Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

Abstract

Background: Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2-3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412-425) was inserted into the hydrophilic loop of sHBsAg.

Results: The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412-425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes.

Conclusions: For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

Keywords: HBV small surface antigen (sHBsAg); Hepatitis C virus (HCV); Leishmania tarentolae; VLP; Vaccine.

Fig. 1

Schematic illustration of the sHBsAg sequence with the foreign HCV E2 412–425 epitope inserted into the “a”-determinant region. The sHBsAg insertion site corresponding to amino acid positions 127 and 128 is marked red. The numbering corresponds to the amino acid positions in the sHBsAg protein. The 412–425 epitope of the HCV E2 glycoprotein is shown in green

Fig. 2

Characterization of the particles expressed in the L. tarentolae system. a Immunofluorescence of recombinant L. tarentolae cells transfected with plasmids expressing 412–425_sHBsAg and sHBsAg. Cells transfected with empty pLEXSY_I-blecherry3 plasmid were used as negative control (NC). The staining was carried out using AP33 mAbs (blue) and anti-HBsAg Abs (green), scale bar = 5 µm. b Western blot analysis of the Leishmania-derived particles in reducing conditions. sHBsAg and 412–425_sHBsAg were treated with PNGase F and detected using the anti-HBsAg specific antibody. G represents the glycosylated and DG deglycosylated form of protein. c Recognition of particles with anti-HBsAg and AP33 Abs in the ELISA tests. ELISA plates were coated with serial dilutions of recombinant L. tarentolae cell lysates containing sHBsAg and 412–425_sHBsAg particles. The dilution factor is depicted on x-axis. For each ELISA assay, the mean from three independent experiments performed in duplicate is shown. The mean A₄₅₀ values and standard deviations are shown on the y-axis. The background from the L. tarentolae wild-type cell lysate in each dilution was subtracted from the obtained results

Fig. 3

OptiPrep density gradient centrifugation of recombinant particles. Seventeen fractions of 0.5 mL each were harvested from the top of the OptiPrep gradient after centrifugation. The aliquots of each fraction were analyzed by SDS-PAGE Coomassie blue staining (CB) and western-blot (WB) with anti-HBsAg Abs. The figure represents the results of fractions 8–10 where the highest amount of particles was detected

Fig. 4

Electron micrograph of chimeric 412–425_sHBsAg particles. After centrifugation on OptiPrep gradient, the particles were negatively stained with uranyl acetate and analyzed by electron microscopy (top). Numerous particles approximately 22 nm in diameter were observed. The exposition of the 412–425 epitope was studied by immunogold labeling using AP33 mAbs and secondary goat anti-mouse Abs conjugated with 6 nm gold particles (bottom). Scale bar 100 nm

Fig. 5

Analysis of cellular response in BALB/c mice immunized with Leishmania-derived particles. Box plot of antigen-specific IFN-γ ELISPOT responses in sHBsAg (right) and 412–425_sHBsAg (left) groups of immunized mice. Splenocytes from six immunized BALB/c mice per group were stimulated with 20 µg/well of 43 different 15-mer overlapping peptides spanning the sHBsAg protein sequence pooled into three groups (P1, P2, P3). The splenocytes were collected 2 weeks after the last immunization. The gray region is the 25–75th percentile; the horizontal lines indicate the mean (dashed) and median (solid) values

Fig. 6

Analysis of the humoral response induced by Leishmania-derived particles in BALB/c mice. a Analysis of the antibody endpoint titers of the pooled mouse antisera specific to the recombinant particles. ELISA plates were coated with 412–425_sHBsAg (right) or sHBsAg (left) particles. b Analysis of the interaction of immune sera with yeast-derived HBsAg proteins. ELISA plates were coated with 5 µg/mL of purified HBsAg protein from P. pastoris (yHBsAg) (right) or commercially available vaccine against HBV (Engerix-B) (left). c Analysis of the antibody response to the 412–425 synthetic peptide. ELISA plates were coated with 20 µg of 412–425 peptide. AP33 mAb was used to estimate concentration of Abs specific to the 412–425 region in serum. Dilution factor of the pooled 412–425_sHBsAg sera and concentration of the AP33 antibody are shown on x-axis (bottom and top, respectively). The 412–425_sHBsAg sera values are represented with bars, the AP33 values are marked with solid line. Mean A₄₅₀ values and standard deviations are shown on the y-axis. The background from the negative control serum in each dilution was subtracted from the obtained results (a, b, c). For each ELISA assay, the mean from three independent experiments performed in duplicate is shown. Asterisks indicate statistical significance (*P < 0.05, paired two-tailed t-test) (a, b). The solid horizontal line (a, b) indicates the cutoff value (three times the mean background value). d Analysis of cross-reactivity of the 412–425_sHBsAg sera to the E1E2 complex from different HCV genotypes. The figure represents western blotting in reducing conditions with 412–425_sHBsAg sera diluted 1:500. As an antigen, extracts of HEK293 cells transfected with plasmids expressing E1E2 glycoproteins from different HCV genotypes were used. Non-transfected HEK293 cell lysate was used as negative control (NC)