope. Mice lacking either Nesp4 or Sun1 were evaluated for hair cell defects and hearing loss. In both Nesp4-/- and Sun1-/- mice, OHCs formed normally, but degenerated as hearing matured, leading to progressive hearing loss. The nuclei of OHCs from mutant mice failed to maintain their basal localization, potentially affecting cell motility and hence the response to sound. These results demonstrate that the LINC complex is essential for viability and normal morphology of OHCs and suggest that the position of the nucleus in sensory epithelial cells is critical for maintenance of normal hearing.

Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in Middle Eastern families.

BACKGROUND: Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity. RESULTS: A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population. CONCLUSIONS: Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.

MicroRNAs and epigenetic regulation in the mammalian inner ear: implications for deafness.

Sensorineural hearing loss is the most common sensory disorder in humans and derives, in most cases, from inner-ear defects or degeneration of the cochlear sensory neuroepithelial hair cells. Genetic factors make a significant contribution to hearing impairment. While mutations in 51 genes have been associated with hereditary sensorineural nonsyndromic hearing loss (NSHL) in humans, the responsible mutations in many other chromosomal loci linked with NSHL have not been identified yet. Recently, mutations in a noncoding microRNA (miRNA) gene, MIR96, which is expressed specifically in the inner-ear hair cells, were linked with progressive hearing loss in humans and mice. Furthermore, additional miRNAs were found to have essential roles in the development and survival of inner-ear hair cells. Epigenetic mechanisms, in particular, DNA methylation and histone modifications, have also been implicated in human deafness, suggesting that several layers of noncoding genes that have never been studied systematically in the inner-ear sensory epithelia are required for normal hearing. This review aims to summarize the current knowledge about the roles of miRNAs and epigenetic regulatory mechanisms in the development, survival, and function of the inner ear, specifically in the sensory epithelia, tectorial membrane, and innervation, and their contribution to hearing.

MicroRNAs are essential for development and function of inner ear hair cells in vertebrates.

MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the 2 inner ear compartments. A conditional knock-out mouse for Dicer1 demonstrated that miRNAs are crucial for postnatal survival of functional hair cells of the inner ear. We identified miRNAs that have a role in the vertebrate developing inner ear by combining miRNA transcriptome analysis, spatial and temporal expression patterns, and bioinformatics. Microarrays revealed similar miRNA profiles in newborn-mouse whole cochleae and vestibules, but different temporal and spatial expression patterns of six miRNAs (miR-15a, miR-18a, miR-30b, miR-99a, miR-182, and miR-199a) may reflect their roles. Two of these miRNAs, miR-15a-1 and miR-18a, were also shown to be crucial for zebrafish inner ear development and morphogenesis. To suggest putative target mRNAs whose translation may be inhibited by selected miRNAs, we combined bioinformatics-based predictions and mRNA expression data. Finally, we present indirect evidence that Slc12a2, Cldn12, and Bdnf mRNAs may be targets for miR-15a. Our data support the hypothesis that inner ear tissue differentiation and maintenance are regulated and controlled by conserved sets of cell-specific miRNAs in both mouse and zebrafish.

Mouse models to study inner ear development and hereditary hearing loss.

Hereditary sensorineural hearing loss, derived from inner ear defects, is the most common hereditary disability with a prevalence of 1 in 1,000 children, although it can be present in up to 15% of births in isolated communities. The mouse serves as an ideal animal model to identify new deafness-related genes and to study their roles in vivo. This review describes mouse models for genes that have been linked with hearing impairment (HI) in humans. Mutations in several groups of genes have been linked with HI in both mice and humans. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. For example, the roles of collagens and tectorins in the tectorial membrane, as well as the necessity of intact links between the hair cell projections, stereocilia and kinocilia, have been discovered in mice. Accurate endolymph composition and the proteins which participate in its production were found to be crucial for inner ear function, as well as several motor proteins such as prestin and myosins. Two systematic projects, KOMP and EUCOMM, which are currently being carried out to create knock-out and conditional mutants for every gene in the mouse genome, promise that many additional deafness-related genes will be identified in the next years, providing models for all forms of human deafness.

Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: implications for cancer immunotherapy.

mAbs to receptor tyrosine kinases such as EGF receptor/ErbB-1 and HER2/ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Ab-receptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.

Organophosphate poisoning: a multihospital survey.

BACKGROUND: Organophosphates are frequently used as insecticides in the household and in agricultural areas, thus posing a risk for accidental exposure. OBJECTIVES: To describe the characteristics, clinical course and outcome of 97 patients admitted to emergency rooms with a diagnosis of acute OP poisoning. METHODS: The clinical details of 97 patients were collected from 6 different hospitals in Israel. Diagnosis of intoxication was based on clinical findings, butyrylcholinesterase levels and, in several cases, the material brought to the hospital. Demographic, intoxication and clinical data were analyzed. RESULTS: The study group comprised 64 men and 33 women whose age range was 1-70 years (mean 19.8 +/- 17.1); more than one-third of the patients were less than 10 years old. Accidental exposure was the cause of intoxication in 51.5% of the patients, and suicide in 20.6% of exposures. Intoxication occurred at home in most patients (67%), and the route of intoxication was oral in 65% of them. The patients arrived at the hospital 20 minutes to 72 hours after intoxication. Nine patients were asymptomatic; 53 presented with mild intoxication, 22 with moderate, and 13 had severe intoxication, 5 of whom died. There was a direct correlation between the degree of inhibition of butyrylcholinesterase levels and the severity of intoxication. Treatment included decontamination and antidotal medication. Duration of hospitalization ranged between 1 and 14 days (average 2.9 days). CONCLUSIONS: Organophosphates may cause severe morbidity and mortality. Medical staff should therefore be aware of the clinical manifestations and the antidotal treatment for this poisoning.