Spray-drying-microencapsulated Minthostachys verticillata essential oil and limonene as innovative adjuvant strategy to bovine mastitis vaccines.

Design of innovative adjuvant strategies with an appropriate safety profile is relevant to developed subunit or inactivated microorganism vaccines for bovine mastitis. Minthostachys verticillata essential oil (EO) has demonstrated ability to stimulate the innate immune response and adjuvant effect similar to Al(OH)3. Here we evaluated the adjuvant effect of EO and its metabolite, limonene (L) alone and microencapsulated by spray-drying, using an inactivated Enterococcus faecium strain bovine-mastitis inducer. The gas chromatography-mass spectrometry analysis showed that microencapsulation process did not alter the EO or L chemistry. Microencapsulated EO (McEO) or L (McL) (2.0, 2.5 and 5.0 mg/ml) decreased the viability of bovine mammary gland epithelial cells in a dose-dependent way. Balb/c mice (n = 32) were subcutaneously inoculated (day 0) and revaccinated (day 14 and 28) with saline solution, inactivated bacteria alone or combined with Incomplete Freund's Adjuvant; EO or L (2.5 mg/ml); McEO or McL (5.0 mg/ml); or microcapsule wall material (Mc) alone (2.5 mg/ml). EO, L, McEO and McL stimulated E. faecium-specific IgG (IgG1 or IgG2a) with opsonizing capacity and increased the proportion of CD4+ and CD8+ T cells producers of IFN-γ. Microencapsulation was an effective strategy to increase the adjuvant potential of EO or L. These new adjuvants deserve further study to evaluate their incorporation into vaccines for bovine mastitis.

Evaluation of the efficacy of polymeric antigen BLSOmp31 formulated in a new cage-like particle adjuvant (ISPA) administered by parenteral or mucosal routes against Brucella ovis in BALB/c mice.

Brucella ovis is an economically important cause of epididymitis in rams worldwide. Polymeric BLSOmp31 was previously identified as a protective immunogen against this pathogen. In this study, BLSOmp31 was formulated with a modified version of ISCOMATRIX adjuvant called ISPA (BLSOmp31/ISPA) and was administered in BALB/C by the subcutaneous and ocular route. The systemic and mucosal immune responses, the opsonic activity of antibodies and the protection conferred against B. ovis were evaluated. BLSOmp31+ISPA injected subcutaneously or by ocular route induced significantly higher IgG antibody levels with a mixed Th1/Th2 profile compared to non-immunized mice. IgA and IgG were detected in sera and nasal, tracheobronchial, vaginal secretions, tears and faeces, from SC immunized mice while in the group immunized by the ocular route a slight increase in both isotypes was mainly observed in all secretions, except in vaginal fluid. Opsonic antibodies stimulated binding and increased uptake of PHrodo™ Green-labelled B. ovis by neutrophils and monocytes. BLSOmp31 administered subcutaneously induced the highest levels of IFN-É£. The ocular immunization not only produced significant levels of this cytokine but also IL-4 compared to non-immunized mice. Both, subcutaneous and ocular routes of immunization, significantly protected against B. ovis infection. These results indicate that BLSOmp31/ISPA administered parenterally or by ocular route is a safe and effective vaccine against B. ovis in the murine model.

Polymeric antigen BLSOmp31 formulated with class B CpG-ODN in a nanostructure (BLSOmp31/CpG-ODN/Coa-ASC16) administered by parenteral or mucosal routes confers protection against Brucella ovis in Balb/c mice.

Previously, we demonstrated that the chimera BLSOmp31 formulated in chitosan microspheres or Poloxamer407-Chitosan administered via the nasal and the ocular mucosa conferred partial protection in sheep against B. ovis. In this work, we tested a new delivery system for mucosal immunization with BLSOmp31 in the murine model to improve the efficacy of previously used formulations. First, we evaluated the protective efficacy against B. ovis induced by BLSOmp31 administered by the subcutaneous route using either BLSOmp31 alone, co-administered with immunostimulatory synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG-ODN) or with CpG-ODN in a nanostructure called Coa-ASC16 compared with BLSOmp31 emulsified in Incomplete Freund Adjuvant. Then, we evaluated the protection conferred by the best performing formulation (BLSOmp31/CpG-ODN/Coa-ASC16) administered by both subcutaneous and ocular routes. BLSOmp31/CpG-ODN/Coa-ASC16 injected subcutaneously did not induce higher IgG antibody levels compared to BLSOmp31 alone or BLSOmp31/CpG-ODN but it did stimulate a mixed immune Th1-Th2 response with the highest levels of IFN-É£ and conferred significant protection against the B. ovis challenge. Although ocular instillation of BLSOmp31/CpG-ODN/Coa-ASC16 showed a similar degree of protection compared to the parenteral route (3.66 and 3.60 logs of protection, respectively), it induced lower levels in serum of specific IgG (with mixed IgG1/IgG2a) and IgA antibodies and, less IFN-É£ and IL-4 than the subcutaneous route. No antibodies were detected in vaginal lavages or saliva. Fecal antigen-specific IgA was slightly higher in mice immunized with BLSOmp31/CpG-ODN/Coa-ASC16 subcutaneously compared with the ocular route. These results indicate that BLSOmp31/CpG-ODN/Coa-ASC16 was a safe and effective vaccine against B. ovis in mice.