Desenvolvimento e caracterização da proteína recombinante ORF2 do vírus da hepatite e genótipo 3

Abstract

Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Since HEV discovery 30 years ago, many cases of waterborne epidemics have been attributed to this virus. These outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with infected fecal material. Hepatitis E is also reported in many industrialized countries probably due to consumption of HEV-positive swine meat or to travelers returning from endemic regions. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV gt3. Viral RNA was isolated from swine feces infected with the native virus. 267 residues from the C-terminal ORF2(394-661) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. The amino acid sequence cloned was aligned against other human or swine HEV gt3 ORF2 sequences, revealing a 98% homology. The expression of ORF2p recombinant protein was achieved with 0.4mM of IPTG and purified by protein liquid chromatography. The method of expression used to produce ORF2p was highly efficient, yielding 10mg of soluble protein per liter of E. coli medium. Wistar rats were inoculated with ORF2p to test its immunogenic properties. In parallel, HEV-negative swine were experimentally infected with HEV gt3. The study of ORF2p immunogenic capacity in rats resulted in a high antibody titration able to recognize both the homologous antigen and the native HEV gt3 ORF2 present in infected stool. Furthermore, anti-HEV antibodies from Cynomolgus monkey recognized ORF2p recombinant protein, underlying its antigenicity. None of the infected piglets showed clinical signs compatible with the disease during the experiment or macroscopic alterations during the necropsy. Despite the overall healthy clinical picture, HEV was detected by PCR 14 days post-infection in 3/4 piglets feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p. According to our results, ORF2p is a valid candidate for the development of diagnostic tools to detect HEV gt3 circulating in Brazil as well as for the design of a vaccine targeting this specific genotype