OBJECTIVETo investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

METHODSTwenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

RESULTSImmunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

CONCLUSIONHBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication

OBJECTIVETo investigate the genetic variation and typing of hepatitis B virus (HBV) in patients with chronic hepatitis B in relation to HBeAg status.

METHODSFluorescence quantitative polymerase chain reaction (PCR) was employed to detect serum HBV DNA in patients with chronic hepatitis B negative for HBeAg. Real-time fluorescent PCR and PCR-reverse dot blot hybridization were used to detect HBV genotypes and mutations, respectively.

RESULTSOf the 389 patients, 214 (55.01%) were positive and 175 (44.99%) were negative for HBV DNA; 102 (26.22%) had a HBV DNA copy number of 1×10(3), and 41 (10.54%) had a copy number of 1×10(4) (Χ(2)=226.6729, P<0.001). Of the 21 patients with a HBV DNA load of 1×10(5), 15 (71.43%) were found to have precore mutations, and 11 (52.38%) had basic core promoter (BCP) mutations; a higher HBV-DNA load was associated with an increased incidence of HBV mutations. In the 214 patients positive for HBV DNA, HBV genotypes A, B, C, D and the mixed type were found in 6 (2.80%), 84 (39.25%), 106 (49.53%), and 7 (3.27%), and 11 (5.14%) patients, who showed precore mutation rates of 16.67% (1 case), 36.90% (31 cases), 44.34% (47 cases), 0, and 0, and BCP mutation rates of 0, 19.05% ( 16 cases), 26.42% (28 cases), 0, and 0, respectively, demonstrating significant differences in HBV mutations between the genotype groups (P<0.001).

CONCLUSIONHBeAg-negative and HBV DNA-positive patients with chronic hepatitis B have a relatively low HBV replication level, and HBV DNA load is associated with HBV mutations. The B and C genotypes are more likely to have HBV mutations in HBeAg-negative patients.

DNA, Viral ; blood ; Female ; Genotype ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; classification ; genetics ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Male ; Mutation ; Promoter Regions, Genetic ; Viral Core Proteins ; genetics ; Viral Load

Adult ; Asian Continental Ancestry Group ; DNA, Viral ; blood ; Female ; Genes, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Protein Isoforms ; genetics ; Viral Core Proteins ; genetics

BACKGROUND: Hepatitis C virus (HCV) core antigen (Ag) levels are known to be well correlating with HCV RNA levels, and may be used as an alternative marker of HCV replication for monitoring the response to HCV treatment. However, the low sensitivity of HCV core Ag assay has been an obstacle for clinical use. In this study, recently developed ARCHITECT HCV Ag assay (Abbott Laboratories, USA) was evaluated for analytical performance and clinical usefulness. METHODS: A total of 109 sera from HCV infected patients including various genotypes of HCV (1b, 2, 2a/2c, 2b, and 3a) and 20 sera from healthy donors were used for evaluating the sensitivity, precision, and linearity of the HCV core Ag assay. The cross reactivity with HIV, hepatitis B virus and myeloma proteins (N=5, each) and correlation with HCV RNA PCR assay were also evaluated. RESULTS: The sensitivity of the HCV core Ag assay was 97.2% (106/109) and there were no false positive results and cross reactivity. The within-run, between-run and between-day CVs were 3.0%, 2.5% and 3.0%, respectively. The levels of HCV core antigen showed a good correlation with those of HCV RNA quantification (r=0.940). The HCV Ag assay showed an excellent linearity in the range from 0.63 to 17,114 fmol/L (r=0.999). CONCLUSIONS: The ARCHITECT HCV Ag assay was good in sensitivity, precision, and linearity and its results well correlated with HCV RNA levels. This assay could be used as a good marker of viral replication for monitoring the therapy response in chronically HCV infected patients.
Chemiluminescent Measurements/*methods ; Cross Reactions ; Genotype ; Hepacivirus/genetics/*immunology ; Hepatitis Antigens/*blood ; Humans ; Polymerase Chain Reaction/*methods ; RNA, Viral/blood ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Viral Core Proteins/*blood

OBJECTIVETo investigate the value of detection of hepatitis C virus core antigen (HCV-cAg) for screening blood donor by using the internal reagent enzyme immunoassay (EIA) and anti-HCV antibody.

METHODSThe first and repeat assays were performed for detection of serum anti-HCV and HCV-cAg ELISA in 3972 donor's serum specimens from August to October of 2004. Twenty-five donors positive for anti-HCV were tested with HCV-cAg EIA kits and the results were compared with the results of HCV RNA determination with RT-PCR method.

RESULTSIn 3972 donor's serum samples, only 1 serum specimen was positive for HCV RNA identification among 10 specimens which were positive for anti-HCV in first assays, and only 1 serum specimens was positive for HCV RNA identification among 12 specimens positive for anti-HCV in repeat assays, only 2 serum specimens were positive HCV RNA identification in 3 specimens which were positive for HCV-cAg assays.

CONCLUSIONThe sensitivity of HCV-cAg ELISA is similar to HCV RT-PCR, but it is much cheaper. Therefore, HCV-cAg ELISA and anti-HCV may be used together to screen blood donor.

Blood Donors ; Enzyme-Linked Immunosorbent Assay ; Hepatitis C Antibodies ; blood ; Hepatitis C Antigens ; blood ; Humans ; RNA, Viral ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Core Proteins ; blood

OBJECTIVETo assess the prevalence of serum anti-F in patients with hepatitis C virus (HCV) infection and the distribution of anti-F.

METHODSThe recombinant protein (HCV-F/GST) was coated onto micro titer plates as antigen. Sera of 120 patients with hepatitis C virus infection, 15 patients with hepatitis B, 3 patients with hepatitis E and 10 normal sera were tested by indirect ELISA for detecting anti-F.

RESULTS82 samples out of the 120 (68%) HCV infected patients exhibited a positive anti-F reaction, showing significant difference from the controls with no HCV infection (P < 0.01). Data from logistic analysis showed that the positive rate of anti-F was higher in patients over 50 year olds (OR = 6.675, 95% CI: 2.407-19.071). Patients of midrange, severe phase and hepatic cirrhosis had higher rate than the others (OR = 2.749, 95% CI: 1.470-5.141).

CONCLUSIONPrevalence and distribution of anti-F in Yixing hepatitis C patients was reported and which might be related to the progression of HCV infection.

Adult ; China ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepacivirus ; immunology ; Hepatitis Antibodies ; blood ; Hepatitis C ; immunology ; Hepatitis C Antigens ; immunology ; Humans ; Male ; Middle Aged ; Prevalence ; Viral Core Proteins ; immunology ; Young Adult

This study was purposed to investigate the feasibility to screen donor with HCV infection by means of HCV-cAg ELISA. The first and repeat assays were performed for detection of serum anti-HCV in 8677 donor's serum specimens from January 2003 to December 2005. All serum anti-HCV specimens with positive anti-HCV from first and repeat assays were finally identified by using HCV-cAg ELISA and HCV RT-PCR methods. The results showed that only 5 serum specimens were positive anti-HCV by HCV-cAg ELISA identification in 29 specimens including 15 specimens with positive ant-HCV in first assays and 14 specimens with positive anti-HCV in repeat assays, the positive rate detected by HCV cAg ELISA was 17.24%. 5 serum specimens were positive anti-HCV by HCV RT-PCR detection also in 29 specimens mentioned above, the positive rate detected by HCV RT-PCR was 17.24% too. It is concluded that sensitivity of HCVcAg ELISA is similar to HCV RT-PCR and may be useful for the early diagnosis of hepatitic C or used as a reliable method to screen donor with HCV infection in blood transfusion medicine.
Blood Donors ; Blood Transfusion ; Enzyme-Linked Immunosorbent Assay ; methods ; Hepacivirus ; isolation & purification ; Hepatitis C Antigens ; blood ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Core Proteins ; blood

OBJECTIVETo examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients.

METHODSEleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose.

RESULTSAfter recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000.

CONCLUSIONOur expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.

Animals ; Antibodies, Viral ; blood ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; blood ; epidemiology ; immunology ; Hepatitis C Antigens ; blood ; immunology ; Humans ; Prevalence ; Rabbits ; Viral Core Proteins ; blood ; immunology ; Viral Envelope Proteins ; immunology

OBJECTIVE: To evaluate the clinical significance of serum hepatitis C virus (HCV) core antigen detected by enzyme linked immunosorbent assay (ELISA). METHODS: The serum HCV core antigen, which was taken from 149 patients with chronic hepatitis C, 20 patients of chronic hepatitis B and 20 health volunteers, was detected by ELISA. Meanwhile, the serum HCV RNA was detected by RT-PCR, and anti-HCV was detected by ELISA. RESULTS: The qualitative HCV core antigen in the serum, which was take from 20 patients of chronic hepatitis B and 20 health volunteers, was negative.The positive percentage of HCV core antigen was 49.66% in the 149 sera of patients with chronic hepatitis C. The coincidence of detective results of HCV RNA and HCV core antigen was 54.36%, without significant difference (P>0.05). The positive percentage of HCV RNA and HCV core antigen in the 149 anti-HCV antibody positive sera samples were 55.03% (82/149) and 49.66% (74/149), respectively, and there was no significant difference (P>0.05). CONCLUSION The qualitative HCV core antigen detected by ELISA has a high specificity. The positive percentage of HCV core antigen in the serum of patients with chronic hepatitis C is 49.66%. HCV core antigen is related to HCV RNA. HCV core antigen may be a useful serum marker which could show HCV viraemia like HCV RNA.
Hepatitis C Antigens ; blood ; Hepatitis C, Chronic ; blood ; Humans ; RNA, Viral ; blood ; Viral Core Proteins ; blood

Hepacivirus ; classification ; genetics ; Hepatitis C ; diagnosis ; Hepatitis C Antibodies ; blood ; Humans ; RNA, Viral ; blood ; Viral Core Proteins ; blood