Fig 1.
Lenvervimab neutralizes HBV infection in culture.
(Upper) HepG2-NTCP cells were infected for 16 h with HBV at 500 Geq/cell in the presence of 0.001~1 microgram/ml of Lenvervimab (shown on the left) or a commercial rabbit polyclonal anti-HBsAg antibody (from Novus Biologicals) (shown on the right). HBsAg secreted into the culture media was measured after 5 days by ELISA. Bars indicate the average value and standard errors of triplicate experiments. P-values were obtained by utilizing the Excel software based on the two-tailed Student’s t-test. P-values indicated are less than 0.05 (*), 0.01 (**), 0.001 (***) and 0.0001 (****), respectively. (Lower) Intracellular viral core protein (HBcAg) after 5 days was examined by immunoblotting with an anti-HBcAg antibody. Values shown below the blot are the intensity of HBcAg scanned by Image J software (NIH) and normalized with that of the loading control Hsp70.
Fig 2.
Lenvervimab binds HBsAg only in nondenaturing conditions.
Aliquots of culture media from Huh7 cells transfected with pHM6-HA-HBs or HBV1.3 were separated on a 1% agarose gel in nondenaturing condition (Two panels shown on the left) or a 15% polyacrylamide gel in denaturing condition (shown on the right). Protein samples transferred to PVDF membrane were probed with Lenvervimab or anti-HA antibody, respectively.
Fig 3.
Lenvervimab binds the S antigens of all viral genotypes.
(A) Aliquots of culture supernatant from transfection of various genotype S clones pHM6-HA-HBs were separated by nondenaturing agarose gels, capillary-transferred to a nitrocellulose membrane and probed with either Lenvervimab (upper), anti-HA (middle) or Dako’s polyclonal anti-HBsAg (lower) antibodies. HBV1.3 was included as a control. Indicated below the image is the quantified signal intensity of each clone relative to that of a genotype D clone (ayw), referred here as wild type (WT). The image is a representative of two independent experiments. (B) Indicated as bars are signals of the Lenvervimab (in dark) and the Dako’s anti-HBsAg antibody (in yellow) after normalization with anti-HA antibody signal. Average and standard errors of duplicate immunoblot analyses are shown.
Table 1.
S gene clones of HBV genotypes investigated in this study.
Fig 4.
HBV S gene mutations investigated in this study.
A schematic diagram of S antigen depicted with the four transmembrane domains and amino acid residues comprising the two loops of dominant antigenic region (Genotype D, ayw). In circles are mutations in the antigenic ‘a’ determinant (in yellow) and mutations (except K160N) in the other region of S that overlap with some viral polymerase mutations conferring resistance to nucleos(t)ide drugs.
Fig 5.
Lenvervimab binds coherently to S antigens of a variety of clinical mutants.
(A, B) S antigens mimicking a variety of clinical mutants were separated by native agarose gel and immunoblotted with Lenvervimab (upper), anti-HA (middle) or polyclonal anti-HBsAg (lower) antibodies. HBV1.3 was included as a control. Indicated below the image is the quantified signal intensity of each clone relative to that of wild-type (Genotype D). (C) Bars indicate specific antibody signals normalized with the anti-HA antibody signal. Average and standard errors of duplicate immunoblot analyses are shown. Lenvervivmab (dark bars) shows consistent binding to all clones, whereas the polyclonal anti-HBsAg antibody (light bars) shows severely reduced binding to mutants at residues 141~148. No antigen was expressed from the C149R clone.
Table 2.
S gene mutations investigated in this study.
Fig 6.
Lenvervimab binds S antigens of drug-resistant variants.
(A) S antigen variations associated with drug-resistant viral polymerase mutations were separated by native agarose gel and immunoblotted with Lenvervimab (upper), anti-HA (middle) or polyclonal anti-HBsAg (lower) antibodies. Lenvervimab binds S antigens of most drug-resistant mutants except for those with mutations at residue E164. Lenvervimab binding was also impaired by mutation at residue K160, which is unrelated to drug resistance but is considered to be an antigenic element.