Oral Application of Recombinant Bacillus subtilis Spores to Dogs Results in a Humoral Response against Specific Echinococcus granulosus Paramyosin and Tropomyosin Antigens

Abstract

Bacillus subtilis is known as an endospore-and biofilm-forming bacterium with probiotic properties. We have recently developed a method for displaying heterologous proteins on the surface of B. subtilis biofilms by introducing the coding sequences of the protein of interest into the bacterial genome to generate a fusion protein linked to the C-terminus of the biofilm matrix protein TasA. Although B. subtilis is a regular component of the gut microflora, we constructed a series of recombinant B. subtilis strains that were tested for their ability to immunize dogs following oral application of the spores. Specifically, we tested recombinant spores of B. subtilis carrying either the fluorescent protein mCherry or else selected antigenic peptides (tropomyosin and paramyosin) from Echinococcus granulosus, a zoonotic intestinal tapeworm of dogs and other carnivores. The application of the recombinant B. subtilis spores led to the colonization of the gut with recombinant B. subtilis but did not cause any adverse effect on the health of the animals. As measured by ELISA and immunoblot, the dogs were able to develop a humoral immune response against mCherry as well as against E. granulosus antigenic peptides. Interestingly, the sera of dogs obtained from immunization with recombinant spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, which represent the infective form of the head of the tapeworms. These results represent an essential step towards the establishment of B. subtilis as an enteric vaccine agent. The application of the recombinant B. subtilis spores led to the colonization of the gut 32 with recombinant B. subtilis but did not cause any adverse effect on the health of the 33 animals. As measured by ELISA and immunoblot, the dogs were able to develop a 34 humoral immune response against mCherry as well as against E. granulosus antigenic 35 peptides. Interestingly, the sera of dogs obtained from immunization with recombinant 36 spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, 37 which represent the infective form of the head of the tapeworms. These results represent 38 an essential step towards the establishment of B. subtilis as an enteric vaccine agent. 39 40

Mouse intestinal microbiota reduction favors local intestinal immunity triggered by antigens displayed in Bacillus subtilis biofilm

“…We first showed that B. subtilis was found in the mice intestine expressing TasA, even after the oral inoculation with recombinant B. subtilis tasA/lux/ TasA-mCherry ([ 23 ] and Table 1 ) and that recombinant vegetative cells were not found in mice feces after 6 days post-inoculation (Additional file 1 : Figure S1a and b). Our data also show that 64–93.5% of the orally applied recombinant B. subtilis spores are retained in the mice gut (Additional file 1 : Figure S1c and d).…”

“…Initial evidence suggested that recombinant B. subtilis spores germinate in the gut of BALB/ c mice without generating adverse effects. Therefore in an attempt to evaluate the effectiveness of our method, based on the display of antigens in biofilms, we used a previously tested recombinant B. subtilis (102-207)EgTrp that elicited a positive humoral immune response in dogs [ 23 ]. The antigen (102-207) EgTrp corresponds to tropomyosin, an immunogenic peptide of Echinococcus granulosus , described previously by Pétavy and collaborators [ 27 , 28 ].…”

“…Therefore, the decrease of the intestinal microbiota of mice, colonized for at least 1000 different bacterial species (40), is a requisite for the settlement of recombinant B. subtilis spores. Previous evidence demonstrates that B. subtilis naturally colonize the gut of humans [ 14 ], dogs [ 23 ], and even grass carps [ 41 ], suggesting that B. subtilis cannot be considered as an allochthonous microorganism. Interestingly, the generated immunoglobulins recognized the EgTrp antigen peptides only when present in a biofilm extract but not the one from the recombinant purified H 6 -(102-278)EgTrp.…”

“…Interestingly, the generated immunoglobulins recognized the EgTrp antigen peptides only when present in a biofilm extract but not the one from the recombinant purified H 6 -(102-278)EgTrp. In this context, we recently showed [ 23 ] that dogs treated orally with recombinant spores of B. subtilis (102-207)Egtrp elicited a humoral immune response recognizing specifically recombinant purified H 6 -EgTrp. Our results suggest that the biofilm TasA-(102-207)EgTrp fusion displayed in the gut mice got selected by immunoglobulins through specific conformation that is not forged by H 6 -EgTrp [ 42 , 43 ].…”

“…A third feature corresponds to its probiotic properties in human and livestock [ 20 – 22 ]. By combining these properties, we recently showed that it is possible, in a dog model, to elicit a local humoral immune response against enteric antigens such as E. granulosus parasite [ 23 ]. In this dog model, the recombinant spores were able to bypass the stomach barrier and then form, after germination, a biofilm in the intestine that displayed an antigen within its matrix, allowing the stimulation of gut-associated lymphoid tissue (GALT) and thereby, eliciting a local humoral immune response.…”