Elsevier

Vaccine

Volume 39, Issue 30, 5 July 2021, Pages 4072-4081
Vaccine

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

https://doi.org/10.1016/j.vaccine.2021.06.010Get rights and content

Highlights

  • Cytoplasmic antigen in LAB could sufficiently induce antigen-specific immunogenicity.

  • Spontaneous release of cytoplasmic antigen from LAB is crucial for vaccine efficacy.

  • Antigen expression level matters rather than its localization for LAB vaccine design.

  • M cell targeting moiety could improve immunogenicity of an antigen in oral vaccine.

Abstract

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.

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].

References (32)

  • C. Czerkinsky et al.

    Mucosal delivery routes for optimal immunization: targeting immunity to the right tissues

    Mucosal Vaccines.

    (2010)
  • A. Miquel-Clopés et al.

    Mucosal vaccines and technology

    Clin Exp Immunol

    (2019)
  • S.H. Kang et al.

    Oral Vaccine Delivery for Intestinal Immunity—Biological Basis, Barriers, Delivery System, and M Cell Targeting

    Polymers.

    (2018)
  • K. Szatraj et al.

    Lactic acid bacteria—promising vaccine vectors: possibilities, limitations, doubts

    J Appl Microbiol

    (2017)
  • A.C. Vilander et al.

    Adjuvant strategies for lactic acid bacterial mucosal vaccines

    Vaccines.

    (2019)
  • A. Wyszyńska et al.

    Lactic acid bacteria—20 years exploring their potential as live vectors for mucosal vaccination

    Appl Microbiol Biotechnol

    (2015)
  • Cited by (9)

    View all citing articles on Scopus
    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.

    View full text