| Background:Hepatitis C virus(HCV) is the most common pathogen associated with post-transfusion hepatitis and sporadic hepatitis and constitutes a worldwide public health problem. Globally, there are approximately 170 million anti-HCV antibodies positive individuals who may develop serious consequences such as cirrhosis and liver cancer. The lack of an effective immune response to acute HCV infection leads to approximately 80% of patients developing chronic HCV infection, while only 20%spontaneously recover from the disease without sequelae. The non-neutralising antibodies detected in HCV patients are directed against the HCr43 protein(encoded by sequences within HCV non-structural region NS3 and the core coding region) and c100-3 antigen(encoded by sequences within HCV non-structural region NS3 and NS4).Most patients with HCV infection produce antibodies targeted against various viral proteins of HCV, and these antibodies can be detected in the serum. Therefore, different immunological antibodies and their level of production play important roles in the prognosis of chronic HCV infection.The status of the immune response to HCV infection cannot be accurately predicted by the HCV antibody test alone. In a previous study, a strong correlation was observed between the affinity of non-neutralising antibodies, such as anti-HBc antibodies, and prognosis in HBV infection. Another study showed that antibody affinity could accurately reflect the activity status of cytomegalovirus infection. Therefore, theevaluation of HCV antibody affinity could lead to an improved assessment of the immune response and provide valuable information for the diagnosis and prognosis of HCV-infected patients.Total antibody activity of HCV is determined by the antibody quantity and binding capacity for antigens(affinity). Currently, clinical laboratories only test HCV antibody total activity without evaluating the antibody quantity or affinity. In the present study,the commonly used HCV antibody total activity test system was used to establish an HCV antibody affinity test as previously described for the anti-HBc affinity test. We then examined the association between the total activity, quantity and affinity of anti-HCV antibodies and the viral load and hepatic inflammation level in HCV-infected patients, which contributes to understand the function of total activity, quantity and affinity of anti-HCV antibodies in disease progress.A large number of studies have examined the relationship between HLA alleles and HCV persistent infection and viral clearance in recent ten years. The immune responses against viruses rely on antigen-processing and antigen-presenting ability of antigen-presenting cells and recognizing ability to those antigen epitopes by immne cells. The immune function against HCV is regulated by the host’s HLA molecules.Therefore, the differences in HLA alleles may strongly influence the outcome of HCV infection. There exsits a strong correlation between HLA class I alleles and spontaneous prognosis, for instance, HLA-A3, HLA-B27 and HLA-Cw*01 are correlated with viral clearance while HLA-B8 is correlated with persistent infection. An epitope in the NS3 region shows high affinity for HLA-DR, indicating the possible role of HLA class II in virus clearance. HLA class II allels are connected with HCV infection prognosis.HLA-DQB1*0301 and HLA-DRB1*1101 are highly correlated with viral clearance.However, the results from these studies are confusing and even conflicting.Objective: Our paper aims to establish the method to detect affinity of anti-HCV antibodies based on multidimensional antibody data analysis and explore the dynamics changes of affinity, content and total activity of anti-HCV antibodies with viral load,body injury and different stages of HCV infection, which proposing a novelmeasurement of antibody affinity and providing a promising understanding into immune response to HCV. Our paper also aims to investigate the relationship between HLA-A,HLA-B, HLA-DRB1 alleles and hepatitis C disease process and the possible mechanism.Methods:1. The rabbits were immuned by subcutaneous injection of recombinant recombinant hepatitis B vaccine for three times. To collect the rabbits’ serum one week later since the first injection and the sesum contained low affinity antibody. To collect the rabbits’ serum two weeks later since the third injection and the sesum contained high affinity antibody. To determine the level of anti-HBs antibody in 2-fold diluted serum by double antigen sandwich ELISA. 2. Serum samples from 106 patients with HCV infection were collected. HCV antibodies were analyzed with chemiluminescence micro-particle immunoassay method, while PCR-fluorescent probe method was used to detect HCV-RNA. ALT, AST and ALB were also measured. The samples were divided into three groups based on ALT, AST level(both normal, either abnormal and both abnomal) and HCV-RNA level(negative, low viral load and high viral load).Differences in affinity, content and total activity of anti-HCV antibodies were analyzed.2. Serum samples from 328 patients with HCV infection were collected. HCV antibodies were analyzed with chemiluminescence micro-particle immunoassay method,while PCR-fluorescent probe method was used to detect HCV-RNA. ALB were also measured. Patients were classified into chronic hepatitis and liver cirrhosis groups. The former was further divided into two groups based on patients’ ALB & RNA level: ALB normal & RNA negative group(the recovery stage of HCV infection) and other group(the progressive stage of HCV infection). Differences in affinity, content and total activity of anti-HCV antibodies were analyzed. 3. Serum samples from 104 patients with HCV infection were collected. HCV antibodies HCV-RNA and ALB were measured as described in part one. DNA samples from HCV-infected patients were collected at random. Sequence based typing(PCR-SBT) was used to detect HLA-A,-B,-DRB1 alleles. The association between the HLA loci and disease status was further confirmed by cross-validation of the 2 sets of analytical results. Differences in affinity,content and total activity of anti-HCV antibodies were analyzed. 4. The interaction between HCV NS3 protein and HLA-DRB1*09,HLA-A*02,HLA-A*33,HLA-B*58,LA-DRB1*07,HLA-DRB1*13 were analyzed by combination of internet searching and Discovery Studio(DS)software. The internet databases include National Center for Biotechnology Information(The National Library of Medicine, NLM), Protein Data Bank(PDB) and PROSITE.Results:1. The antibody affinity of the female rabbit after the first immunization was0.522. The affinity of the second time was 0.826 and that of the third time was 0.896.The antibody affinity of the male rabbit after the first immunization was 0.816. The affinity of the second time was 0.846 and that of the third time was 0.972. The affinity was gradually increased, which was in accord with the regular pattern of antibody affinity maturation. 2. The patients in the group with normal levels of ALT and AST demonstrated significantly lower antibody content than that of the other two groups(p<0.05). Conversely, antibody affinity in this group was higher than that of the other two groups(p<0.05). There were no significant differences in total antibody activity between three groups(p>0.05). 3. The 53 patients with normal ALT and AST were further divided into two groups according to the HCV RNA level(HCV RNA-negative group and HCV RNA-positive group). The total antibody activity and antibody content were both significantly lower in the HCV RNA-negative group than in the HCV RNA-positive group(p<0.05). However, the antibody affinity in the HCV RNA-negative group was significantly higher than that in the HCV RNA-positive group(p<0.05). 4. Based on the results of 106 patients the total antibody activity and antibody content were significantly lower in the HCV RNA-negative group than in low viral load group and high viral load group(p<0.05), although the antibody affinity in RNA-negative group was significantly higher than that measured in low viral load group and high viral load group(p<0.05). 5. The relationship between dependent variable(positive HCV-RNA) and covariates(affinity, quantity and total activity) was analyzed using logistic regression. HCV-RNA level was negatively correlated with affinity(p <0.05). 6. Affinity in ALB normal & RNA negative group was significantlyhigher than that in other hepatitis and cirrhosis groups(p < 0.05) while content in ALB normal & RNA negative group was significantly lower than that in other hepatitis and cirrhosis groups(p < 0.05). Total activity was not significant different among ALB normal & RNA negative group, other hepatitis and cirrhosis groups(p > 0.05). 7. The area under ROC curve for affinity and content were 0.816 and 0.811 respectively,showing high diagnostic performance and higher than traditional antibody detection(the area under ROC curve was 0.581). The cut off value of affinity for diagnosing sustained HCV replication was 0.873. The higher the affinity was, the less the virus replicated. 8.High affinity(≥0.873) ratio of chronic hepatitis with ALB normal &RNA negative was significantly higher than other hepatitis and cirrhosis groups(p < 0.05). 9. The lgRNA level of HCV-RNA(+) serum was significantly decreased after neutrolized by high antibody affinity serum(p < 0.05). 10. 8 HLA-loci were identified to have significant differences between HCV patients and healthy control(p < 0.05). The frequencies of HLA-A*02, HLA-B*39, HLA-DRB1*09, HLA-DRB1*11 in HCV patients were lower than those in healthy control, whereas HLA-A*33, HLA-B*58, HLA-DRB1*07,HLA-DRB1*13 in HCV patients were higher than those in healthy control. 11. Of these8 HLA-loci, HLA-B*58 and HLA-DRB1*09 showed significant differences in ALB level between patients with and without the allele(p < 0.05). Only the p value of the cross-validation for HLA-DRB1*09 was less than 0.05, while others showed no significance. 12. The HLA-A gene frequency(high resolution) was positively related with antibody affinity, indicating that the higher the frequency was, the stronger the antibody affinity was. 13. Comparing the differences of affinity of anti-HCV antibodies in patients who carrying HLA-A*02,HLA-B*39,HLA-DRB1*09,HLA-DRB1*11,HLA-A*33,HLA-B*58,HLA-DRB1*07,HLA-DRB1*13 alleles, the ranking sequence was as following: HLA-A*33 > HLA-B*58 > HLA-DRB1*07 > HLA-A*02 >HLA-DRB1*09 > HLA-DRB1*13. HLA-B*39 and HLA-DRB1*11 were not analyzed due to the limited cases(the former had only 1 case and the latter had 4 cases). 14. The ranking sequence of affinity of anti-HCV antibodies to HCV NS3 analyzed from bioinformatics aspect was as following: HLA-B*58 > HLA-A*33 > HLA-DRB1*09 >HLA-DRB1*07 > HLA-A*02 > HLA-DRB1*13.Conclusions : 1. Successfully established the multidimensional antibody data analysis method of anti-HCV antibody. The affinity, content and total activity of anti-HCV antibodies may reflect hepatic cell injury and viral load in patients infected with HCV and could serve as valuable information for clinical applications. 2. The affinity of anti-HCV antibody is closely connected with the disease condition based on multidimensional antibody data analysis and it is better than traditional laboratories which only detect antibody total activity. The affinity of anti-HCV antibody may play an indicationg role in the prognosis of HCV infection with high diagnositic efficiency and determination of antibody affinity is helpful for doctors to predict the prognosis of HCV infection. 3. There was a connection between HLA polymorphism and the disease process of HCV infection and the affinity of anti-HCV antibody. The high the gene frequency is(more conservative), the more possibility the body produces high affinity antibody. |
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