Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route
Introduction
Compared with injected vaccines, mucosal vaccines are more promising to defend against pathogenic infection at its initiation sites via induction of the mucosal immune response because most pathogens that cause epidemic diseases generally infect through mucosal routes [1], [2], [3]. In this regard, various mucosal compartments, including gastrointestinal, respiratory, and urogenital tracts, have been tested as vaccine administration sites [1], [3]. The oral route is frequently adapted for mucosal vaccine delivery because orally administered vaccines can stimulate mucosal immunity through gut-associated lymphoid tissue (GALT) such as Peyer’s patches (PP) in the gastrointestinal tract (GIT) which is a major infection sites for many pathogens [4].
However, there are still challenges to developing an efficient oral vaccine. After its ingestion, an oral vaccine should be protected from the low gastric pH and digestive enzymes during transit in the GIT and effectively guided to contact the lymphocytes in GALT for the effective induction of mucosal immunity [4]. The application of recombinant lactic acid bacteria (LAB), producing heterologous protein antigens originating from target pathogens, is a promising approach for oral vaccine delivery formulation to overcome the problems mentioned above [5], [6], [7]. Currently, research on the utilization of recombinant LAB as mucosal vaccines can be summarized by three strategies according to the localization of heterologous protein antigens: ‘cytoplasmic expression (CE)’, ‘cell surface display (SD)’, and ‘extracellular secretion (ES)’ [7], [8], (Supplementary Fig. 1A - C). In contrast to the SD and ES strategies, in which the protein antigens are exposed to GALT directly in the GIT environment, the CE strategy, in which the protein antigens are enclosed by the bacterial cell wall, has been relatively overlooked for its potential efficacy as an oral vaccine. Depending on the strains, orally administered recombinant LAB could pass through the GIT or colonize the intestinal mucosa, potentially exposing their antigens to the GIT environment on both occasions. Some portion of the antigen-expressing LAB are also assumed to be captured and phagocytosed by antigen presenting cells (APCs), such as dendritic cells (DCs) in the GALT after entering via M cells (microfold cells), the antigen collecting portals in the GIT, subsequently facilitating the induction of antigen-specific mucosal immunity as an oral vaccine [6]. However, in the case of the CE strategy, relatively lower efficacy as a mucosal vaccine is expected compared to the other two strategies since the cytoplasmic antigens in LAB seldom have a chance to contact B cells. Nevertheless, the exact immunization mechanism of each LAB vaccine strategy is not fully understood, and the superiority in efficacy as a mucosal vaccine among the three strategies is still questionable because studies showing high efficacy as a mucosal vaccine with the CE strategy have also been reported, especially through the oral route [7], [9], [10]. For the recombinant LAB with the CE strategy, the high induction level of the antigen-specific immune response after oral immunization could not be explained without exposure of the cytoplasmic protein antigens to B cells before phagocytosis of the LAB themselves by DCs in GALT. Therefore, we hypothesized that the extracellular release of antigens from the LAB cytoplasm into the intestinal lumen prior to GALT access could occur and be crucial to achieving high efficacy as a mucosal vaccine—even in the CE strategy.
In this research, we constructed three recombinant Lactobacillus plantarum (LP) strains with the CE strategy that expressed a model protein antigen, BmpB (Brachyspira membrane protein B) [11], in the cytoplasm, with or without an M cell-targeting peptide moiety (CKS9), which we previously validated to facilitate delivery of its conjugated molecules through M cells [12], [13], [14]. Then we compared the immunogenicity of the recombinant LP strains through their oral administration into mice to elucidate the hypothesis (Supplementary Fig. 1D - F). If the model antigens were released from the LP cytoplasm into the intestinal lumen before entering the M cells, the model antigen with CKS9 could show stronger antigen-specific immune responses compared with others due to its M cell-targeting property. In contrast, if the recombinant LPs access GALT without any exposure of their cytoplasmic antigens in the GIT environment, a significant difference would not be observed among the recombinant LPs in their immunization efficacy.
As a result, differential efficacies in their mucosal immune responses with those recombinant LPs have been observed, which supports the hypothesis that cytoplasmic antigens of recombinant LAB with the CE strategy could be released into the GIT environment and subsequently influence antigen-specific immune responses as oral vaccines.
Section snippets
Bacterial strains, plasmids, reagents, and growth conditions
Brachyspira hyodysenteriae B204 was obtained from the National Veterinary Research and Quarantine Service (NVRQS) in Korea and was cultured on tryptic soy blood agar plates (Difco, USA) containing spectinomycin (400 μg/ml) using the GasPakTM EZ Anaerobe Pouch System (BD Biosciences, USA) at 37 °C. The lactic acid bacteria (LAB) Lactococcus lactis IL1403 and Lactobacillus plantarum 25 (LP 25) were used as host cells for plasmid preparation and heterologous expression of the model antigen BmpB,
Expression of recombinant BmpBs in E. coli and LAB
A model protein antigen, BmpB originated from B. hyodysenteriae, was heterologously expressed in E. coli and LAB with or without peptide moieties (SSP-G4S for P-BmpB or CKS9-G4S for M−BmpB) on its N-terminus. In the E. coli system, three His6-tagged recombinant proteins, BmpB, P-BmpB, and M−BmpB, were expressed as soluble proteins under the pET21a (+) vector in the cytoplasm of E. coli BL21 (DE3) (Fig. 2A-B) and purified by a single step with Ni2+-affinity column chromatography (Fig. 2C).
Discussion
Most strains of LAB are regarded as ‘generally recognized as safe (GRAS)’. Some strains can colonize the intestinal mucosa and stimulate mucosal immunity by enhancing the innate immune response and influence the physiology of the immune cells within GALT as commensal bacteria in GIT [21], [22]. Genetically modified LAB to produce foreign protein antigens could also survive in harsh environments, such as low gastric pH, bile acids, and intestinal digestive enzymes, during their transit in the
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) [grant number 316005-5].
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- 1
Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea.
- 2
Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
- 3
Davis Center for Regenerative Biology and Medicine, MDI Biological Laboratory, Bar Harbor, ME 04609, USA.