Oral Recombinant Lactobacillus Vaccine Targeting the Intestinal

Ma et al. Microb Cell Fact (2018) 17:20 https://doi.org/10.1186/s12934-018-0861-7 Microbial Cell Factories RESEARCH Open Access Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus Sunting Ma1†, Li Wang1†, Xuewei Huang1, Xiaona Wang1, Su Chen1, Wen Shi1, Xinyuan Qiao1,2, Yanping Jiang1, Lijie Tang1,2, Yigang Xu1,2* and Yijing Li1,2* Abstract Background: Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) has led to serious economic losses to the swine industry worldwide. In this study, an oral recombinant Lactobacillus casei vaccine against PEDV infection targeting the intestinal microfold (M) cells and dendritic cells (DCs) for delivering the core neutralizing epitope (COE) of PEDV spike protein was developed with M cell-targeting peptide (Col) and dendritic cell-targeting peptide (DCpep). The immunogenicity of the orally administered recombinant strains was evaluated. Results: After immunization, signifcantly higher levels of anti-PEDV specifc IgG antibodies with PEDV neutralizing activity in the sera and mucosal sIgA antibodies in the tractus genitalis, intestinal mucus, and stools were detected in mice orally administered with the recombinant strain pPG-COE-Col-DCpep/L393, which expressed DCpep and Col targeting ligands fused with the PEDV COE antigen, compared to mice orally immunized with the recombinant strain pPG-COE/L393 without the DCpep and Col targeting ligands. Moreover, in response to restimulation with the PEDV COE antigen in vitro, a signifcant diference in splenocyte proliferation response and Th2-associated cytokine IL-4 level was observed in the group of mice orally immunized with pPG-COE-Col-DCpep/L393 (p < 0.05) compared to the groups of mice that received pPG-COE-Col/L393 and pPG-COE-DCpep/L393, respectively. Conclusions: The intestinal M cells- and DCs-targeting oral delivery of genetically engineered Lactobacillus express- ing the COE antigen of PEDV can efciently induce anti-PEDV mucosal, humoral, and cellular immune responses via oral administration, suggesting a promising vaccine strategy against PEDV infection. Keywords: Lactobacillus, Porcine epidemic diarrhea virus, Dendritic cell-targeting peptide, Microfold cell-targeting peptide, Oral immunization Background virus (PEDV). PEDV, a member of the Coronaviridae Porcine epidemic diarrhea (PED) characterized by vom- family, is an enveloped, single-stranded RNA virus whose iting, diarrhea, and dehydration, is a highly contagious genome comprises fve open reading frames (ORFs) intestinal disease caused by porcine epidemic diarrhea encoding an accessory protein and four structural pro- teins [1]. Of these, the spike on the surface of PEDV medi- *Correspondence: [email protected]; [email protected] ates binding and fusion between the virus and cells. In †Sunting Ma and Li Wang are joint frst author addition, the neutralizing epitope region of PEDV (COE) 1 College of Veterinary Medicine, Northeast Agricultural University, Mu Cai has been shown to induce virus neutralization antibodies Street No. 59, Xiang Fang District, Harbin, People’s Republic of China Full list of author information is available at the end of the article and is used as a potential candidate immunogen against © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ma et al. Microb Cell Fact (2018) 17:20 Page 2 of 12 PEDV [2, 3]. PEDV has caused serious economic losses A combination of targeting to DCs and M cells simulta- to the swine industry in Europe, America, Australia, and neously may further strengthen and improve the antigen especially Asia. Currently, commercial vaccines, which uptake and presentation. mostly include inactivated vaccines [4] and attenuated Terefore, in the present study, genetically engineered PEDV vaccines, have provided some protection, but their Lactobacillus casei 393 (L. casei 393, L393) strains, were efcacy has been poor [5, 6]. Since PEDV causes mainly constructed to constitutively express the COE of PEDV intestinal infections [7], secretory IgA (sIgA), which can with either one or both of DCpep and Col on their cell bind microbes and toxins in the intestine to prevent their surface. Te immunogenicity of these recombinant adherence to epithelial cells [8], is desirable for defend- strains was then investigated by oral administration in ing the mucosa against PEDV. Tus, research on vaccina- mice. tion inducing a more efcacious sIgA-based protective mucosal immunity is urgently needed to prevent PEDV Methods infections. Virus, bacteria, plasmids, and cell line Oral mucosal vaccination has the great advantage of PEDV HLJ-2012 was isolated from the intestines of pig- improving practicality for mass vaccination during pan- lets with severe diarrhea in Heilongjiang, China, and was demics, along with ease of production and administra- maintained in our laboratory. Lactobacillus casei ATCC tion. Above all, local protective mucosal immunity which 393 was cultured in de Man Rogosa and Sharpe (MRS) is pivotal in response to PEDV invasion is generally not broth at 37 °C. Te constitutive expression plasmid pPG- induced with parenteral vaccination [9]. Induction of T7g10-PPT, previously constructed by our laboratory, mucosal immunity should be focused on the interaction contained the HCE strong constitutive promoter, T7g10 between the antigen and lymphocytes within the mucosa. transcriptional enhancer, PgsA anchor from Bacillus sub- One promising approach involves the use of live recom- tilis for stabilizing the heterologous protein in the cell binant Lactobacillus which can colonize the gastroin- membrane (surface-displaying), and the rrnBT1T2 ter- testinal tract and compete with pathogens for mucosal minator [23], and was used to construct COE plasmids as binding sites [10]. Cell wall associated or secreted fac- described below. tors from some Lactobacillus strains can enhance innate immune responses and epithelial barrier function, modu- Construction of recombinant Lactobacillus strains late the intestinal micro-environment, regulate immune A recombinant expression plasmid was constructed as cell behavior, and elicit release of cytokines [11]. In addi- shown in Fig. 1. Nucleic acid manipulations and cloning tion to natural immune-stimulating adjuvants with weak procedures were performed according to standard proce- immunogenicity [12] and capacity to survive the gastric dures. Total virus RNA was extracted using a commer- acid and digestive enzymes, several Lactobacillus strains cial kit (Fastagen, Shanghai, China). First-strand cDNA have been applied for the delivery of heterologous anti- was transcribed with Revert Aid M-MlV reverse tran- gens to trigger mucosal immune responses against patho- scriptase (Fermentas, New York, USA). Briefy, RNA was gens [13–15]. incubated with 2 μL (10 µmol L−1) of reverse primer for Although a live recombinant Lactobacillus vaccine can COE for 10 min at 75 °C. Subsequently, 1 mL of RNase propagate in the porcine intestine, only a small amount Inhibitor (20 g mL−1), 2 mL of dNTP mix, and 1 mL of of antigen can reach the efective immune sites such as RevertAid M-MuLV reverse transcriptase were added, Peyer’s patches (PPs) [16]. To increase the bioavailabil- mixed gently, centrifuged at 1000×g, and incubated at ity of the immunogen of interest, the addition of den- 42 °C for 2 h. Te reaction was terminated by heating dritic cell-targeting peptide (DCpep) and microfold cell at 70 °C for 5 min, and the cDNA template was used for (M cell)-targeting peptide (Col) to this oral Lactobacil- polymerase chain reaction (PCR). Primers designed on lus delivery system has been proposed. Microbial sam- the spike gene of PEDV HLJ-2012 (GenBank Accession pling commonly includes two principal mechanisms: No. JX512907.1) are listed in Table 1. Primers named (1) M cells, predominantly localized at PPs can transfer COE-F1 and COE-R1 were used to obtain COE gene the antigen to DCs and macrophages for processing and without targeting peptide. Ten we use COE gene as PCR (2) DCs pierce the epithelial layers to sample the anti- template, COE-F1 and COE-R2 as primers to get COE- gen directly [17]. Previous studies have demonstrated Col-DCpep. Te reverse primer named COE-R2 con- that DCpep directs the protective antigen to DCs using tained the sequence of targeting peptides to fuse DCpep Lactobacillus as a vector to induce the desired intesti- and Col with the C terminus of COE. Te fexible linker nal and systemic immune response [18–20]. Further- (GGGGS) was inserted between the two targeting pep- more, M cell ligand-fused antigens have been shown to tides and fexible amino acids (GS) were inserted between enhance the specifc mucosal immune response [21, 22]. COE and targeting peptide. SWISS-MODEL was used Ma et al. Microb Cell Fact (2018) 17:20 Page 3 of 12 Fig. 1 Construction diagram of recombinant plasmid pPG-COE-Col-DCpep. HCE strong constitutive promoter; T7g10

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