Bacillus toyonensis amplifies the immunogenicity of an experimental recombinant tetanus vaccine in horses.

Probiotic microorganisms can stimulate an immune response and increase the efficiency of vaccines. For example, Bacillus toyonensis is a nonpathogenic, Gram-positive bacterium that has been used as a probiotic in animal supplementation. It induces immunomodulatory effects and increases the vaccine response in several species. This study aimed to evaluate the effect of B. toyonensis supplementation on the modulation of the immune response in horses vaccinated with recombinant Clostridium tetani toxin. Twenty horses were vaccinated twice, with an interval of 21 days between doses, and equally divided into two groups: the first group was supplemented orally for 42 days with feed containing viable spores of B. toyonensis (1 × 108) mixed with molasses (40 ml), starting 7 days before the first vaccination; the second (control) group received only feed mixed with molasses, starting 7 days before the first vaccination. Serum samples were collected to evaluate the humoral immune response using an in-house indirect enzyme-linked immunosorbent assay (ELISA), and peripheral blood mononuclear cells (PBMCs) were collected to evaluate cytokine transcription (qPCR). For the specific IgG-anti-rTENT titer, the supplemented group had ELISA values that were four times higher than those of the control group (p < 0.05). The supplemented group also showed higher ELISA values for the IgGa and IgGT sub-isotypes compared to the control group. In PBMCs stimulated with B. toyonensis, relative cytokine transcription of the supplemented group showed 15-, 8-, 7-, and 6-fold increases for IL1, TNFα, IL10 and IL4, respectively. When stimulated with a vaccine antigen, the supplemented group showed 1.6-, 1.8-, and 0.5-fold increases in IL1, TNFα, and IL4, respectively, compared to the control group. Horses supplemented with B. toyonensis had a significantly improved vaccine immune response compared to those in the control group, which suggests a promising approach for improving vaccine efficacy with probiotics.

Immunomodulatory effect of Lacticaseibacillus casei CB054 supplementation in calves vaccinated against infectious bovine rhinotracheitis.

Probiotics are live microorganisms that, confer health benefits to the host when supplemented in adequate amounts. They can promote immunomodulation by inducing phagocyte activity, leukocyte proliferation, antibody production, and cytokine expression. Lactic acid bacteria (BAL) are important probiotic specimens with properties that can improves ruminant nutrition, productivity and immunity. The aim of the present study was to evaluate the immunomodulatory effect of the supplementation with Lacticaseibacillus casei CB054 in calve vaccinated against bovine infectious rhinotracheitis (IBR). Calve were vaccinated with a commercial IBR vaccine, on day 0 and received a booster dose on day 21. L. casei CB054 was orally administered (4 ×109 UFC) for 35 days, while a non-supplemented control group received Phosphate Buffer Saline (PBS). Stimulation of bovine splenocytes with L. casei CB054 markedly enhanced mRNA transcription levels of cytokines IL2, IL4, IL10 and IL17 genes. Calves supplemented with L. casei CB054 showed significantly higher (p < 0.05) specific anti-BoHV-1 IgG levels, higher serum neutralization, as well as higher mRNA transcription for IL2, IL4, IL10 and IL17 genes in Peripheral Blood Mononuclear Cells (PBMCs) comparing with control calves. Supplemented calve had an average weight gain of ∼14 kg more than non-supplemented during the experimental period. These results suggest that L. casei CB054 supplementation increase immunogenicity of a commercial IBR vaccine in cattle and improve weight gain.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae.

OBJECTIVES: Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid. RESULTS: The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD. CONCLUSIONS: These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

Recombinant bovine IL17A acts as an adjuvant for bovine herpesvirus vaccine.

The Bovine herpes virus type 5 glycoprotein D (gD) is essential for viral penetration into host permissive cells. The Herpes virus gD glycoprotein has been used for bovine immunization, being efficient in reduction of viral replication, shedding and clinical signs, however sterilizing immunity is still not achieved. Recombinant subunit vaccines are, in general, poorly immunogenic requiring additional adjuvant components. Interleukin 17A (IL17A) is a pro-inflammatory cytokine produced by T helper 17 cells that mediate mucosal immunity. IL17 production during vaccine-induced immunity is a requirement for mucosal protection to several agents. In this study, we investigated the potential of a recombinant IL17A to act as an adjuvant for a recombinant BoHV-5 glycoprotein D vaccine in cattle. Three cattle groups were divided as: group 1) rgD5 + alumen + rIL-17A; 2) rgD5 + alumen; and 3) PBS + alumen. The cattle (3 per group) received two doses of their respective vaccines at an interval of 21 days. The group that received rIL17 in its vaccine formulation at the 7th day after the prime immunization had significant higher levels of specific rgD-IgG than the alumen group. Addition of rIL17 also led to a significant fold increase in specific anti-rgD IgG and neutralizing antibodies to the virus, respectively, when compared with the alumen group. Cells stimulated with rIL17A responded with IL17 transcription, as well IL2, IL4, IL10, IL15, Bcl6 and CXCR5. Our findings suggest that the rIL17A has adjuvant potential for use in vaccines against BoHV-5 as well as potentially other pathogens of cattle.

Bacillus Toyonensis BCT-7112T Spores as Parenteral Adjuvant of BoHV-5 Vaccine in a Murine Model.

Bacterial spores of the genus Bacillus are being evaluated as adjuvant molecules capable of improving the immune response to vaccines. In this study, we investigate whether subcutaneously administered spores of B. toyonensis BCT-7112T could enhance a vaccine immune response in mice. Three groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: 40 µg of recombinant glycoprotein D (rgD) from bovine herpesvirus type 5 (BoHV-5) adsorbed in 10% aluminum hydroxide (alum) without B. toyonensis spores (group 1) and B. toyonensis (1 × 106 viable spores) + 40 µg of rgD adsorbed in 10% alum (group 2); and B. toyonensis (1 × 106 viable spores) without rgD (group 3). Group 2 showed significantly higher titers (P < 0.05) of total specific serum IgG, IgG2a, and neutralizing antibodies, when compared with the groups 1 and 3. A significantly higher (P < 0.05) transcription level of cytokines IL-4, IL-12, and IFN-γ was observed in splenocytes from mice that received the B. toyonensis spores in the vaccine formulation. In addition, stimulation of the macrophage-like cell line RAW264.7 with spores of B. toyonensis markedly enhanced the cell proliferation and mRNA transcription levels of IL-4, and IL-12 cytokines in these cells. Our findings indicated that the subcutaneous administration of B. toyonensis BCT-7112T spores enhanced the humoral and cellular immune response against BoHV-5 in mice.

Association of Bacillus toyonensis spores with alum improves bovine herpesvirus 5 subunit vaccine immune response in mice.

Spores of the genus Bacillus are molecules capable of increasing the vaccine adjuvanticity. Bovine herpesvirus type 5 (BoHV-5) is responsible for meningoencephalitis that causes important economic losses in cattle. BoHV-5 glycoprotein D (gD) is a target of vaccine antigen and plays an important role in host cell penetration. The present study aimed to evaluate the adjuvanticity of Bacillus toyonensis (B.t) spores, live and heat-killed, associated with a vaccine formulated with aluminum hydroxide (alum) and the recombinant BoHV-5 glycoprotein D (rgD) in an experimental murine model. Six experimental groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: rgD (40 µg) + live spores (2 × 109 CFU); rgD + killed spores; rgD + live spores + alum (2.0 mg); rgD + killed spores + alum; rgD + alum, and rgD + PBS. Mice from rgD + live spores group showed an increase in rgD IgG titers from the 21st day until the end of the experiment. The groups of live and killed spores, associated to alum, had similar levels of IgG titers with no significant difference between each other; however, by the 14th and 28th day until the end of the experiment, presented higher IgG titers in comparison to the rgD + alum group. Moreover, increased serum levels of IgG1, IgG2a, and IgG2b were detected in mice that received spores in the vaccine formulation. The spores associated with alum groups showed neutralizing BoHV-5 antibodies and high mRNA transcription of the cytokines IFN-γ (66-fold), IL-17 (14-fold), and IL-12 (2.8-fold). In conclusion, our data demonstrated that the B. toyonensis spores, live or killed, associated with alum increased the adjuvanticity for BoHV-5 rgD in mice, suggesting the use of B. toyonensis spores as a promising component for vaccine formulations.