Qingbing Zheng

Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors

BACKGROUND & AIMS This study aimed at investigating mutations in the hepatitis B surface protein (HBsAg) in occult hepatitis B virus (HBV) infection (OBI) and their influence on viral antigenicity and phenotype. METHODS The characteristics of 61 carriers with OBI (OBI group), 153 HBsAg(+) carriers with serum HBsAg ≤ 100 IU/ml (HBsAg-L group) and 54 carriers with serum HBsAg >100 IU/ml (HBsAg-H group) from 38,499 blood donors were investigated. Mutations in the major hydrophilic region (MHR) of the viral sequences were determined. Thirteen representative MHR mutations observed in OBI sequences were antigenically characterized with a panel of monoclonal antibodies (MAbs) and commercial HBsAg immunoassays and functionally characterized in HuH7 cells and hydrodynamically injected mice. RESULTS Of 61 OBI sequences, 34 (55.7%) harbored MHR mutations, which was significantly higher than the frequency in either the HBsAg-L (34.0%, p=0.003) or the HBsAg-H group (17.1%, p<0.001). Alterations in antigenicity induced by the 13 representative MHR mutations identified in the OBI group were assessed by reacting recombinant HBV mutants with 30 different MAbs targeting various epitopes. Four out of the 13 mutations (C124R, C124Y, K141E, and D144A) strongly decreased the analytical sensitivity of seven commercial HBsAg immunoassays, and 10 (G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R) significantly impaired virion and/or S protein secretion in both HuH7 cells and mice. CONCLUSIONS MHR mutations alter antigenicity and impair virion secretion, both of which may contribute to HBsAg detection failure in individuals with OBI.

Novel Double-Antigen Sandwich Immunoassay for Human Hepatitis B Core Antibody

ABSTRACT A novel diagnostic immunoassay testing procedure for hepatitis B virus core antibody (anti-HBc) using homogeneous purified full-length hepatitis B virus core antigen (HBcAg) capsids obtained from Escherichia coli was compared with Abbott Architect anti-HBc chemiluminescent microparticle immunoassay (CMIA; indirect method) against a library of specimens. A monoclonal anti-HBc neutralization confirmatory assay was then used to determine the degree of discordance between specimens. The new assay was found to be superior in both sensitivity and specificity.

Evaluation of a rapid test for detection of H5N1 avian influenza virus

The performance of H5 Dot ELISA, a rapid test for detection of avian H5N1 influenza virus, was evaluated using 30 H5N1 strains belonging to 10 major genetic groups of H5N1 influenza virus, 14 strains of non-H5N1 influenza virus and 652 field samples collected from healthy and diseased chickens from markets and poultry farms. The detection limit of the test for all 30 strains of H5N1 virus was < or = 0.1 hemagglutinin (HA) units and the test yielded a negative result when tested against 100 HA units of the non-H5N1 viruses. The test gave a positive result for 87 of the 106 poultry samples from which H5N1 virus was isolated by culture and 3 of 546 culture-negative poultry samples. Compared with virus culture, the overall prediction rate of the test was determined to be 96.6%; the positive prediction rate was 96.7% and negative prediction rate, 96.6%. The false positive rate was 0.5% and false negative rate 17.9%. Considering that the test is also convenient to use, it was concluded that H5 Dot ELISA is suitable for field use in the investigation of H5N1 influenza outbreaks and surveillance in poultry.

Virus-mimetic nanovesicles as a versatile antigen-delivery system

Significance The previously unidentified virus-mimetic nanovesicles (VMVs) described in this manuscript consist of phospholipid derived from mammalian cell plasma membrane, recombinant protein anchored to cell membrane via the route of signal peptide sorting, and surfactants capable of controlling the VMV size and strength, which allows the VMVs to display functional polypeptides or maintain the correct conformation of protein antigen. The protein integrated into VMV by its hydrophobic transmembrane peptide has more modifications, such as glycosylation, than proteins in conventional subunit vaccines. Moreover, many viral envelope glycoproteins can be genetically engineered onto VMV liposomal surface so as to mimic the properties and conformational epitopes of natural virus. VMV provides an effective, straightforward, and tunable approach against a wide range of emerging enveloped viruses. It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses.

A rapid test for the detection of influenza A virus including pandemic influenza A/H1N1 2009

A new rapid diagnostic test for detection of influenza A virus was evaluated with four sets of experiments: first, a comparison with a commercial diagnostic kit against a panel of virus strains was conducted; second, the kit was tested against a collection of 40 strains of influenza A virus isolated from five different host species and 26 strains of other respiratory viruses used as controls; third, the kit was tested against specimens collected in the field obtained from human and chicken; and fourth, the kit was tested against the novel pandemic influenza A/H1N1 2009 clinical specimens obtained from admitted to hospital patients. The test kit displayed a sensitivity of 88% for both human specimens and avian specimens. The corresponding specificity was 99.3% for human specimens and 96.5% for avian specimens. This test kit may be useful for rapid diagnosis of influenza A virus.

Serological survey of antibodies to influenza A viruses in a group of people without a history of influenza vaccination

A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the haemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than those for seasonal H1N1. Samples positive for antibodies to the pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p <0.01) at 1 year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.

A multimechanistic antibody targeting the receptor binding site potently cross-protects against influenza B viruses

A cross-lineage therapeutic antibody potently targets the receptor binding site of influenza B virus via multiple mechanisms. An antibody to battle flu B Although it circulates globally and is prevalent enough to warrant inclusion in the seasonal influenza vaccine, influenza B is far less well studied than its cousin, influenza A, and therapeutics are lacking. Shen et al. have now generated a potent antibody that inhibits diverse strains of influenza B virus. The antibody recognizes the receptor binding site in hemagglutinin, a region critical to viral entry, and was shown to be therapeutically effective in mice and ferrets. This antibody could be widely deployed to treat or prevent influenza B infection around the world. Influenza B virus causes considerable disease burden worldwide annually, highlighting the limitations of current influenza vaccines and antiviral drugs. In recent years, broadly neutralizing antibodies (bnAbs) against hemagglutinin (HA) have emerged as a new approach for combating influenza. We describe the generation and characterization of a chimeric monoclonal antibody, C12G6, that cross-neutralizes representative viruses spanning the 76 years of influenza B antigenic evolution since 1940, including viruses belonging to the Yamagata, Victoria, and earlier lineages. Notably, C12G6 exhibits broad cross-lineage hemagglutination inhibition activity against influenza B viruses and has higher potency and breadth of neutralization when compared to four previously reported influenza B bnAbs. In vivo, C12G6 confers stronger cross-protection against Yamagata and Victoria lineages of influenza B viruses in mice and ferrets than other bnAbs or the anti-influenza drug oseltamivir and has an additive antiviral effect when administered in combination with oseltamivir. Epitope mapping indicated that C12G6 targets a conserved epitope that overlaps with the receptor binding site in the HA region of influenza B virus, indicating why it neutralizes virus so potently. Mechanistic analyses revealed that C12G6 inhibits influenza B viruses via multiple mechanisms, including preventing viral entry, egress, and HA-mediated membrane fusion and triggering antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity responses. C12G6 is therefore a promising candidate for the development of prophylactics or therapeutics against influenza B infection and may inform the design of a truly universal influenza vaccine.

Differential diagnosis of pandemic (H1N1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay

Abstract A sensitive and convenient immunoassay that can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from seasonal influenza virus can play an important role in the clinic. In the presented study, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus, was developed. After laboratory determination of the sensitivity and specificity characteristics, the performance of this assay was evaluated in a cohort of 904 patients with influenza-like illness. All seven strains of pH1N1 virus tested were positive by pH1N1 ELISA, with an average lower detection limit of 103.0 ± 0.4 tissue culture infective dose (TCID)50/mL (or 0.009 ± 0.005 HA titre). Cross-reaction of the assay with seasonal influenza virus and other common respiratory pathogens was rare. In pH1N1-infected patients, the sensitivity of the pH1N1 ELISA was 92.3% (84/91, 95% CI 84.8–96.9%), which is significantly higher than that of the BD Directigen EZ Flu A + B test (70.3%, p <0.01). The specificity of pH1N1 ELISA in seasonal influenza A patients was 100.0% (171/171, 95% CI 97.9–100.0%), similar to that in non-influenza A patients (640/642, 99.7%, 95% CI 98.9–100.0%). The positive predictive value for pH1N1 ELISA was 97.7% and the negative predictive value was 99.1% in this study population with a pH1N1 prevalence of 10.1%. In conclusion, detection of HA of pH1N1 virus by immunoassay appears to be a convenient and reliable method for the differential diagnosis of pH1N1 from other respiratory pathogens, including seasonal influenza virus.

Evaluation of a new rapid influenza A diagnostic test for detection of pandemic (H1N1) 2009 and seasonal influenza A virus

BACKGROUND Rapid influenza A diagnostic tests (RIDTs) play an important role in the clinical setting, especially in the influenza post-pandemic era with three influenza A viruses in circulation. OBJECTIVES Determine the sensitivity and specificity of a new RIDT (FluA Dot) by comparison with BD Directigen EZ FluA+B and CDC rRT-PCR. STUDY DESIGN Two sets of experiments were conducted to determine the performance of the new test. (1) Serial dilutions of eight pandemic (H1N1) 2009 (pH1N1) isolates, five seasonal H3N2 isolates, five seasonal H1N1 isolates and three recombinant nucleoproteins were tested by FluA Dot assay, Directigen EZ FluA+B test and CDC real-time RT-PCR. (2) Using CDC rRT-PCR as the gold standard, the clinical sensitivity and specificity of the FluA Dot and Directigen EZ FluA+B were evaluated in nasopharyngeal swab (NPS) specimens of 807 patients presenting with influenza-like illness. RESULTS The average analytical sensitivity of FluA Dot (0.06 ng/mL for recombinant nucleoproteins and 2.16 ± 0.85 log 10 TCID(50) for viruses) was approximately 10-fold higher than Directigen EZ FluA+B (1-2 ng/mL for recombinant nucleoproteins and 3.54 ± 0.81 log 10 TCID(50) for viruses), and was approximately 10-fold lower than the CDC rRT-PCR (1.09 ± 0.69 log 10 TCID(50) for viruses). Among 807 NPS specimens tested, the sensitivities and specificities of FluA Dot were 91.1% (95%CI: 86.7-94.4%)/99.7% (95%CI: 98.7-99.9%), and the Directigen EZ FluA+B were 71.9% (95%CI: 65.7-77.6%)/99.8%(95%CI: 99.0-99.9%). CONCLUSION The new test (FluA Dot) exhibit higher sensitivity than Directigen EZ FluA+B both in pH1N1 and seasonal influenza A detection. The promising RIDT can play important roles in influenza diagnosis and therapy.

Properties and Therapeutic Efficacy of Broadly Reactive Chimeric and Humanized H5-Specific Monoclonal Antibodies against H5N1 Influenza Viruses

ABSTRACT Highly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.