RESUMEN
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.
Asunto(s)
Proteínas de la Cápside/genética , Virus de la Hepatitis A/inmunología , Hepatitis A/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Adulto , Proteínas de la Cápside/inmunología , Brotes de Enfermedades , Europa (Continente)/epidemiología , Evolución Molecular , Hepatitis A/inmunología , Hepatitis A/virología , Antígenos de Hepatitis A/genética , Antígenos de Hepatitis A/metabolismo , Virus de la Hepatitis A/genética , Homosexualidad Masculina , Humanos , Masculino , Persona de Mediana Edad , Cuasiespecies , Análisis de Secuencia de ARN/métodos , VacunaciónRESUMEN
Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.
Asunto(s)
Epítopos/inmunología , Antígenos de Hepatitis A/inmunología , Virus de la Hepatitis A/inmunología , Hepatitis A/diagnóstico , Hepatitis A/inmunología , Proteínas Recombinantes/inmunología , Vacunas contra Hepatitis Viral/inmunología , Secuencia de Aminoácidos , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Orden Génico , Antígenos de Hepatitis A/química , Antígenos de Hepatitis A/genética , Antígenos de Hepatitis A/aislamiento & purificación , Virus de la Hepatitis A/genética , Anticuerpos Antihepatitis/sangre , Anticuerpos Antihepatitis/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Variación Genética , Genoma Viral , Virus de la Hepatitis A , Hepatitis A , Juego de Reactivos para Diagnóstico , Animales , Hepatitis A/diagnóstico , Hepatitis A/genética , Hepatitis A/metabolismo , Antígenos de Hepatitis A/genética , Antígenos de Hepatitis A/metabolismo , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/crecimiento & desarrollo , Virus de la Hepatitis A/metabolismo , HumanosRESUMEN
OBJECTIVE: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods. RESULTS: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition. CONCLUSIONS: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.
Asunto(s)
Antígenos de Hepatitis A/genética , Vacunas contra la Hepatitis A/inmunología , Virus Vaccinia/genética , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Formaldehído/farmacología , Vectores Genéticos , Antígenos de Hepatitis A/inmunología , Humanos , Propiolactona/farmacología , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Severe structural constraints in the hepatitis A virus (HAV) capsid have been suggested as the reason for the lack of emergence of new serotypes in spite of the occurrence of complex distributions of mutants or quasispecies. Analysis of the HAV mutant spectra under immune pressure by the monoclonal antibodies (MAbs) K34C8 (immunodominant site) and H7C27 (glycophorin binding site) has revealed different evolutionary dynamics. Populations composed of complex ensembles of mutants with very low fitness or single dominant mutants with high fitness permit the acquisition of resistance to each of the MAbs, respectively. Deletion mutants were detected as components of the mutant spectra: up to 61 residues, with an average of 19, and up to 83 residues, with an average of 45, in VP3 and VP1 proteins, respectively. A clear negative selection of those replacements affecting the residues encoded by rare codons of the capsid surface has been detected through the present quasispecies analysis, confirming a certain beneficial role of such clusters. Since these clusters are located near or at the epitope regions, the need to maintain such clusters might prevent the emergence of new serotypes.
Asunto(s)
Variación Antigénica/genética , Proteínas de la Cápside/genética , Antígenos de Hepatitis A/genética , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/inmunología , Selección Genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Cápside/inmunología , Codón/genética , Evolución Molecular Dirigida , Epítopos/genética , Epítopos/inmunología , Evolución Molecular , Virus de la Hepatitis A/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
Knowledge of the molecular biology of hepatitis A virus (HAV) has increased exponentially since its identification. HAV exploits all known mechanisms of genetic variation to ensure survival, including mutation and genetic recombination. HAV has been characterized by the emergence of different genotypes, three human antigenic variants and only one major serotype. This paper reviews the genetic variability and molecular epidemiology of HAV. Its evolutionary mechanisms are described with particular emphasis on genetic recombination and HAV mutation rate. Genotypic classification methods are also discussed.
Asunto(s)
Variación Genética , Virus de la Hepatitis A Humana/genética , Secuencia de Aminoácidos , Variación Antigénica , Evolución Biológica , Canadá/epidemiología , Europa (Continente)/epidemiología , Genotipo , Hepatitis A/epidemiología , Antígenos de Hepatitis A/genética , Virus de la Hepatitis A Humana/clasificación , Humanos , Incidencia , Epidemiología Molecular , Datos de Secuencia Molecular , Mutación , ARN Viral/genética , Recombinación Genética , Alineación de Secuencia , América del Sur/epidemiologíaRESUMEN
The construction of poliovirus chimera is an useful way to investigate the antigenic structure conformation of poliovirus. In this research, the poliovirus chimera containing a piece of antigenic fragment of hepatitis A virus was constructed for analysis of poliovirus antigenic structure conformation. Depending on the characterization of this chimera, the conformation of poliovirus neutralization antigenic site I in VP1 was analyzed, and that the possible influence of hepatitis A virus antigenic fragment inserted to the structure conformation of poliovirus antigen was also investigated.
Asunto(s)
Antígenos Virales/inmunología , Quimera , Poliovirus/genética , Animales , Antígenos Virales/química , Células Cultivadas , Antígenos de Hepatitis A/genética , Humanos , Poliovirus/inmunología , Conejos , TransfecciónRESUMEN
The nucleotide and amino acid sequences of a Chinese hepatitis A virus Long-Jia (LJ) strain were compared with that of HM175, MBB and LA strains in structural genes (nt 630-3049). The most extensive nucleotide homology was identified between LJ and MBB strains. The identity rates of nucleotide were 95.4%, 96.7% and 91.4%, respectively. Variation rates of amino acid were 0.91%, 0.91% and 2.98%, respectively. A total of 23 amino acid differences located in whole capsid region between LJ and LA strain, especially in VP1. Only 7 amino acid differences located in VP2 and VP3 between LJ and HM175/MBB strain. Restriction enzyme sites increased 10, 13 and 30 sites in 56 restriction enzymes tested, and decreases 15, 1 and 27 sites, respectively. BstE II (nt 2810) and Pvu I (nt 2013) were the peculiar sites of LJ strain. Hind III, Pst I and Sac I sites were identical among the four strains. After structural gene (nt 745-2993) of HM175 strain was replaced by LJ strain, the complete hepatitis A virus cDNA open reading frame was inserted into pJSA1175 (vaccinia virus expression vector) downstream of promoter 7.5 k. Hepatitis A virus antigen expressed was 1:16 in titer by sandwich ELISA. Band-patterns of anti-VP0, anti-VP1 and anti-VP1, 2, 3, were as same as that of HM175 strain and natural hepatitis A virus antigen by Western blot analysis.
