Adenovirus F protein as a delivery vehicle for botulinum B

Abstract

Background: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination.

Results: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50.

Conclusion: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Figure 1

Nasal or i.m. immunization with Hcβtre-Ad2F induces enhanced serum IgG and mucosal IgA anti-Hcβtre Ab responses. BALB/c mice were immunized by the (A,B) i.n. or (C,D) i.m. route with equimolar concentrations of Hcβtre (25 μg) or Hcβtre-Ad2F (50 μg), either alone or with combination with cholera toxin (CT), as a mucosal adjuvant, on days 0, 7, and 14. Sera and fecal extracts were collected at weekly intervals beginning on day 14 post-primary immunization. Fecal IgA (A,C) and serum IgG (B,D) Ab titers against Hcβtre were determined by ELISA. The addition of vaccine-targeting molecule, Ad2F, in the absence and the presence of CT, improved mucosal Ab responses when compared to mice immunized with Hcβtre only. Without adjuvant, both groups induced weak IgG Abs responses. Data are expressed as mean ± SEM of three different experiments (n = 15 mice). *, p ≤ 0.001; **, p ≤ 0.05 represent the significant differences in IgA and IgG anti-Hcβtre levels between mice immunized with Hcβtre-Ad2F and Hcβtre with or without CT.

Figure 2

Nasal immunization with Hcβtre-Ad2F plus CT stimulates an enhanced nasal wash secretory (S)-IgA and elevated serum IgG1 and IgG2a anti-Hcβtre Ab titers. (A) BALB/c mice were nasally immunized with Hcβtre-Ad2F or Hcβtre plus CT, as described in Figure. 1. Three weeks after final immunization, S-IgA Ab levels in nasal washes were determined by ELISA. Mice vaccinated nasally with Hcβtre-Ad2F + CT showed significantly greater levels of Hcβtre-specific S-IgA when compared with mice nasally dosed with Hcβtre + CT. Data represent the mean ± SEM (n = 15 mice). *, p ≤ 0.001 represent the statistical differences in Hcβtre-specific S-IgA Abs between mice immunized with Hcβtre-Ad2F + CT vs. mice immunized with Hcβtre + CT. Serum IgG subclass anti-Hcβtre Ab responses on day 35 from (B) i.n. and (C) i.m. immunized mice (n = 7 mice/group) with Hcβtre-Ad2F or Hcβtre plus CT were evaluated. Significant elevations in (B,C) IgG1 and (C) IgG2a Ab titers were observed by the Hcβtre-Ad2F plus CT-immunized mice versus the Hcβtre plus CT-immunized mice: *, p ≤ 0.001.

Figure 3

Nasal immunization with Hcβtre-Ad2F plus CT enhances (A) IgA AFC responses in nasal passages (NPs) and NALT and (C) IgG AFC responses in head and neck LNs (HNLNs), submaxillary glands (SMGs), spleens, and Peyer's patches (PPs) when compared with Hcβtre plus CT. Four weeks post-primary immunization, mononuclear cells from selected lymphoid tissues were evaluated by using B cell ELISPOT assay to determine the number and distribution of (A,C) Hcβtre-specific and total (B,D) IgG and IgG AFCs. Data are expressed as mean ± SEM of three separate experiments (n = 15 mice) of AFC/1 × 10⁶ lymphocytes. *, p ≤ 0.001; **, p ≤ 0.05 represents the statistical differences in number of IgA and IgG AFCs in individual tissues between mice immunized with Hcβtre-Ad2F and Hcβtre with CT.

Figure 4

I.m. immunization with Hcβtre-Ad2F plus CT enhances the number of IgG AFCs in peripheral LNs (PLNs) HNLNs, and PPs when compared to mice immunized with Hcβtre plus CT. Four weeks post-primary immunization, mononuclear cells from selected lymphoid tissues were examined using B cell ELISPOT assay to determine the number and distribution of (A, C) Hcβtre-specific and (B, D) total IgA and IgG AFCs. Data are expressed as mean ± SEM of three separate experiments (n = 15 mice) of AFC/1 × 10⁶ lymphocytes. *, p ≤ 0.001; **, p ≤ 0.05 represents the significant differences in number IgG AFCs in individual tissues between mice immunized with Hcβtre-Ad2F and Hcβtre with CT.

Figure 5

Cytokine responses by i.n.-immunized mice with Hcβtre and Hcβtre-Ad2F plus CT show enhanced Th1-and Th2-type cell responses. Three weeks after final immunization total lymphocytes from (A) spleens, (B) HNLNs, and (C) MLNs of Hcβtre plus CT-or Hcβtre-Ad2F plus CT-immunized mice were isolated and cultured with Hc/B, OVA, or media only for 2 days, and then CFC responses were measured by cytokine-specific ELISPOT. Immune lymphocytes were evaluated for IFN-γ, IL-4, IL-5, IL-10, and IL-13 CFCs/1 × 10⁶ lymphocytes. Data represent the mean ± S.E.M. of three experiments (n = 15 mice). p ≤ 0.001; **, p ≤ 0.05.

Figure 6

Cytokine responses by i.m.-immunized mice with Hcβtre and Hcβtre-Ad2F plus CT show enhanced Th1-and Th2-type responses. Three weeks after final immunization, total lymphocytes from (A) spleens, (B) HNLNs, and (C) MLNs of Hcβtre plus CT-or Hcβtre-Ad2F plus CT-immunized mice were isolated and cultured with Hc/B, OVA, or media only for 2 days, and then CFC responses were measured by cytokine-specific ELISPOT. Immune lymphocytes were evaluated for IFN-γ, IL-4, IL-5, IL-10, and IL-13 CFCs/1 × 10⁶ lymphocytes. Data represent the mean ± S.E.M. of three experiments (n = 15 mice). p ≤ 0.001; **, p ≤ 0.05.