title: Evaluation of antigenicity and cell mediated immunity of hepatitis E virus patients: using non radioactive MTT assay.
abstract: Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [3 H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher ( P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.
authors.affiliation: Department of Virology, PGIMER, Chandigarh, India.
authors.forename: M
authors.initials: M
authors.lastname: Majumdar
authors.forename: R
authors.initials: R
authors.lastname: Ratho
authors.forename: Y
authors.initials: Y
authors.lastname: Chawla
authors.forename: M P
authors.initials: MP
authors.lastname: Singh
mesh_terms: D000328:Adult; D000956:Antigens, Viral / Q000276:immunology*; D049109:Cell Proliferation; D003584:Cytological Techniques / Q000379:methods*; D005260:Female; D016751:Hepatitis E / Q000276:immunology*; D016752:Hepatitis E virus / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014764:Viral Proteins / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; C400971:ORF2 protein, Hepatitis E virus; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D014764:Viral Proteins; C022616:thiazolyl blue