Application Of Small Interfering RNA In Anti-HCV Infection And Induction Of Antibody Response To HCV Envelope Protein 2 In Mice

Hepatitis C virus (HCV), the only member of the hepacivirus genus, family Flaviviridae, is a major cause of posttransfusional and non-A non-B hepatitis. HCV infects more than 170 million people worldwide, among which 40 million are in China. About 70% patients fail to clear the virus and contract persistent infection that frequently leads to chronic liver disease, including cirrhosis and hepatocellular carcinoma. The HCV genome is a positive-stranded 9.6 kb RNA molecule consisting of a single ORF, which is flanked by 5'and 3'UTR. The HCV ORF encodes a single large precursor polyprotein that is about 3000 aa in length which is cleaved by both cellular and viral proteases into structural and nonstructural proteins and is posttranslationally modified to produce at least ten different proteins: core, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.Detailed analyses of HCV have been hampered by the lacks of stable small animal model for a long time. In the recent two years, although the infectious HCV cell model has been established, it is restricted in only one genotype, which brought difficulties to HCV preventing and curing research. The current therapy for HCV infection is PegIFN-αin combination with ribavirin. Although the sustained viral response (SVR) rate for HCV genotype 2 and 3 is more than 75%, it is no more than 50% for HCV genotype 1, the most widespread type in the world. This treatment is expensive and has side effects. Since HCV was cloned, some European and American developed countries have been always trying to develop its vaccine, but so far yet, there is no effective vaccine available. Thus, the development of new therapies and effective vaccine for HCV infection is of great clinical and economic significance.1. Application of Small interfering RNA in anti-HCV infectionRNA interference (RNAi) is a phenomenon or mechanism that small double-stranded RNA (small interfering RNA, siRNA) induces homologous mRNA degradation and leads to gene silencing. Since it was found in 2000, RNAi has been used in functional genomics, gene therapy and antiviral research. Anti-viral effects of siRNAs have been demonstrated in many studies.The first step of cell entry for virus is to attach to the receptors on the target cell surface. In this step the virus envelope protein plays an important role. The exact mechanism of HCV cell entry is still not very clear. A series of conclusive evidences show that the HCV envelope glycoprotein 2 could bind to a variety of target cell surface receptors, among which CD81 is most important. CD81, also known as TAPA-1, is a member of the tetraspanin family, containing four transmembrane domains. It is a 26kD nonglycosylated cell surface protein and absolutely necessary for the HCV cell entry. So interfering with the event of E2 and CD81 binding could be used as an anti-HCV therapy. In this part, we chose HCV E2 protein and the receptor CD81 as the objects of study to explore the anti-HCV function of the siRNAs targeting these two genes.RNA interference of E2 gene: Firstly, we used a simple and direct-viewing method to screen effective siRNAs. We chose EGFP as reporter gene to construct a recombinant plasmid expressing EGFP and HCV E2 gene in fusion. According to the HCV sequence we designed and transcribed in vitro 2 E2 specific siRNAs. By transfecting the plasmid and siRNAs simultaneously into the HEK 293T cells and then observing the green fluorescence intensity by fluorescence microscope we can get initial judgment on the silencing effects of the siRNAs. Then flow cytometry, Western blot and FQ-PCR were used to prove the effectiveness of the siRNAs on protein and mRNA levels respectively. The two E2-specific siRNAs could inhibit E2 expression effectively, and E2-siRNA1 is more effective than E2-siRNA2. The results on different levels were consistent with each other. Then these two E2-specific siRNAs were transfected into HCV genomic replicon cells HepG2-HCV and 48h later HCV RNA levels were detected by FQ-PCR. The results showed that these two siRNAs could reduce the HCV level by 70.5% and 55.4% respectively.RNA interference of CD81 gene: Six siRNAs, four targeting open reading frame (ORF) and two targeting 3'NTR of CD81 mRNA were designed and in vitro transcribed to explore the possibility of silencing this gene in HEK 293T cells. The results showed that these six siRNAs could reduce CD81 expression to different extents, and two siRNAs targeting 3'NTR were most effective. Then these six siRNAs were introduced into human hepatoma cell line Huh7.5 cells, and the cell surface expression level of CD81 were examined by flow cytometry. The results showed that the six siRNAs could reduce the Huh7.5 cell surface expression of CD81 to different extents and the changing trends were entirely consistent with those obtained on HEK 293T cells. The two siRNAs targeting 3'NTR were most effective, which could decrease the Huh7.5 cell surface expression of CD81 by 77% and 82% respectively. Afterwards, the CD81 siRNA expression plasmid pGCsi-CD81 was constructed and transfected into Huh7.5 cells and then screened with G418. About 2-3 weeks later, resistant cell clones appeared. Individual clones were picked and cultured and then the cell surface expression of CD81 was analyzed by flow cytometry. The results showed that compared with the negative control (the pooled clones obtained after screening the Huh7.5 cells transfected with scramble siRNA expressing plasmid pGCsi-scramble), the cell surface expression of CD81 of all clones were reduced, but to different levels. The lowest one was reduced to 8%. After that, two cell clones with the CD81 expression levels mostly reduced were infected with HCV pseudoparticles (HCVpp) and the infection rates were examined by flow cytometry (count the green fluorescent cell population). The results showed that compared with the negative control, the infection rates of the cloned cells were decreased by 90% and 72% respectively. By drawing cell growth curves with MTT assay and analyzing the growth activity of the two cloned cells, we found that compared with the control cells, the cloned cells with CD81 stably silenced showed cell growth inhibition, and the lower the expression of CD81, the greater the degree of growth inhibition.In this part of experiments, the E2-specific siRNAs could inhibit HCV RNA replication and the CD81-specific siRNAs could inhibit HCVpp cell entry. All these prove that siRNAs whether targeting HCV genome or targeting host cell receptor - - could play a very good role in the anti-HCV therapy and siRNA-based antiviral therapy has a great prospect. In the RNAi study of eukaryotic cell genes, researchers often choose ORF as a target. While this study suggests that when interfering with the hCD81 gene 3 'NTR is also a good target. Moreover the obtained Huh7.5 cell lines with CD81 expression stably silenced will display a vital role in the CD81 biological function research and the study of HCV cell entry.2. Induction of antibody response to HCV envelope protein 2 in miceHCV envelope protein 2 (E2) is the key protein to mediate HCV infection and to induce antibody response. It is also the most important candidate in HCV vaccine development. The hypervariable region 1 (HVR1) which is 27 amino acid residues in length and resides in the amino-terminal of E2 protein is the most variable region in HCV proteins. 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 there are other conservative conformational and linear neutralizing epitopes in HCV E2 protein. In this study we constructed some eukaryotic plasmids to express the carboxyl-terminal truncated E2 protein with or without HVR1. Then the plasmids were used to immune mice and the relationship between HVR1 antibody and the total E2 antibody on amount and neutralizing activities were analyzed.Plasmids expressing different types of HCV E2 protein of H77 strain (genotype 1a) and J4 strain (genotype 1b) were constructed. They included plasmids E2746 which express HCV E2 protein in whole length, plasmids E2661 which express HCV E2 with carboxyl-terminal amino acid residues truncated from aa661 and plasmids E2661? which express HCV E2 with HVR1 truncated simultaneously. Plasmids E2661 and E2661? were fused with 6×histidine coding sequence in the carboxy-terminal of E2 gene. HEK 293T cells were transfected with these six plasmids and then the culture supernatant and cell lysate were analyzed by 6×His monoclonal antibody, E2 polyclonal antibody and E2 conformation-dependent monoclonal antibody. The results showed that the plasmids E2661 and E2661? expressed E2 proteins secretionarily and HVR1 did not affect the expression, secretion and spatial conformation of E2 protein. We relatively quantitated the E2 protein in the cell lysate and cell culture supernatant and then analyzed by ELISA the bindings between E2661 and E2661? of H77 and J4 strains and the fusion protein Trx (thioredoxin)-CD81 (LEL) (large extracellular loop)expressed by E. coli. The results showed that HVR1 did not affect the bindings between E2 protein and CD81 LEL; the affinity between the E2 in cell lysate and CD81 LEL is significantly higher than that between the E2 secreted into cell culture supernatant and CD81 LEL; the affinity between the E2 of H77 strain and CD81 LEL was significantly higher than that between the E2 of J4 strain and CD81 LEL. The plasmids pcDNA3.1-1a746, pcDNA3.1-1a661 and pcDNA3.1-1a661Δexpressing E2 protein of HCV H77 strain and the empty vector pcDNA3.1 were injected intramuscularly into BALB/c mice (six mice/group, 200μg/mouse /injection, inject once every 2 weeks, totally 3 times). Blood samples were collected regularly by retro-orbital puncture from immunized mice. The serum anti-E2 antibodies were analyzed using culture supernatants or lysates of the E2 expression plasmids transfected HEK 293T cells. The anti-HVR1 antibodies were analyzed by the Trx-HVR1 fusion protein expressed by E. coli. Results: only one in the 1a746 immunized mice become anti-E2 and anti-HVR1 positive. After the second immunization 5 in the 1a661 and 2 in the 1a661Δimmunized mice become anti-E2 positive and all the mice immunized by 1a661Δbecome anti-HVR1 positive. After the third immunization, 6 in the 1a661 and 3 in the 1a661Δimmunized mice become anti-E2 positive. The serum anti-E2 level in 1a661 group was significantly higher than that of 1a661Δgroup. In 1a661 group, the HVR1 antibody levels were not parallel with those of E2 antibody. In analyzing mouse serum antibodies, the results obtained by using 1a661Δor E2661 of J4 strain were similar to those obtained by using 1a746 or 1a661.From analyzing the neutralizing effects of mouse serum to HCVpp, we found that the neutralizing effects of the serum from the 1a661 immunized mice were not correlated to the total serum anti-E2 levels but showed a positive correlation with anti-HVR1. The neutralizing effects of the serum from the 1a661Δimmunized mice were almost parallel with the total serum anti-E2 levels, but for 1a661 and 1a661Δsera with similar anti-E2 levels, the former had a much stronger neutralizing effect than the latter. Because the CD81 expression level influences HCV infection efficiency, we observed the effects of the CD81 up-regulation of Huh7.5 cells on the neutralizing effects of mouse serum. We used lentivirus expressing CD81 to infect Huh7.5 and then analyzed the neutralizing effects of the sera on these cells. The results showed that the neutralizing effects of 1a661Δserum decreased significantly and the neutralizing effects of 1a661 serum decreased too but at a lower rate and overall the neutralizing effects were still parallel with anti-HVR1 levels.These results suggest :①HVR1 has no influence on HCV E2 protein expression, secretion and spatial conformation.②In addition to HVR1, there exist exactly other neutralizing epitopes in E2 protein.③Although anti-HVR1 is only a small part of the total anti-E2 in the sera immunized by secreted truncated E2 protein, it is the main part of the neutralizing antibodies. Comparing to the neutralizing antibodies targeting epitopes outside HVR1, the neutralizing effects of anti-HVR1 were not influenced by the CD81 expression level on target cells.④The neutralizing antibodies targeting epitopes outside HVR1 block HCVpp infection mainly through blocking the binding between E2 and CD81.⑤E2 proteins of H77 and J4 strains have many cross antigens.⑥For HCV E2 DNA vaccine secretionary expression is conducive to induce antibody response.⑦HVR1 not only could induce neutralizing antibody as a B-cell epitope, but also contains important helper T cell epitope, which could significantly enhance the E2 protein immunogenicity. Summary:1. We screened six effective HCV E2 or hCD81 gene specific siRNAs by using a simple and direct-viewing method. E2 specific siRNAs could effectively inhibit the HCV RNA replication in the HCV replicon cell line HepG2-HCV, while CD81 specific siRNAs could inhibit HCVpp cell entry. All of these results laid a foundation for RNA interference in anti-HCV therapy.2. We constructed plasmids expressing whole length or carboxy-terminal truncated HCV E2 protein with or without HVR1 and used them to immune mice and then analyzed the antibody response and the neutralizing effects of the serum to HCVpp. The results showed that in the total antibodies induced by the secretionarily expressed E2, anti-HVR1 played an important role, and the target cell surface expression of CD81 has very little influence on its neutralizing effects. The antibodies against epitopes outside HVR1 are superior to anti-HVR1 in quantity, but their neutralizing effects are inferior to anti-HVR1. In addition, HVR1 is not only a neutralizing epitope, but also an important helper T-cell epitope to enhance the immunogenicity of E2 protein. These results are of great theoretical significance for HCV vaccine development.