Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Abstract

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (K_d ∼10^-12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Keywords: ELISA; Fab; antibody engineering; core antigen (HBcAg); e-antigen (HBeAg); hepatitis B virus (HBV, Hep B); phage display; single-domain antibody; surface plasmon resonance (SPR); viral immunology.

Figure 1.

Structures of rHBcAg and rHBeAg. A, sequence schematics for HBcAg and HBeAg. In addition to the assembly domain (residues 1–149), HBcAg has an arginine-rich nucleic acid-binding domain (residues 150–183). HBeAg shares the assembly domain with HBcAg but has a 10-residue propeptide (magenta) and a potentially variable carboxyl terminus, as discussed in the text. B, atomic structure of rHBcAg dimer (residues 1–149 only) in surface representation. The two chains are joined at Cys-61 by an intermolecular disulfide bond. C, atomic structure of rHBeAg dimer (residues (−10)–149) in surface representation. Each chain has a Cys-(-7)–Cys-61 intramolecular disulfide bond. D, atomic structure of rHBcAg (capsid, T = 4 symmetry) assembled from 120 rHBcAg dimers. The structure is considered analogous to HBcAg (nucleocapsid, core-antigen). One dimer is highlighted. E, rHBeAg dimer shown at the same scale as rHBcAg in D. The differences in conformation, size, and polymeric state are all not adequately communicated by the terms HBcAg and HBeAg.

Figure 2.

Screening the affinity and specificity of Fabs. Microtiter plates were coated with 10 μg/ml dimeric rHBeAg, HBcAg capsid, or rHBsAg, washed, blocked, and then treated with 2 μg/ml of the indicated Fab. Following additional washing the bound Fab was detected with anti-human IgG as described under “Experimental procedures.” The samples indicated (−) were probed with two different HBV-negative human plasmas. This survey was performed twice, with similar results.

Figure 3.

Sedimentation equilibrium of rHBeAg-Fab e13 complex. The panels show absorbance (bottom panel) and residuals (upper panel). Open circles show the UV absorbance gradient in the centrifuge cell. The solid line indicates the calculated fit for an ideal single species. Residuals show the difference between the fitted and experimental values as a function of radial position. The determined molecular weight 127,000 (±1500) is indicated, and this compares with a molecular weight of 130,867 calculated for a complex of 2 Fab e13 and one rHBeAg dimer.

Figure 4.

Secondary antibody pairing. A sandwich ELISA was used that incorporated Mab e6-coated plates for antigen capture and Fabs e13, e21, or e38 for detection. Anti-human IgG-HRP was used to generate the absorbance signal at 450 nm. The assay was performed over the range of 0–1 μg/ml rHBeAg. The experiment was performed three times with similar results.

Figure 5.

Epitope mapping using surface plasmon resonance. A, Biacore sensograms indicating binding kinetics generated from immobilized Fab e13 (ligand) titrated with analyte mixtures containing a fixed amount of rHBeAg (1.5 μm) and a variable amount of Fab me6 (0–133 nm). B, immobilized Fab me6 (ligand) titrated with analyte mixtures containing either a fixed amount (0.4 μm) of rHBeAg and a variable amount of Fab e13, or a fixed amount (1.5 μm) rHBeAg and a variable amount of Fab me6. The steady-state maximum binding is plotted as a function of analyte Fab concentration (abscissa). A and B, ordinate scales indicate SPR response in response units (RU).

Figure 6.

ELISA response curve with rHBeAg. With the optimized sandwich ELISA (supplemental Fig. S4), rHBeAg is titrated over the concentration range 0–100 μg/ml (0–5.6 μm). The experiment was performed in triplicate, and a sigmoidal fit to the data is shown with an EC₅₀ ∼12 nm.

Figure 7.

Calibration curves, rHBeAg and WHO standard. Using the sandwich ELISA (supplemental Fig. S4), calibration curves for the HBeAg WHO reference sample from the Paul-Ehrlich-Institut (PE IU/ml) and rHBeAg were prepared where the S/CO ratio was determined as described under “Experimental procedures.” From the plots, 1 PE IU/ml corresponds to ∼0.05 μg/ml (2.8 nm) rHBeAg. The experiment was performed in triplicate, and the individual plots and linear fittings are shown in supplemental Fig. S6.

Figure 8.

Immunoaffinity purification of HBeAg from HBV patient plasma. A, SDS-PAGE/Western blot of rHBeAg run under reducing (R) conditions (+DTT) and non-reducing conditions (O). The position of the reduced monomer is indicated with M, and the oxidized monomer with the higher mobility with M*. HBeAg was immunoaffinity-purified from an individual HBV-positive patient (see “Experimental procedures”) and analyzed under reducing (+R) and non-reducing conditions (+O). An HBV-negative plasma sample was analyzed under reducing conditions (−R). B, SDS-PAGE with Coomassie Blue staining: rHBeAg analyzed under reducing (lane 1) and non-reducing conditions (lane 2) and rHBcAg analyzed under reducing (lane 3) and non-reducing conditions (lane 4). Because of the intramolecular disulfide bond (Cys-(−7)–Cys-61) in rHBeAg, the protein in lane 2 has a slightly higher mobility than the reduced form, although due to the intermolecular disulfide bond (Cys-61–Cys-61) in rHBcAg, the protein in lane 4 has a substantially lower mobility than the reduced from. The molecular weights of a standard protein mixture are indicated.