Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle

Abstract

In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1β and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.

Keywords: Adjuvants; Bovine tuberculosis; Mycobacterium bovis; Vaccine.

Figure 1. Selection of the ID83 fusion protein for vaccination studies

(A) Responder frequency of 22 TB reactor animals to 54 recombinant proteins from the IDRI protein repository. Filled bars denote responses to novel IDRI fusion proteins. (B) Responder frequency of 22 TB reactor and between 5 and 9 (depending on the protein) BCG vaccinated animals to the 6 IDRI fusion proteins. In both graphs, the responder frequency is based on IFN-γ production as the readout.

Figure 2. Cytokine production by bovine MDDC following stimulation with GLA and R848

Bovine MDDC were cultured for 24 hours in the presence of increasing concentrations of GLA, R848 or a mixture of GLA and R848. Quantification of (A) TNF-α, (B) MIP-1β, (C) IL-10 and (D) IL-12 was performed using a cytokine multiplex assay. Data shown is representative of two experiments.

Figure 3. Immunisation with ID83 induces cellular and humoral immune responses

Cattle (four per group) were immunised at week 0 and week 4 as detailed in the material and methods. Blood samples were taken at 2 weekly intervals for assessment of (A) ID83-specific whole blood IFN-γ production, (B) ID83-specific PBMC proliferation, and (C) ID83-specific IgG serum antibody levels. Median responses for each group are plotted. * p<0.05, ** p<0.01, *** p<0.001, nonparametric ANOVA with Dunn’s Multiple Comparison Test (versus the PBS control group at each time point).

Figure 4. Immunisation with ID83/ISA70 induces production of ID83-speficic IgG₂ serum antibodies

Cattle (four per group) were immunised at week 0 and week 4 as detailed in the material and methods, and blood samples taken at 2 weekly intervals for assessment of (A) ID83-specific IgG₁, or (B) ID83-specific IgG₂ serum antibody levels. Median responses for each group are plotted. * p<0.05, ** p<0.01, *** p<0.001, nonparametric ANOVA with Dunn’s Multiple Comparison Test (versus the PBS control group at each time point).