ABSTRACT
<p><b>BACKGROUND</b>Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles (VLPs) have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris (P. pastoris) expression system for production of dengue virus type 1 (DENV-1) VLPs and evaluate the humoral and cellular immune response of this particle in mice.</p><p><b>METHODS</b>A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response.</p><p><b>RESULTS</b>Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4.</p><p><b>CONCLUSIONS</b>P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.</p>
Subject(s)
Animals , Male , Mice , Antibodies, Neutralizing , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Dengue Virus , Genetics , Allergy and Immunology , Metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Mice, Inbred BALB C , Pichia , Genetics , Metabolism , T-Lymphocytes , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To analyse the relationships among HBV genotypes, HBV DNA levels and histopathological features of the livers of familial grouped hepatitis B patients in the Tianjin area.</p><p><b>METHODS</b>One hundred familial grouped hepatitis B (CHB) patients were enrolled in this study. Thirty-five of the 100 patients were chronic asymptomatic HBsAg carriers (ASC) and 65/100 were mild CHB patients. Their HBV genotypes and HBV-DNA levels were detected and liver biopsies were performed for analyzing the pathological features.</p><p><b>RESULTS</b>Seven patients were of HBV genotype B (7%), and most of them had a HBV DNA level in the middle 10(3-5) copies/ml (57.14%). The histopathological features of the livers were of different degrees of injury. Eleven patients HBV was of genotype BC (11%); their HBV DNA levels were from 10(3-5) copies/ml (45.45%) to 10(6-7)copies/ml (36.36%). Their liver pathology showed slight or severe injuries (< or = G2 90.91%, < or = S(2) 81.82%). Eighty-two patients HBV was of genotype C (82%), and among the 82, 29 were ASC and 53 were CHB. Among the ASC, most of them had a high HBV DNA level (72.41%), and all of them had different degrees of liver injury. Among the CHB, their HBV DNA levels were 10(6-7) copies/ml (39.62%) and more than or equal 10(8) copies/ml (49.06%). The liver histopathological features were > or = G(2) in 38 patients (71.70%), and > or = S(2) in 25 patients (47.17%).</p><p><b>CONCLUSIONS</b>(1) In the majority, HBV of the family gathered hepatitis B patients living in Tianjin is of genotype C and they have a high HBV-DNA level and severe liver pathological injuries. These features of the family gathered hepatitis B patients are the main factors causing the unfavorable prognosis of the patients. (2) There is inflammation of different degrees in the livers and high HBV DNA levels in all the family gathered ASC patients. Antiviral therapy should be planned according to the pathological features in patients livers. (3) Liver biopsies should be performed routinely before their antiviral therapy.</p>