Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies.

BACKGROUND: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. OBJECTIVE: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. METHODS: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. RESULTS: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. CONCLUSION: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

Influence of Pattern Recognition Receptor Ligands on Induction of Innate Immunity and Control of Hepatitis B Virus Infection.

Failure of current therapies to cure chronic hepatitis B has led to renewed interest in therapies that stimulate the host immune system. APOBEC3 (A3) family enzymes have been shown to induce mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) leading to inhibition of HBV transcription and replication. Pattern recognition receptor (PRR) agonists have been reported to suppress HBV, but it is unclear whether these agonists induce A3 gene expression in hepatocytes. We, therefore, evaluated whether PRR signaling activates the expression of A3 genes and other innate immunity genes and restricts HBV infection. HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were infected with HBV and treated with various PRR agonists. The level of HBV infection was subsequently assessed by measurement of HBV biomarkers, including HBV DNA, cccDNA, HBs, and HBe antigens in infected hepatocytes. Among all tested PRR ligands, only Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW significantly inhibited hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV DNA, and cccDNA, whereas R848 and lipopolysaccharide (LPS) only showed significant inhibition on HBsAg and HBeAg, but not virus DNA. CpG and Pam3CSK4, on the other hand, had no significant inhibitory effect on any of the HBV infection parameters. Moreover, Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW were the only ligands that significantly increased IL-8 secretion. Interestingly, HBV infection reduced IL-8 secretion induced by Poly(I:C)-HMW and to a lesser extent Poly(I:C)-HMW/LyoVec. Poly(I:C)-HMW/LyoVec had a significant effect on increasing the expression level of A3F, A3G, A3H, TLR3, RIG-1, and MDA5 genes. Our data suggest that PRR agonists may control HBV infection through different mechanisms. The RIG-1 and MDA5 agonist, Poly(I:C)-HMW/LyoVec, seems to downregulate HBV infection through induction of A3 genes.

Immunoreactivity pattern of monoclonal antibodies against Hepatitis B vaccine with global Hepatitis B virus genotypes.

Hepatitis B surface antigen (HBsAg) specific monoclonal antibodies (mAbs) are potentially valuable therapeutic and diagnostic tool. We have previously established and characterized a panel of mAbs derived from immunized BALB/c mice with a yeast-derived recombinant HB vaccine subgentoype A2 and HBsAg subtype adw2. This study was conducted to evaluate the reactivity pattern of this anti-HBs mAbs panel with various genotypes and subgenotypes of HBV using the first WHO HBV genotype reference panel containing 15 serum samples representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H. Ten out of 21 anti-HBs mAbs were able to strongly recognize all gentopye/subtypes of HBsAg provided in the WHO reference panel. However, 10 out of 21 anti-HBs mAbs showed a moderate to profound loss of reactivity with HBV genotypes/HBsAg subtypes D2/ayw3, E/ayw4, F2/adw4, and H/adw4. Two mAbs from the second group displayed a profoundly reduced reactivity with only 1 out of 3 C2/adr genotype/subtype samples. The amino acid alignment of these 3 samples showed that this particular sample contains amino acid substitution at residue 127, which is located inside "a" determinant. This amino acid substitution, which profoundly affected the reactivity of anti-HBs antibodies, has been previously reported only in D/ayw3, E/ayw4, F/adw4, and H. Interestingly, the amino acid alignment of the samples in this WHO panel showed that P127T substitution can also be found in C2/adr. Comparing amino acids sequences inside the antigenic loop (AGL) showed that D2/ayw3 contains a T118A/P127T double substitution, E/ayw4 contains P127L/T140S, F2/adw4 contains P127L/T140S/ F158L, and H/adw4 contains P127L substitution. Therefore, amino acid variability at positions 118, 127, 140, and 158 was found to cause significant loss of reactivity with anti-HBs mAbs. Since HBsAg variability in different genotypes of HBV can profoundly affect the reactivity of anti-HBs mAbs, analytical sensitivity for HBsAg assays should be considered based on the circulating and common HBV variants in the relevant countries.