Prokaryotic Expression Of HAV 3C Proteinase And The Application For Its Antigenicity

Current diagnostic assays, such as the Abbott HAVAB test, used to determine exposure to HAV detect antibodies only to the structural proteins and as a result are not able to distinguish between a natural infection and vaccination with an inactivated virus. Hepatitis A virus (HAV) natural infection can stimulate the production of antibodies to structural and nonstructural proteins of the virus, while vaccination with an inactivated vaccine produces antibodies exclusively to the structural proteins and the level of these antibodies induced by attenuated live vaccine were limited to a certain one. Therefore, an ELISA was developed that is specific for antibodies to the nonstructural protein of HAV and thus serves to document the occurrence of viral replication. It is conformed that antibodies to 3Cpro (a viral nonstructural protein) was detected in the serum of all primates experimentally infected with virulent HAV and in the serum of naturally infected humans. The 3Cpro is the only nonstructural protein of HAV to date that has been expressed from a recombinant vector to yield a functional protein. The recombinant 3Cpro was chosen for the ELISA because it can be recovered with a high yield from Escherichia coli as an Inclusion-Body protein that can be purified by standard biochemical methods.The 3Cpro gene of HAV was cloned into a multicopy expression vector with a fragment of Thio following the T7 promoter which controls the expression of the insertion element in Escherichia coli.The resulting plasmid construction produced a fusion protein containing 3Cpro and Thio as an inclusion-body constituting above 40% of total cellular proteins. The protein was purified by an anion-exchange chromatography named DEAE following an affinity chromatography and was certified by SDS-PAGE to apparent homogeneity.In this study, we determined whether the ELISA could detect anti-3C IgM in sera from acute-phase patients that had been experimentally and clinically identified as a natural infection and compared the results with those from the children that had been immunized with an attenuated live vaccine before long.Our data indicate that the ELISA using HAV 3Cpro as the antigen can accurately detect antibodies to a nonstructural protein and therefore distinguish an immune response to a natural infection from vaccination with an attenuated live vaccine, let alone with an inactive vaccine. In addition, the ELISA appears to provide a useful and simple method for the detection of limited replication in cases in which virus is not excreted to detectable levels and disease does not occur and thus are of utility of the diagnosis of Hepatitis A in situations in which vaccination is widespread. This assay may facilitate a better understanding of the role of the nonstructural antibodies in the immune response to HAV infection.