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.KEYWORDS antibody, astrovirus, capsid, receptor binding, structure, vaccines, virus neutralization T he Astroviridae family is comprised of two genera, Mamastrovirus and Avastrovirus, which infect mammalian and avian species, respectively (1). Members of the Avastrovirus genus cause a variety of disease manifestations, growth defects, and mortality in poultry (2). Members of the Mamastrovirus genus cause infections in humans and a wide range of mammals (3). Human astroviruses (HAstVs) are classified into eight canonical serotypes (HAstV-1 to ) within the Mamastrovirus genogroup 1 (4), where HAstV-1 is the predominant serotype worldwide (5, 6). HAstV is a leading cause of viral diarrhea in children, immunocompromised individuals, and the elderly (7). There are approximately 3.9 million cases of viral diarrhea due to HAstV in the United States every year (8). In addition, highly divergent strains of HAstV have recently been attributed to encephalitis in immunocompromised individuals (9-11).

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.

Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology.

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Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify that proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348.  Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP34 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication.