Fig 1.
Representative trehalose glycolipids, trehalose dibehenate (TDB), the natural product brartemicin, and a lipophilic derivative thereof, C18Brartemicin (C18Brar).
TDB and C18Brar are potent ligands for the C-Type lectin, Mincle.
Table 1.
Vaccine groups and formulation of vaccines.
Fig 2.
Illustration showing synthesis of C18Brar.
To prepare C18Brar, 2,2’,3,3’,4,4’-hexa-O-trimethylsilyl-α,α’-D-trehalose S1 [56] and 4-octadecyloxy-benzoic acid S2 [38] were treated with N-ethyl-N’-(3-dimethyaminopropyl)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP), to form the diester S3 in an 59% yield. The trimethylsilyl ether protecting groups were subsequently removed with acidic Dowex to give C18Brar in 95% yield. Characterization data was consistent with that previously reported [38].
Table 2.
Proportion of animals showing vaccination site reactions (lumps).
Fig 3.
IgG antibody responses to M. haemolytica and M. ovipneumoniae formulated in different adjuvants.
Mean (+SE) serum IgG antibody responses to M. haemolytica and M. ovipneumoniae in animals vaccinated with a mixture of M. haemolytica and M. ovipneumoniae whole cell antigens formulated with different adjuvants or given antigens (Ags) alone at week 0 and 3 (timing of vaccinations shown by arrows). Antibody responses were measured in serum samples collected at weeks 0, 3, 6, 9, and 13 using ELISA. Different alphabetical letters indicate significant differences (P < 0.05) between the groups, while the same letter indicates no significant difference between groups.
Fig 4.
IgG antibody responses to M. ovipneumoniae antigen promoted by C18Brar adjuvant persist long-term.
Mean (+SE) serum IgG antibody responses to M. ovipneumoniae at 34 weeks after initial vaccination in animals vaccinated with either Antigens alone (Ags), antigens formulated with Quil-A (QA), Emulsigen-D (ED), Aldhydrogel + QuilA (AGA), or C18Brar. Antibody responses were measured in serum samples using ELISA. Significant differences are represented by * P = < 0.05 in antibody responses compared to Ags alone group. NS = non-significant.
Fig 5.
Antibody responses to leukotoxin in the vaccinated animals.
Serum antibody responses to M. haemolytica leukotoxin in animals vaccinated with a mixture of M. haemolytica and M. ovipneumoniae whole cell antigens formulated with different adjuvants or given antigens (Ags) alone. Antibody responses were measured in serum samples collected at weeks 0 and 6 using ELISA. An individual animal is represented by ‘●’ and significant differences are represented by * = < 0.05, ** = < 0.01 compared to Ags alone group. NS = non-significant.
Fig 6.
Cytokine responses to M. haemolytica and M. ovipneumoniae whole cell antigens in a whole blood stimulation assay.
Mean (+SE) IFN-γ, and IL-17A responses to PBS (Nil), M. haemolytica (MH) and M. ovipneumoniae (MO) whole cell antigens at week 0 (W0) and week 6 (W6). Blood samples from the animals vaccinated with Antigen alone (Ags), Quil-A (QA), Emulsigen-D (ED), Alhydrogel + QuilA (AQA), and C18Brar were stimulated for 40 h with M. haemolytica and M. ovipneumoniae whole cell antigens. Cytokine release was measured by ELISA and the reported values were calculated from the values for antigen stimulation where PBS was used as a control. For IL-17A, significant differences are represented by * = P < 0.05 compared to Ags alone group at week 6, while no significant differences between groups were observed for IFN-γ levels.
Fig 7.
Relative expression of immune responsive genes.
Whole blood samples were collected from animals before and after vaccination of animals with either a mixture of M. haemolytica and M. ovipneumoniae whole cell antigens alone, or antigens formulated with Quil-A (QA), Emulsigen-D (ED), Alhydrogel + Quil-A (AQA), or C18Brar adjuvants. Blood cultures were stimulated in vitro with either PBS, M. haemolytica (MH), or M. ovipneumoniae (MO) at a final protein concentration of 14.3 and 10 μg/mL, respectively, for 24 h. Total RNA was prepared from these samples and gene expression was measured by Nanostring nCounter. Total RNA counts for each gene were normalised against the geometric mean counts of the three house-keeping genes (GUSB, RPL15, HPRT1). The gene expression for both pre- and post-vaccination (week 6) were obtained by dividing counts for antigen-stimulation by counts for PBS. Change in the expression of immune responsive genes are shown as the ratios of normalised RNA counts post-vaccination and normalised RNA counts pre-vaccination. The mean ratio of each group (n = 12) is presented here. Different letters written in superscript indicate significant differences (P < 0.05) between the groups, while the same letter indicates no difference.