Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model

Abstract

Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

Fig 1

Research protocol.

Fig 2

Sequence map for Helicobacter pylori 26995 katA (corresponding to C terminus). Solid and broken lines beneath nucleotides highlight overlapping C-terminal-KatA-encoding nucleotide sequences 1 to 6, cloned into HBsAg VLPs. The numerical order of clones is 1 to 6 (Table 1); reference, NCBI accession no. AAD07923.1 (GI:2314010).

Fig 3

Western blot analysis showing immunoreactivity of HBsAg-KatA clones probed with a polyclonal rabbit anti-KatA antiserum. Lane 1, molecular size (MW) marker (kDa); lane 2, negative control; lane 3, HBsAg VLP control; lane 4, reactant HBsAg-KatA construct 1; lane 5, nonreactant HBsAg-KatA construct 2; lane 6, nonreactant HBsAg-KatA construct 3; lane 7, reactant HBsAg-KatA clone 4; lane 8, reactant HBsAg-KatA construct 5; lane 9, nonreactant HBsAg-KatA construct 6. Arrows highlight positive constructs.

Fig 4

Electron micrographs illustrate secretion-competent recombinant HBsAg-KatA VLPs prepared as described in Material and Methods. Panel A shows VLPs from construct 4, which are composed of HBsAg-S subunits of the expected 38-kDa size. Panel B illustrates VLPs derived from construct 1, which are comprised of HBsAg-S subunits apparently larger in size than the expected 43 kDa (see Fig. 3).

Fig 5

KatA-specific IgG antibody response in serum of mice following subcutaneous vaccination with recombinant HBsAg-KatA constructs (VLP-KatA) or recombinant HBsAg (VLP) alone using alhydrogel as an adjuvant. Serum IgG titers were determined by ELISA and measured at days 21, 33, and 41 post-primary vaccination. Results are shown as average antibody titers detected post-primary vaccination, with the standard error of the mean represented as a bar. The rise in VLP-KatA IgG titers 41 days postvaccination in serum from mice vaccinated with VLP-KatA was significantly greater than that of the titer observed for mice vaccinated with VLP alone (P < 0.05).

Fig 6

KatA-specific IgG1 and IgG2a antibody responses in serum of mice following subcutaneous vaccination with recombinant HBsAg-KatA constructs (VLP-KatA) or KatA alone (KatA) using alhydrogel as an adjuvant. Results are shown as the average antibody titers detected at 41 days post-primary vaccination, with the standard error of the mean represented as a bar. The increase in the IgG2a antibody titer induced by VLP-KatA compared to results for vaccination with KatA alone was significant (P < 0.05). The increase in the IgG1 titer induced by KatA alone was significantly greater than that induced with VLP-Kat (P < 0.05).

Fig 7

Bacterial numbers of H. pylori viewed per microscopic field in Giemsa-stained stomach tissue sections of recombinant HBsAg-KatA (VLP-KatA)-vaccinated mice challenged with the H. pylori SS1 strain, as described in Materials and Methods. Controls were recombinant HBsAg alone (VLP), KatA, and alhydrogel adjuvant alone. Bacterial load is expressed as bacterial load per microscopic field examined. The reduction in the bacterial load following vaccination with VLP-KatA compared to that with adjuvant alone was significant (*) [F value, 4.883, with df (3,36); VLP-KatA vs. alhydrogel has a t value of 3.748 with df 36 and a P value of <0.005).