Structure and antigenicity of the divergent human astrovirus VA1 capsid spike.

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

Mouse and human immune responses share neutralization epitopes of HAstV-VA1.

Astroviruses are highly divergent and infect a wide variety of animal hosts. In 2009, a genetically divergent human astrovirus (HAstV) strain VA1 was first identified in an outbreak of acute gastroenteritis. This strain has also been associated with fatal central nervous system disease. In this work, we report the isolation of three high-affinity neutralizing monoclonal antibodies (Nt-MAbs) targeting the capsid spike domain of HAstV-VA1. These antibodies (7C8, 2A2, 3D8) were used to select individual HAstV-VA1 mutants resistant to their neutralizing activity and a HAstV-VA1 triple mutant that escapes neutralization from all three Nt-MAbs. Sequencing of the virus genome capsid region revealed escape mutations that map to the surface of the capsid spike domain, define three potentially independent neutralization epitopes, and help delineate four antigenic sites in human astroviruses. Notably, two of the escape mutations were found to be present in the spike sequence of the HAstV-VA1-PS strain isolated from an immunodeficient patient with encephalitis, suggesting that those mutations arose as a result of the immune pressure generated by the patient's immunotherapy. In agreement with this observation, human serum samples exhibiting strong neutralization activity against wild-type HAstV-VA1 had a 2.6-fold reduction in neutralization titer when evaluated against the triple-escape HAstV-VA1 mutant, suggesting that both mouse and human antibody responses target shared neutralization epitopes. The isolated Nt-MAbs reported in this work will help to characterize the functional domains of the virus during cell entry and have the potential for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1. IMPORTANCE: Human astroviruses (HAstVs) have been historically associated with acute gastroenteritis. However, the genetically divergent HAstV-VA1 strain has been associated with central nervous system disease. In this work high-affinity neutralizing monoclonal antibodies directed to HAstV-VA1 were isolated and characterized. The proposed binding sites for these antibodies and for neutralizing antibodies against classical HAstVs suggest that there are at least four neutralization sites on the capsid spike of astroviruses. Our data show that natural infection with human astrovirus VA1 elicits a robust humoral immune response that targets the same antigenic sites recognized by the mouse monoclonal antibodies and strongly suggests the emergence of a variant HAstV-VA1 virus in an immunodeficient patient with prolonged astrovirus infection. The isolated Nt-MAb reported in this work will help to define the functional sites of the virus involved in cell entry and hold promise for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1.

Immune response to SARS-CoV-2 variants after immunization with different vaccines in Mexico.

There is limited information on the antibody responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in subjects from developing countries with populations having a high incidence of co-morbidities. Here, we analysed the immunogenicity of homologous schemes using the ChAdOx1-S, Sputnik V, or BNT162b2 vaccines and the effect of a booster dose with ChAdOx1-S in middle-aged adults who were seropositive or seronegative to the SARS-CoV-2 spike protein before vaccination. The study was conducted post-vaccination with a follow-up of 4 months for antibody titre using enzyme-linked immunosorbent assay (ELISA) and pseudovirus (PV) neutralization assays (PNAs). All three vaccines elicited a superior IgG anti-receptor-binding domain (RBD) and neutralization response against the Alpha and Delta variants when administered to individuals with a previous infection by SARS-CoV-2. The booster dose spiked the neutralization activity among individuals with and without a prior SARS-CoV-2 infection. The ChAdOx1-S vaccine induced weaker antibody responses in infection-naive subjects. A follow-up of 4 months post-vaccination showed a drop in antibody titre, with about 20% of the infection-naive and 100% of SARS-CoV-2 pre-exposed participants with detectable neutralization capacity against Alpha pseudovirus (Alpha-PV) and Delta PV (Delta-PV). Our observations support the use of different vaccines in a country with high seroprevalence at the vaccination time.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Structure and immunogenicity of the murine astrovirus capsid spike.

Human astroviruses (HAstVs) are small, non-enveloped icosahedral RNA viruses that are a significant cause of diarrhoea in young children. Despite their worldwide prevalence, HAstV pathogenesis studies and vaccine development remain challenging due to the lack of an animal model for HAstV infection. The recent development of a murine astrovirus (MuAstV) infection model in mice provides the opportunity to test proof-of-concept vaccines based on MuAstV antigens. To help establish a system in which an astrovirus capsid spike-based vaccine could be tested in vivo, we designed and produced a recombinant MuAstV capsid spike protein based on predicted secondary structure homology to HAstV spike proteins. The recombinant MuAstV spike can be expressed with high efficiency in Escherichia coli and retains antigenicity to polyclonal antibodies elicited by MuAstV infection. We determined the crystal structure of the MuAstV spike to 1.75 Å and assessed its structural conservation with HAstV capsid spike. Despite low sequence identity between the MuAstV and HAstV spikes and differences in their overall shapes, they share related structural folds. Additionally, we found that vaccination with MuAstV spike induced anti-MuAstV-spike antibodies, highlighting that the recombinant spike is immunogenic. These studies lay a foundation for future in vivo MuAstV challenge studies to test whether MuAstV spike can be the basis of an effective vaccine.

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G (H. C. Bergeron, J. Murray, A. M. Nuñez Castrejon, et al., Viruses 13:352, 2021, https://doi.org/10.3390/v13020352). However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. IMPORTANCE Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

Structures of Two Human Astrovirus Capsid/Neutralizing Antibody Complexes Reveal Distinct Epitopes and Inhibition of Virus Attachment to Cells.

Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk for contracting severe disease. However, no vaccines exist to combat human astrovirus infection. Evidence points to the importance of antibodies in protecting healthy adults from reinfection. To develop an effective subunit vaccine that broadly protects against diverse astrovirus serotypes, we must understand how neutralizing antibodies target the capsid surface at the molecular level. Here, we report the structures of the human astrovirus capsid spike domain bound to two neutralizing monoclonal antibodies. These antibodies bind two distinct conformational epitopes on the spike surface. We add to existing evidence that the human astrovirus capsid spike contains a receptor-binding domain and demonstrate that both antibodies neutralize human astrovirus by blocking virus attachment to host cells. We identify patches of conserved amino acids which overlap or border the antibody epitopes and may constitute a receptor-binding site. Our findings provide a basis for developing therapies to prevent and treat human astrovirus gastroenteritis. IMPORTANCE Human astroviruses infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies block astrovirus infection. Here, we determined the crystal structures of the astrovirus capsid protein in complex with two virus-neutralizing antibodies. We show that the antibodies bind to two distinct sites on the capsid spike domain, however, both antibodies block virus attachment to human cells. Importantly, our findings support the use of the human astrovirus capsid spike as an antigen in a subunit-based vaccine to prevent astrovirus disease.

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in infants and the elderly. Currently, no vaccine or effective treatment exists for RSV. The RSV G glycoprotein mediates viral attachment to cells and contributes to pathogenesis by modulating host immunity through interactions with the human chemokine receptor CX3CR1. Antibodies targeting the RSV G central conserved domain are protective in both prophylactic and postinfection animal models. Here, we describe the crystal structure of the broadly neutralizing human monoclonal antibody 3G12 bound to the RSV G central conserved domain. Antibody 3G12 binds to a conformational epitope composed of highly conserved residues, explaining its broad neutralization activity. Surprisingly, RSV G complexed with 3G12 adopts a distinct conformation not observed in previously described RSV G-antibody structures. Comparison to other structures reveals that the RSV G central conserved domain is flexible and can adopt multiple conformations in the regions flanking the cysteine noose. We also show that restriction of RSV G flexibility with a proline mutation abolishes binding to antibody 3G12 but not antibody 3D3, which recognizes a different conformation of RSV G. Our studies provide new insights for rational vaccine design, indicating the importance of preserving both the global structural integrity of antigens and local conformational flexibility at antigenic sites, which may elicit a more diverse antibody response and broader protection against infection and disease.IMPORTANCE Respiratory syncytial virus (RSV) causes severe respiratory infections in infants, young children, and the elderly, and currently, no licensed vaccine exists. In this study, we describe the crystal structure of the RSV surface glycoprotein G in complex with a broadly neutralizing human monoclonal antibody. The antibody binds to RSV G at a highly conserved region stabilized by two disulfide bonds, but it captures RSV G in a conformation not previously observed, revealing that this region is both structured and flexible. Importantly, our findings provide insight for the design of vaccines that elicit diverse antibodies, which may provide broad protection from infection and disease.

Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization.

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

Structural Basis for Escape of Human Astrovirus from Antibody Neutralization: Broad Implications for Rational Vaccine Design.

Human astroviruses are recognized as a leading cause of viral diarrhea worldwide in children, immunocompromised patients, and the elderly. There are currently no vaccines available to prevent astrovirus infection; however, antibodies developed by healthy individuals during previous infection correlate with protection from reinfection, suggesting that an effective vaccine could be developed. In this study, we investigated the molecular mechanism by which several strains of human astrovirus serotype 2 (HAstV-2) are resistant to the potent HAstV-2-neutralizing monoclonal antibody PL-2 (MAb PL-2). Sequencing of the HAstV-2 capsid genes reveals mutations in the PL-2 epitope within the capsid's spike domain. To understand the molecular basis for resistance from MAb PL-2 neutralization, we determined the 1.35-Å-resolution crystal structure of the capsid spike from one of these HAstV-2 strains. Our structure reveals a dramatic conformational change in a loop within the PL-2 epitope due to a serine-to-proline mutation, locking the loop in a conformation that sterically blocks binding and neutralization by MAb PL-2. We show that mutation to serine permits loop flexibility and recovers MAb PL-2 binding. Importantly, we find that HAstV-2 capsid spike containing a serine in this loop is immunogenic and elicits antibodies that neutralize all HAstV-2 strains. Taken together, our results have broad implications for rational selection of vaccine strains that do not contain prolines in antigenic loops, so as to elicit antibodies against diverse loop conformations.IMPORTANCE Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. In this study, we investigated how several strains of HAstV are resistant to a virus-neutralizing monoclonal antibody. We determined the crystal structure of the capsid protein spike domain from one of these HAstV strains and found that a single amino acid mutation induces a structural change in a loop that is responsible for antibody binding. Our findings reveal how viruses can escape antibody neutralization and provide insight for the rational design of vaccines to elicit diverse antibodies that provide broader protection from infection.

Functional and evolutionary insight from the crystal structure of rubella virus protein E1.

Little is known about the three-dimensional organization of rubella virus, which causes a relatively mild measles-like disease in children but leads to serious congenital health problems when contracted in utero. Although rubella virus belongs to the same family as the mosquito-borne alphaviruses, in many respects it is more similar to other aerosol-transmitted human viruses such as the agents of measles and mumps. Although the use of the triple MMR (measles, mumps and rubella) live vaccine has limited its incidence in western countries, congenital rubella syndrome remains an important health problem in the developing world. Here we report the 1.8 Å resolution crystal structure of envelope glycoprotein E1, the main antigen and sole target of neutralizing antibodies against rubella virus. E1 is the main player during entry into target cells owing to its receptor-binding and membrane-fusion functions. The structure reveals the epitope and the neutralization mechanism of an important category of protecting antibodies against rubella infection. It also shows that rubella virus E1 is a class II fusion protein, which had hitherto only been structurally characterized for the arthropod-borne alphaviruses and flaviviruses. In addition, rubella virus E1 has an extensive membrane-fusion surface that includes a metal site, reminiscent of the T-cell immunoglobulin and mucin family of cellular proteins that bind phosphatidylserine lipids at the plasma membrane of cells undergoing apoptosis. Such features have not been seen in any fusion protein crystallized so far. Structural comparisons show that the class II fusion proteins from alphaviruses and flaviviruses, despite belonging to different virus families, are closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. By contrast, in the absence of this constraint, the strictly human rubella virus seems to have drifted considerably into a unique niche as sole member of the Rubivirus genus.

Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes.

Chromatin structure plays a pivotal role in facilitating proper control of gene expression. Transcription factor (TF) binding of cis-elements is often associated with accessible chromatin regions. Therefore, the ability to identify these accessible regions throughout plant genomes will advance understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with fluorescence-activated nuclei sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it requires less than 50 000 nuclei for accurate identification of accessible genomic regions. SUMMARY: Application of ATAC-seq to sorted nuclei identifies accessible regions genome-wide.

Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization.

Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease. IMPORTANCE: Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

Structural, Mechanistic, and Antigenic Characterization of the Human Astrovirus Capsid.

UNLABELLED: Human astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies. IMPORTANCE: Astroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus capsid proteins. Fitting these structures into previously determined low-resolution maps of astrovirus allowed us to characterize the molecular surfaces of immature and mature astroviruses. Our studies provide the first evidence that astroviruses undergo viral RNA-dependent assembly. We also provide new insight into the molecular mechanisms that lead to astrovirus maturation and infectivity. Finally, we show that both capsid proteins contribute to the adaptive immune response against astrovirus. Together, these studies will help to guide the development of vaccines and antiviral drugs targeting astrovirus.

Crystal structure of the avian astrovirus capsid spike.

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.

Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease.

Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.

Structural basis for the activity and specificity of the immune checkpoint inhibitor lirilumab.

The clinical success of immune checkpoint inhibitors has underscored the key role of the immune system in controlling cancer. Current FDA-approved immune checkpoint inhibitors target the regulatory receptor pathways of cytotoxic T-cells to enhance their anticancer responses. Despite an abundance of evidence that natural killer (NK) cells can also mediate potent anticancer activities, there are no FDA-approved inhibitors targeting NK cell specific checkpoint pathways. Lirilumab, the most clinically advanced NK cell checkpoint inhibitor, targets inhibitory killer immunoglobulin-like receptors (KIRs), however it has yet to conclusively demonstrate clinical efficacy. Here we describe the crystal structure of lirilumab in complex with the inhibitory KIR2DL3, revealing the precise epitope of lirilumab and the molecular mechanisms underlying KIR checkpoint blockade. Notably, the epitope includes several key amino acids that vary across the human population, and binding studies demonstrate the importance of these amino acids for lirilumab binding. These studies reveal how KIR variations in patients could influence the clinical efficacy of lirilumab and reveal general concepts for the development of immune checkpoint inhibitors targeting NK cells.

Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Antigenic determinants of HAstV-VA1 neutralization and their relevance in the human immune response.

Astroviruses are highly divergent and infect a wide variety of animal hosts. In 2009, a genetically divergent human astrovirus (HAstV) strain VA1 was first identified in an outbreak of acute gastroenteritis. This strain has also been associated with fatal central nervous system disease. In this work, we report the isolation of three high-affinity neutralizing monoclonal antibodies (Nt-MAbs) targeting the capsid spike domain of HAstV-VA1. These antibodies (7C8, 2A2, 3D8) were used to select individual HAstV-VA1 mutants resistant to their neutralizing activity and also select a HAstV-VA1 triple mutant that escapes neutralization from all three Nt-MAbs. Sequencing of the virus genome capsid region revealed escape mutations that map to the surface of the capsid spike domain, define three potentially independent neutralization epitopes, and help delineate four antigenic sites in rotaviruses. Notably, two of the escape mutations were found to be present in the spike sequence of the HAstV-VA1-PS strain isolated from an immunodeficient patient with encephalitis, suggesting that those mutations arose as a result of the immune pressure generated by the patient's immunotherapy. In accordance with this observation, human serum samples exhibiting strong neutralization activity against wild-type HAstV-VA1 had a 2.6-fold reduction in neutralization titer when evaluated against the triple-escape HAstV-VA1 mutant, indicating shared neutralization epitopes between the mouse and human antibody response. The isolated Nt-MAbs reported in this work will help characterize the functional sites of the virus during cell entry and have the potential for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1. Importance: Human astroviruses (HAstVs) have been historically associated with acute gastroenteritis. However, the genetically divergent HAstV-VA1 strain has been associated with central nervous system disease. This work isolated high-affinity neutralizing monoclonal antibodies directed to HAstV-VA1. The proposed binding sites for these antibodies, together with previously reported sites for neutralizing antibodies against classical HAstVs, suggest the existence of at least four neutralization sites on the capsid spike of astroviruses. Our data show that natural infection with human astrovirus VA1 elicits a robust humoral immune response that targets the same antigenic sites recognized by the mouse monoclonal antibodies and strongly suggests the emergence of a variant HAstV-VA1 virus in an immunodeficient patient with prolonged astrovirus infection. The isolated Nt-MAb reported in this work will be helpful in defining the functional sites of the virus involved in cell entry and hold promise for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1.

Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity.

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.