Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus.

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na+-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF50: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.

Long-term HEV carriers without antibody seroconversion among eligible immunocompetent blood donors.

Hepatitis E virus (HEV) is emerging as a potential threat to the safety of blood transfusions. In many countries and regions endemic for HEV, such as China, blood donors are not routinely tested for HEV infection. In this study, 11747 eligible blood donors were screened for anti-HEV immunoglobulin M (IgM)/immunoglobulin G (IgG) and HEV RNA and antigen in China. Twenty-four donors who were positive for both HEV antigen and RNA were followed for ≥ 70 days, and none of these donors reported clinical hepatitis or illness. At least 1 follow-up sample was provided by 17 donors, including 10 with viremia and/or antigenemia for ≥ 70 days and 3 with antigen and RNA positivity for >90 days. Fourteen of the 17 donors did not present with an obvious serologic response during the follow-up period. These results differed from previous reports, in which viremia lasted for 68 days and elicited an antibody response. These donors showed atypical HEV infection progression that differed from that of hepatitis E patients. The presence of these donors presents a challenge for transfusion transmission screening.

[Prevalence of Parvovirus B19 Infection in Chinese Xiamen Area Blood Donors].

OBJECTIVE: To estimate the prevalence of parvovirus B19 infection in Chinese Xiamen area blood donors. METHODS: Blood samples from blood donors were tested for detection of parvovirus B19 DNA and antibody. The direct sequencing and genetype analysis of B19 DNA positive samples were performed. RESULTS: Six out of 10452 samples were B19 DNA positive. The viral loads of the 6 samples were between 3.59×102-1.07×104 IU/ml; the positive rate of B19-IgM was 4.64%(50/1078) and B19-IgG was 16.79%(181/1078). The positive rate of B19-IgG increased with ages, and was not related with the sex. CONCLUSION: The overall prevalence of parvovirus B19 infection in blood donors is lower in Chinese Xiamen area than that in other areas, however, there is still a certain percentage of viremia in donors and the attention should be paid to blood safety in the future work.

[Epidemiologic Survey of Blood Donors with HBsAg⁻/HBV DNA⁺ in Chinese Xiamen Area].

OBJECTIVE: To understand the characteristics of infections from blood donors with HBsAg⁻/HBV DNA⁺ in Xiamen area. METHODS: Donors in Xiamen area were assayed by routine ELISA and those with negative results were tested by nucleic acid amplification testing (NAT). HBsAg⁻/HBV DNA⁺ samples were tested by quantitative detection of HBV DNA. Epidemiological analysis and following up examination were conducted in HBsAg⁻/HBV DNA⁺ donors. RESULTS: Out of 130659 samples 113 were tested as HBsAg⁻/HBV DNA⁺ and with a rate of 0.09%. Among those, 62 samples were tested by quantitative detection of HBV DNA. All of the quantitative results were less than 1 × 10³ IU/ml and 93.5% (58/62) of which were less than 100 IU/ml. The possitive rate of HBsAg⁻/HBV DNA⁺ donors rose with ages. The possitive rate in male donors was higher than that in female and was lower in highly educated ones. Students and public servants had a lower positive rate. CONCLUSION: The possitive rate of HBsAg⁻/HBV DNA⁺ donors is higher in Xiamen and the distribution of possitive donors has certain epidemiological characteristics. It is necessary to mobilize and recruit more people with a lower rate of HBsAg⁻/HBV DNA⁺ infection.

Molecular characteristics of occult hepatitis B virus from blood donors in southeast China.

The characteristics of 30 carriers with occult hepatitis B virus (HBV) infection (OBI) were compared with those of 30 individuals diagnosed as being HBV carriers at the time of blood donation, 60 asymptomatic carriers, and 60 chronic hepatitis patients. The prevalence of genotype C was significantly higher in carriers with OBIs than in any other HBsAg-positive (HBsAg(+)) group (P < 0.001). Specific amino acid substitutions in the regions from amino acids 117 to 121 and amino acids 144 to 147 located in the major hydrophilic region of the S gene were associated with carriers with OBIs (P < 0.01 for carriers with OBIs versus HBsAg(+) donors, carriers with OBIs versus HBsAg(+) asymptomatic carriers, and carriers with OBIs versus HBsAg(+) chronic hepatitis patients). G145R was the major variation in the HBV isolates responsible for local occult HBV infections.

[Quantitation of HTLV-I proviral load using real-time quantitative PCR with Taqman MGB probe].

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.

[Surveillance for occult HBV infection and HBsAg variants in blood donors].

Occult hepatitis B virus (HBV) infection status of blood donors in a southern city in China was investigated by immunological assays and nucleic acid testing. Overall, 17 (0.19%, 95% CI: 0.11%-0.30%) of the 9023 HBsAg negative samples were found to be positive for the presence of HBV DNA. "A" epitope sequences were obtained from 14 among them. Mutation(s) in aa124-aa147 existed in 6 (42.9%, 6/14) samples and 4 (66.7%, 4/6)were G145R mutation. Ratio of genotype C in occult donors (10/17) was statistically higher than HBs-positive donors (0/15, P<0.01), which implied that HBV genotype C leaded to occult infection more easily.

Specific cellular immune response in hepatitis E patients.

AIMS: To evaluate the specific T cell response together with IgM anti-hepatitis-E-virus (HEV) antibodies in acute hepatitis E (HE) patients. METHODS: Blood samples were collected from 11 HE patients every week and assayed for routine blood investigation after onset of disease until their convalescence. Peripheral blood mononuclear cells were separated from some of the blood samples (1-3 samples per patient) and tested for specific T cell response by enzyme-linked immunosorbent spot assay and IgM anti-hepatitis E virus by enzyme-linked immunosorbent assay. RESULTS: A particulate HEV capsid protein, HEV 239, effectively stimulated the response of T cells from HE patients infected by type 1 or type 4 HEV. In acute HE, a burst of HEV-specific cellular immune response occurred, which decreased along with the decreasing IgM anti-HEV antibody titre and normalization of liver function. CONCLUSIONS: HEV open reading frame 2 amino acids 368-606 can effectively stimulate the HEV-specific T cell response in vitro; the specific T cell response decreases along with convalescence and may play a role in the pathogenesis of acute HE and recovery.

Immune response induced by a different combined immunization of HBsAg vaccine.

AIMS: To evaluate the immune responses induced by different combined immunizations of HBsAg protein vaccine (P), recombinant vaccinia virus vaccine (V) and DNA vaccine (D). METHODS: Balb/c mice were primed by one of the three HBsAg vaccines P, V or D and boosted by the same or another, thus nine immune combinations were constructed. Titers of anti-HBsAg IgG and their sub-isotypes were determined by ELISA. Specific cellular immune responses were determined by calcein-release assay. RESULTS: V could induce the quickest humoral immune response with the geometrical mean titer of 1:10(1.6) at week 2 after prime immunization. The antibody titer primed by P (including PP, PV, PD) mounted up to the highest after the first boost. Antibody induced by PP was more polarized to Th2 while the other groups induced balanced Th1/Th2 response. Among all the groups, VD and DV induced the strongest CTL response. After the fourth boost, the specific lysis ratio was 64 and 71% separately at an E:T ratio of 1:1. CONCLUSIONS: P was the most potent for inducing humoral immune response while the weakest for CTL response. D was a poor immunogen to induce specific antibody production. Among all the immune combinations, DV and VD induced the strongest CTL response in Balb/c mice.

Difference of T cell and B cell activation in two homologous proteins with similar antigenicity but great distinct immunogenicity.

The candidate particulate hepatitis E vaccine, HEV 239, has been shown to be an efficacious vaccine in primates, and clinical study to date shows it to be safe and immunogenic for humans. The antigenicity of HEV 239 is virtually identical to its N-terminal 26 amino acids truncated protein, E2, which is not particulate but soluble. However, HEV 239 is over 200 times more immunogenic than E2. In present study, several events underlying this dramatic immunogenicity difference have been addressed. (1) HEV 239 can efficiently evoke a vigorous and predominant T cell response while E2 cannot induce detectable T cell response; (2) the dominant T cell epitopes in HEV 239 are identified, and both are also contained integrally in E2; (3) priming mice with Th epitope peptide can partially rescue the weak immunogenicity of E2 in alum adjuvant and (4) HEV 239 but not E2 can induce significant antibody response in athymic mice, which indicates that HEV 239 can directly activate B cell more efficiently. These results contribute to a better understanding of the mechanisms involved in the significant high immunogenicity of particulate antigen and may provide knowledge for the rational design and development of future vaccines.

[Immune response induced by combinant immunization of different HBsAg vaccines in mice].

AIM: To investigate the specific humoral and cellular immune response induced by prime-boost immunization of HBsAg protein vaccine (P), recombinant vaccinia virus vaccine (V) and DNA vaccine (D) in mice. METHODS: Groups of BALB/c mice were primed by one of the three vaccines P, V or D and boosted by another vaccine at 2, 5, 8 and 11 week later, thus 9 immune combinations were made: PP, PV, PD, VP, VV, VD, DP, DV and DD. Serum samples were collected at week 2, 5, 8 and 11 and levels of anti-HBsAg IgG antibodies and their sub-isotypes were determined. Seven days after every boost, spleen cells of vaccinated mice were separated and the specific CTL lysis ratio of P815S cells were determined. RESULTS: Among the three HBsAg vaccines P, V and D, V could induce the quickest humoral immune response. The memory humoral immune response induced by P was the strongest. D induced the weakest antibody titer. The ratio of specific IgG1/IgG2a indicated that antibody induced by PP was more polarized to Th2. The other groups induced balanced Th1/Th2 immune response. Among all the groups, VD and DV induced the strongest CTL response, and the specific lysis ratio of P815S cells was 71% and 64%, respectively. CONCLUSION: The experimental results suggested that among all the immune combinations, PV, PD, VP and VD can induce better humoral immune response while DV and VD can induce stronger CTL response in BALB/c mice.

Hydrophobicity of reactive site loop of SCCA1 affects its binding to hepatitis B virus.

AIM: To investigate the role of SCCA2 and other SCCA1 molecules in the process of hepatitis B virus (HBV) binding to mammalian cells. METHODS: SCCA1 and SCCA2 were isolated from HepG2. Binding protein (BP) genes were obtained through PCR. Recombinant baculoviruses expressing SCCA1, SCCA2, BP, and different mutants were constructed and utilized to infect mammalian cells to investigate the binding ability of infected cells to HBV. RESULTS: A SCCA1 gene (A1) was isolated from HepG2, but it appeared to lack the binding ability of infected cells to HBV. Two mutants, A1-BP and BP-A1, were constructed by interchanging the carboxyl terminal of A1 and BP. Cells expressing A1-BP showed an increased virus binding capacity, but not BP-A1. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them were found in the reactive site loop (RSL) of SCCA1. Primary structure assay revealed that the hydrophobicity of BP and AJ515706 in this domain was strong, but A1 was relatively weak. Changing the aa349 of A1 from low hydrophobic glutamic acid to high hydrophobic valine enhanced HBV binding. In contrast, HBV binding was reduced by changing the aa349 of BP from valine to glutamic acid. CONCLUSION: The results suggest that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.

[Hydrophobicity of reactive site loop of SCCA1 affects its binding to HBV].

Squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors, includes several variants. It was reported that expression of two SCCA1 (BP and AJ515706) in cells results in increased binding of HBV to these cells by the interaction of the expressed BP and AJ515706 with HBV pre-S1 domain. In this study, a SCCA1 (A1) was isolated from HepG2, but it appears to lack this ability. A possible role of two mutants, A1-BP and BP-A1, constructed by interchanging the carboxyl terminal of A1 and BP, was investigated. Cells expressing A1-BP rather than BP-A1 showed an increased virus binding capacity. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them in the reactive site loop (RSL) of SCCA1. Primary structure analysis revealed that the hydrophobicity of BP and AJ515706 in this domain is higher than that of A1. Changing the aa349 of A1 from low hydrophobic glutamic acid to high hydrophobic valine enhanced HBV binding. In contrast, changing the aa349 of BP from valine to glutamic acid reduced HBV binding. Our finding suggests that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.

[Detection of hepatitis E virus on a blood donor and its infectivity to rhesus monkey].

OBJECTIVE: To understand the infectivity and pathogenicity of the plasma of hepatitis E virus (HEV) viremia to primate animals. METHODS: RNA fragment of HEV genotype IV was detected on one healthy donor who was positive for anti-HEV IgM and negative for anti-HEV IgG. Then 10 ml plasma from above donor was transfused to rhesus monkey to observe its infectivity and pathogenicity. RESULTS: Acute hepatitis E was developed in rhesus monkey who accept HEV RNA positive plasma. It was confirmed by virological, immunological, biochemical and histopathological data. CONCLUSION: Acute hepatitis E can be induced by plasma transfusion of HEV viremia, which indicate the possibility of transfusion transmitted hepatitis E