Hypervariable Region 1 Inhibits Cross-neutralizing Antibodies' Production Induced By Hepatitis C Virus Envelope Protein 2

Hepatitis C virus (HCV), the only member of the hepacivirus genus, family Flaviviridae, is a major cause of posttransfusional non-A and non-B hepatitis. HCV infects more than 170 million people worldwide, among which 40 million are in China. About 85% patients fail to clear HCV and contract persistent infection which frequently leads to chronic liver disease, including cirrhosis and hepatocellular carcinoma. The current therapy for HCV infection is peg-IFNαin combination with ribavirin, but not all genotypies of HCV infected patients could receive the sustained viral response. Therefore, what needs dealing with urgently is developing vaccines to protect healthy persons as well as clear HCV from infected patients. Unfortunately, there is no effective vaccine available until now.HCV E2 envelope protein extends from aa 384-746 of the polyprotein, containing a N-terminal ectodomain and a C-terminal hydrophobic anchor domain. E1 and E2, is thought to form a heterodimer stabilized by non-covalent interactions as envelope of the HCV particles. As an envelope protein, E2 is thought to play a major role in virus attachment to the target cell by binding to receptors on the cell plasma membrane followed by membrane fusion and entry. Meanwhile, as E2 may elicit production of neutralizing antibodies against the virus, it was considered a major candidate for anti-HCV vaccine development. However, the most variable region of HCV (HVR-1) is also located within the N-terminal 27 residues (aa 384-411) of E2, it is also widely recognized as an epitope to which the neutralizing antibody binds. The high variation of HVR1 was considered to be a bottleneck in HCV vaccine development. However, in recent years, virus neutralizing tests based on HCV pseudoparticles (HCVpp) and HCV cell culture model (HCVcc) found that HVR1 is not the only neutralizing epitope and parts of E2 other than HVR1 might be involved in the induction of virus neutralizing antibodies.In our previous research, we constructed some eukaryotic plasmids to express the carboxyl-terminal truncated E2 protein with or without HVR1. Then these plasmids were used to immune mice and the relationship between HVR1 antibody and the total E2 antibody on amount and neutralizing activities were analyzed. What we found was 1. Secretionary expression of E2 is more effective to induce antibody response. 2. In addition to HVR1, there exist exactly other neutralizing epitopes in E2 protein. 3. anti-HVR1 is the main part of the neutralizing antibodies. 4. HVR1 attenuates the cross-reaction of antibody induced by E2. These results implies us whether we can enhance the immunogenicity of E2 by deleting HVR1 which might cover other conserved neutralizing epitopes existing in other regions of E2? If this alteration of E2 could induce effective cross-neutralizing humoral immune response, it could provide us a new strategy on developing HCV vaccine. However, DNA immunization might be interfered with confounding factors induced by other elements exist on the expression vector, which could mislead us. Protein vaccine using purified proteins as immunogens, can avoid these problems. Therefore, from this perspective, in this research, we constructed soluble E2 protein and soluble HVR1-deleting E2 expression plasmids, transfected into CHO cells and establish the E2 proteins'expression system. Based on affinity chromatography, E2 proteins were separated and purified from cell cultural supernatant, their conformation, function and immunogenicity was investigated and compared with each other. Our study was aimed to confirm the conclusion obtained from DNA immunization by E2 protein immunization and further explore the potential use of HVR1-deleting E2 protein in protective HCV vaccine.1. Construction of HCV E2-human IgG Fc fusion protein eukaryotic expression systemIn this part of our research, first, we constructed HCV E2-Fc/E2Δ-Fc fusion protein expression plasmids. This kind of fusion strategy was convenient for affinity chromatography of proteins. Next, we inserted glutamine synthetase gene into the downstream of target gene, by adding glutamine synthetase inhibitor (MSX) into the cell culture medium, recombinant CHO cell lines with high expression levels of fusion proteins could be selected. On the other hand, glutamine can be produced by cell metabolism of amines glutamine, exhibiting better growth and proliferation in the medium without glutamine; this might avoid cell injury induced by accumulation of ammonia, and greatly facilitate the follow-up of cell culture and protein purification. Then, these plasmids were transfected into HEK 293T cells to detect the expression levels of E2-Fc/E2Δ-Fc proteins in the supernatant based on ELISA and Western blotting. Results showed that these two proteins were expressed well and had got similar levels. After that, these two plasmids as well as another negative control plasmid- GS-EGFP were transfected into CHO cells respectively. Recombinant CHO cell clones were screened out under the pressure of MSX, these clones were then picked out and undertaken limited dilution to select clones with higher expression levels of target proteins. In order to improve space and medium utilization as well as reduce the mixed composition in FBS containing cell culture supernatant, serum-free protein-free medium adaptation were performed on these adherent cell lines, FBS concentration of cultural medium was decreased step by step, cells were transferred to suspension state instead. After adaptation, the suspension cell culture system was further enlarged, through 75T to 250ml flask with the small amount of amplification, and finally achieves the perfusion culture in 2L cellbags on Wave Bioreactor EH2/10 cell culture system. Cell density increased from 2×105cells/ml to the final 2×107cells/ml, cell viability was maintained above 80% and the secretion of protein was also stable at 3-4mg/L after perfusion. This part of our study has provided sufficient material for the following protein function analysis. Of course, the way to improve serum protein-free adaption efficiency and optimize the process of cell culture system enlargement still needs further exploration.2. Purification and identification of E2-Fc fusion proteinAs an intermediate steps between protein expression and function analysis, the separation and purification of target proteins play an important role. In this part of the experiment, we used the cultural supernatant collected from HCV E2-Fc fusion protein secreting suspension cell as raw materials, through the clarification step to remove most of the impurities, followed by concentration step based on ultrafiltration system, QuixStand? Benchtop System and hollow fiber ultrafiltration column, to reach a 10 times concentration and remove the molecules less than 30KD. Then, the ultrafiltrate was further purified with protein A affinity column HiTrap? Protein A HP Columns and purification system-?KTApurifier System (GE). During the purification process, through optimizing the column pressure, elution conditions collection, about 3-4mg of pure protein could be obtained from per liter culture supernatant, comparable with the reported E2-Fc fusion protein purification efficiency (2mg/L). Then, we exchanged the buffer of crude protein sample to PBS with ultrafiltration centrifuge column by way of low-temperature low-speed centrifugation, and further enrichment of the samples (3-5 times). Finally, we detected the concentration of protein samples by BCA protein quantitative method, and performed native-PAGE and Western blotting to confirm the conformation of protein samples maintain correct after ultrafiltration, purification, buffer exchange and other which can be used the following function analysis and animal experiments. At the same time, two crude purified proteins were undertaken glycosylation by glycosidase EndoH, PNGase F treatment, results suggest that glycosylation of both crude purified protein remained similar with their natural state. ELISA binding assay and pull down assays showed that E2-Fc, E2Δ-Fc could bind with human CD81 LEL, E2Δ-Fc exhibited higher binding activity than E2-Fc. Then, through flow cytometry, we investigated that both proteins can bind to HCV susceptible cell Huh7.5 cells well and had no much difference with each other. Further, we examined the binding activity between crude protein and CHO cells with expression of human CD81, SR-BI molecules respectively. Results showed that HVR1 deletion, binding efficiency between E2 protein and CHO-SR-BI was greatly decreased but slightly enhanced when binding with CHO-CD81. Next, we performed immunoprecipitation with lysis of Huh7.5 cells after E2 protein binding process to detect molecules that mediated E2 protein and cell binding. As a result, E2-Fc were able to precipitate CD81 and SR-BI, but E2Δ-Fc could co-precipitate little SR-BI and more CD81 when comparing with E2-Fc. This was consistent with report that HVR1 is important in mediating E2 and cell binding by affinity with SR-BI on target cells. Through infection assays of HCVcc and HCVpp, we observed concentration-dependent efficiency of crude pure fusion protein in inhibition of HCVcc and HCVpp infection. About 25-30μg of protein could be sufficient to completely suppress the infection of HCV infectious particles exhibiting no much strain specificity.3. Characteraztion of immunogenecity enhacement by HCV HVR1 deletion in purified E2-Fc fusion proteinThis section, we immunized BALB/c mice with crude purified proteins. Through the strategy of immunization for 3 times at week 0, 3 and 8 with a dose of 10μg once, serum was collected at week 2, 7 and 10 for anti-HVR1 and anti-E2ΔHVR1 antibody detection. In E2-Fc immunizing group, 5 in 10 were anti-HVR1 and anti-E2ΔHVR1 positive, another 4 were anti-E2ΔHVR1 positive only. It was supposed that Fc segment fused with the E2 protein affected the HVR1 epitopes exposure. Similar with results of DNA immunization, E2Δ-Fc protein induced higher anti-E2Δantibody levels. With the same quantity of total IgG, anti-HVR1 positive serum exhibted weaker neutralizing activitiy against the infection of J6/JFH1 HCVcc and Con1 HCVpp, but no much different from anti-HVR1 negative serum in inhibiting the infection of H77 HCVpp. These results confirmed the conclusions that anti-HVR1 had got strong neutralizing activity against the attack of HCV comes from the same strain but weak cross-neutralizing activity against the virus of other strains or genotypes.In summary, we have established a eukaryotic cell culture system to produce secretory HCV E2-Fc and E2Δ-Fc fusion protein and purified the target proteins. Based on E2-Fc and E2Δ-Fc, we investigated the roles of HVR1 in structure, function and immunogenicity of E2 protein. No obvious conformational changes were found in HVR1-deleting E2 protein. Although maintaining major function of E2 protein, HVR1-deleting E2 does decrease affinity with SR-BI, suggesting HVR1 be important in E2 binding with SR-BI. In terms of the immunogenicity, HVR1-deleting E2 protein induced higher cross-neutralizing antibodies protecting Huh7.5 cells from HCVpp and HCVcc infection in vitro. These results indicated protective humoral immune response could be induced by HVR1-deleting E2 protein, which provide a promising approach for the development of prophylactic vaccines against HCV infection.