RESUMO
Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies-F4, F6, F7, and F9-are reported that potently neutralize HAV at 50% neutralizing concentration values (neut50) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC50) of approximately 1 µM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development.
Assuntos
Anticorpos Neutralizantes/ultraestrutura , Desenho de Fármacos , Vírus da Hepatite A/imunologia , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Anticorpos Monoclonais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais , Antígenos Virais , Capsídeo/metabolismo , Simulação por Computador , Epitopos , Antígenos da Hepatite A/metabolismo , Antígenos da Hepatite A/ultraestrutura , Vírus da Hepatite A/patogenicidade , Vírus da Hepatite A/ultraestrutura , Humanos , Piperazinas/metabolismo , Piperazinas/farmacologia , Ligação ProteicaRESUMO
BACKGROUND: A huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016-2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure. METHODS: We performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines. FINDINGS: HAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation. INTERPRETATION: Our data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group.
Assuntos
Proteínas do Capsídeo/genética , Vírus da Hepatite A/imunologia , Hepatite A/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Adulto , Proteínas do Capsídeo/imunologia , Surtos de Doenças , Europa (Continente)/epidemiologia , Evolução Molecular , Hepatite A/imunologia , Hepatite A/virologia , Antígenos da Hepatite A/genética , Antígenos da Hepatite A/metabolismo , Vírus da Hepatite A/genética , Homossexualidade Masculina , Humanos , Masculino , Pessoa de Meia-Idade , Quase-Espécies , Análise de Sequência de RNA/métodos , VacinaçãoRESUMO
The analysis of recently published data on hepatitis A virus (HAV) genome clinical features, molecular diagnostic value and cell culture propagation are reviewed. The growing need in the study of the genetic diversity of HAV isolates and the search of its possible new antigenic variants are underlined. The results of the cultivation of different HAV strains are analyzed for possible application in vaccine and diagnostic kit production.
Assuntos
Variação Genética , Genoma Viral , Vírus da Hepatite A , Hepatite A , Kit de Reagentes para Diagnóstico , Animais , Hepatite A/diagnóstico , Hepatite A/genética , Hepatite A/metabolismo , Antígenos da Hepatite A/genética , Antígenos da Hepatite A/metabolismo , Vírus da Hepatite A/genética , Vírus da Hepatite A/crescimento & desenvolvimento , Vírus da Hepatite A/metabolismo , HumanosRESUMO
The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in Escherichia coli. HAV pentamers (14S) self-assembled into capsids (70S) in vitro. The antibodies induced by these structures recognized and neutralized HAV.
