RESUMO
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.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Humanos , Animais , Anticorpos Neutralizantes/imunologia , Camundongos , Epitopos/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Mamastrovirus/imunologia , Mamastrovirus/genética , Mutação , Infecções por Astroviridae/imunologia , Infecções por Astroviridae/virologia , Testes de NeutralizaçãoRESUMO
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.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Vacinas , Adulto , Pessoa de Meia-Idade , Humanos , SARS-CoV-2 , México/epidemiologia , Vacina BNT162 , Estudos Soroepidemiológicos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Imunização , Vacinação , Imunidade , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
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 those 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 VP33 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. IMPORTANCE 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. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.
Assuntos
Infecções por Astroviridae , Proteínas do Capsídeo , Mamastrovirus , Precursores de Proteínas , Infecções por Astroviridae/virologia , Células CACO-2 , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Humanos , Espaço Intracelular/virologia , Mamastrovirus/fisiologia , Precursores de Proteínas/metabolismo , Tripsina/metabolismoRESUMO
Although human astroviruses (HAstVs) are important agents of gastroenteritis in young children, the studies aimed at characterizing their biology have been limited, in particular regarding their cell entry process. It has been shown that HAstV serotype 8 enters human cells by a classical clathrin-mediated endocytosis pathway; however, the cell receptor or other cell entry factors that may be relevant for an efficient viral infection are unknown. In this work we used a far-Western blotting approach to identify cellular proteins that interact with the recombinant capsid spike proteins of HAstV serotypes 1, 2, and 8, synthesized in Escherichia coli. We identified the 72 kDa protein disulfide isomerase A4 (PDIA4) as a binding partner for HAstV-1 and -8 spikes, but not for the HAstV-2 spike. In agreement with this observation, the PDI inhibitor 16F16 strongly blocked infection by HAstV serotypes 1 and 8, but not serotype 2. RNA interference of PDIA4 expression selectively blocked HAstV-8 infectivity. We also showed that the PDI activity does not affect virus binding or internalization but is required for uncoating of the viral genome.
Assuntos
Infecções por Astroviridae/metabolismo , Infecções por Astroviridae/virologia , Interações Hospedeiro-Patógeno , Mamastrovirus/fisiologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Desenvelopamento do Vírus , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Mamastrovirus/efeitos dos fármacos , Ligação Proteica , Internalização do VírusRESUMO
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.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Antígenos Virais/imunologia , Infecções por Astroviridae/imunologia , Mamastrovirus/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Infecções por Astroviridae/virologia , Células CACO-2 , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Variação Genética , Humanos , Imunização , Mamastrovirus/genética , Camundongos , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle.