Study On Impact Of Hepatitis B Virus Gene Mutations On T Cell Immunological Response

Hepatitis B virus (HBV) infection can lead to a series of spectrums of liver diseases including asymptomatic carrier (AsC), chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). There are approximately 300 million chronic HBV carriers worldwide. In China, there are also about 110 million HBV carriers, out of which 30 million patients will show hepatitis B infection. Moreover, 300 thousand patients died of HBV related diseases in our country every year. Currently, there is no ideal drug to eliminate HBV completely from human body, therefore chronic HBV infection has been a significant health problem at present.HBV is a kind of DNA virus more likely to grow mutation than other virus, and for instance genotype and sub-genotype are the common patterns of HBV mutation. The sequence diversity between HBV genotypes is identified as more than 8% of full HBV genome. Moreover the diversity in geographic and racial distribution is also one of the most important characteristic for HBV genotype. Meanwhile, some site mutations impacting on the biological function of HBV have been known already.Epitope is the principal motive power which can activate specific cell immune response, and is reckoned on the core of immune recognition and activation. When the mutation site is just located in the epitope sequence, it will be possible to impact on the host responsibility to HBV. So far, most epitopes of HBV were identified by the researchers from Europe and America, where genotype A and D are mainly prevalent. For example, HBcAg18-27-FLPSDFFPSV (C18-27V), known as a classic HLA-A* 0201 restricted epitope, was identified from the prevalent HBV strains of in Europe, while the prevalent HBV strains in China are quite different from Europe. Because C18-271, instead of C18-27V, might be popular in China and it can lead to structural change of the HBcAg18-27 epitope. Respecting the diversity of genotype and sub-genotype, it will be very significant to identify new HBV epitopes based on the new virology background and to analyze the regularity of cell immune response with the new epitope system in China.Objective:To explore the molecular epidemiological characteristic and cell immune response diversity of HBcAg18-27V/I mutants in Chinese dominant genotypes strains; to study the characteristic of T cell response in HBV infected patients with overlapping peptides covering genotype B and C genome and to screen the available population to indentify the HBV specific epitope; to compare the difference of T cell responses in patients with genotype B and C, or with mutated and wild type group.Methods:1. Matched and compared 30 HBV sequences from GenBank and 20 HBV sequences sequenced by our lab through DNASIS, and analyzed the characteristics of C18-27V/I mutant distribution; analyzed the C gene region of genotype B and C, and then constructed a PCR-RFLP method to screen C18-27V/I mutants; compared the affinity of C18-27V to HLA molecules with C18-27I through bio-informatics software.2. Selected 160 blood samples randomly from the serum library for our study on national distribution of HBV genotypes; measured the samples with our PCR-RFLP method and analyzed the epidemiological characteristics of C18-27V/I mutant;3. Under the C18-27I background of our country, selected 57 inpatients with chronic hepatitis B in Nanfang hospital; took their blood and isolated PBMCs; measured the frequency and function to compare difference between C18-27I specific T lymphocyte and C18-27V's through tetramer staining, proliferation assay in vitro and ELISPOT assay, respectively; 4. For 57 chronic hepatitis B patients, measured the clinic testing parameter, such as ALT, TBil, HBV DNA; typed their HBV genotype; analyzed the relationship between frequency of C18-27V/I specific T cell and the parameters above;5. Based on the dominant prevalence of genotype B and C in China, designed the overlapping peptides referring to 20 HBV full sequences from GenBank; designed the mixtured peptides pools to test T cell response under the HBV virological background in China;6. Selected 38 inpatients with HBV infection in DID, Nanfang hospital, took blood and isolated their PBMCs; bleed 10 healthy donors as normal control; grouped patients into 5 parts, such as HBeAg-negative CHB, HBeAg-positive CHB, liver failure, liver cirrhosis and acute HBV infection; compared intergroup difference of IFN-γsecreting function through ELISPOT assay;7. Analyzed the ELISPOT results of 38 samples; compared the capabilities of inducing IFN-γsecreting among 10 peptide pools;8. Tested HBV genotype of 38 patients with HBV infection and analyzed difference of Tcell response between patients of the genotype B and the genotype C;9. Tested HBV pre-C mutation(nt1896) and BCP mutation (ntl762/1764) of 38 patients with HBV infection and analyzed difference of T cell response between patients with mutation and wild type strains;10. Collected clinic data of 38 patients, such as ALT, TBil, HBV DNA; typed; analyzed the relationship between T cell response and the parameters above;Results:1. Through matching HBV sequences from GenBank, HBV strains with C18-27I showed great higher prevalence than C18-27V in genotype B and C strains; however, C18-27V strains showed highly prevalent in other genotypes;2. A PCR-RFLP approach was successfully constructed and was convenient and available to screen C18-27V/I mutants in genotype B and C area;3. C18-27I prevalence showed 98.12% (157/160) in our investigation of 160 genotype B and C samples;4. Through C18-27V/I tetramer staining to compare frequency of C18-27V/I specific T cell respectively, it showed that the mean frequency of C18-27I specific T cell was more than that of C18-27V in periphery blood under the background of C18-27I patients(0.335±0.479% vs 0.221±0.392%, P=0.012);5. In proliferation assay induced by C18-27V/I peptide, both peptides can induce proliferation of C18-27I specific T cell (2-10folds), and totally C18-27I peptide showed higher capability than C18-27V, moreover, cross reaction of C18-27V peptide to C18-27I specific T cell was also confirmed through this assay;6. It showed negative result of IFN-γELISPOT ex vivo induced neither by C18-27V nor by C18-27I peptide in 12 HLA-A2~+ chronic hepatitis B patients;7. 282 overlapping synthetic peptides(18mers overlapped by 10 residues and spanned the whole HBV genome) were designed and 10 mixing peptide pools, with 22-34 peptides in each pool, were also designed and setup to classify different HBV genotypes and different gene regions as following: pool 1 for X gene region, pool 2 for C gene region, pool 3 to pool 5 for S gene region and pool 6 to pool 10 for P gene region;8. 38 inpatients with HBV infection in Nanfang hospital and 10 healthy donors grouped into 7 parts, including 12 HBeAg-negative CHB patients, 11 HBeAg-positive CHB patients, 5 acute liver failure patients, 8 liver cirrhosis-cancer patients, 2 acute HBV infection patients, 4 health donor with special HBV marker and 6 health control donors; the mean spot forming cell in group 7 was 2.208±3.924 SFC/well, and based on the G7 results, the positive cutoff value was identified as 10 SFCs;9. The difference of total ELISPOT spots(10 pools SFCs in sum) distribution among 7 groups(G1-G7) showed statistic significance (x~2=15.277, P=0.018) ; the difference of total ELISPOT spots distribution among 4 groups with chronic HBV infection(G1-G4) showed no statistic significance (x~2=6.5, P=0.088) ,but the number of positive wells among the 4 groups showed statistic significance (X~2=9.2, P=0.027) ; further comparison between G1 and G2, not only the difference in the number of positive wells, but also in the number of patients with positive well showed statistic significance; (z=-2.345, P=0.019; x~2=5.856, P=0.016)10. In the groups with HBV infection (G1-G5), there were 36 samples with serum HBV DNA quantifing results (36/38); it showed that the positive correlation between total SFCs and ALT (CC=0.352, P=0.030), while it showed no significant correlation between total SFCs and log DNA(CC=-0.114, P=0.495);11. Between 23 patients with genotype B and 9 patients with genotype C, the former showed higher total ELISPOT spots than the latter (z=-2.517, P=0.012) ; genotype B patients also showed higher SFCs than genotype C patients in S gene region specific pools(pool 3-pool 5)( z=-2.194, P=0.028), and it was the same tendency in P gene region specific pools(pool 6-pool 10) (z=-2.479, P=0.013); however, there was no significant difference in C gene region pool and X gene region pool (z=-1.635, P=0.102; z=-0.728, P=0.467) ;12. It showed that there was no statistic significance in the total SFCs and the C gene region pool SFCs between mutation group (pre-CA83 and BCP)and wild type group, but the number of SFCs in mutation group showed higher tendency than wild type group;Conclusion:1. C18-271 is the dominant virological background instead of C18-27V in China, and under this background, there are signifanct differences in the frequency and function of C18-27I specific T cell with C18-27V specific T cell in periphery blood; so if C18-27V is cited without being considered virologic background, it will result in some errors;2. The capability of T cell response in patients with genotype B is higher than genotype C, which suggests an important evidence to elucidate the mechanism why genotype B patients have a higher HBeAg seroconversion rate than genotype C and why genotype B patients have a better response to antiviral therapy than genotype C.