Modulation Of Immune Response By Hepatitis C Virus E2 N-glycosylation And Interactions Between E2 And Molecular Chaperone

Hepatitis C virus genome encodes two membrane-associated envelope glycoproteins (El and E2). Both El and E2 are heavily modified by N-linked glycosylation. Glycans on the glycoprotein are the target molecules, which are recognized and bound by cells. There is little doubt that glycans have many protective, stablizing conformation of virus and barrier function. They also impact T cells functions, humoral immune, modulate humoral immune and cellular immune responses. Most immune molecules whose synthesis, folding, maturing, processing, locating, translocation are affected by N-glycans. However, the effect of N-linked glycosylation on immunological responses to HCV infection is largely unknown. In order to address this question, two single and one double N-glycosylation HCV E2 mutants(N177NT, N193ST, N177NT/N193ST) were constructed and studied. In these E2 mutants, the Asn codon was replaced by a Tyr codon in its glycosylation sites (Asn-X-Thr/Ser).We used recombinant DNA of wild type E2 and N-glycosylation site mutants E2 as DNA vaccines in our research. The common problem exsisted in DNA vaccine research is the limitation of lasting time in observed objects. It's usually to inject DNA plasmid repeatly or do injection accompany with adjuvant, such as IL-2. DNA (100ug/100ul/each) of wild type and mutant HCV envelope glycoprotein E2 had been injected into BalB/C mice accompany with IL-2 or not, Group N.S is as blank and vector pcDNA3.l as negative control. We injected mice for three times in tibias muscles with ten days intervals. According to some published papers, anti-HCV envelope protein E2 immunity in mouse reached the climax 50 days after the first immunization. We used indirect ELISA method to detect the secretion and titers of specific antibodies. After the anti-coagulant processing by heparin, the collected vein blood samples were measured by flow cytometer for the change of CD4+, CD8+ Tlymphocyte in immunized mouse peripheral blood. We collected spleens of mice and harvested supernatant of splenocytes sitmulated by ConA(10mg/ml) for 72 hours. The sectretion of IFN-y and IL-4 was determined by the ELISA kits. In order to preliminarily explore the relationship and interactions between E2 protein glycosylation and molecular chaperone calnexin(CNX), we constructed both wild type and several mutants together with CNX into eukaryotic expression vector and co-transfected them into Hela cells. The interactions between E2 and CNX were investigated by confocal laser microscopy.Results showed that we have constructed two single site and one double sites N-glycosylation HCV E2 mutants successfully.Wild type E2 DNA was ligated into prokaryotic vector pGEX-KG and wild type/mutant E2 into eukaryotic vector pcDNA3.1(-)/Myc-His B. The results indicated that wild-type E2 could significantly stimulate humoral and cellular immune responses(antibody liters: 1:800-1:100), while the E2 mutants (N193ST) significantly decreased mice humoral response in terms of both seroconversion rates and antibody productions. It was found that both wild type and the N177NT mutant and double sites mutant N177NT/N193ST of E2 stimulated the increasing of the CD4+T cells. Meanwhile, the glycosylation site N193ST mutant gave a much weaker CD4+ T cells cellular response than that of the E2 wild type. There is no significant difference between the results from CD8+T cells quantities. The secretion of IFN-y was greatly increased and there were no significant changes of group N193 ST. From cotransfection results of E2 and CNX, it showed that E2 could co-localize with CNX in ER.These results suggested that glycosylation of the E2 protein play significant roles in immune responses. We can conclude that N193ST is a very important antigen epitope recognized by B cells and CD4+T cells. Maybe the change of its structure affects E2 humoral immune response and cellular immune response: only a little change will decrease specific antibody level and only a little effects on CD4"IT cells level and there is slight decrease in IFN-y secretion compared with other...