Quadrivalent hemagglutinin and adhesion expressed on Saccharomyces cerevisiae induce protective immunity against Mycoplasma gallisepticum infection and improve gut microbiota.

Mycoplasma gallisepticum (MG) infection causes infectious respiratory diseases in poultry, causing economic losses to the poultry industry. Therefore, this study aims to develop a safe, convenient, and effective multivalent recombinant Saccharomyces cerevisiae vaccine candidate and to explore its potential for oral immunization as a subunit vaccine. Mycoplasma gallisepticum Cytadhesin (MGC) and variable lipoprotein and hemagglutinin (vlhA) are associated with the pathogenesis of MG. In this study, a quadrivalent recombinant Saccharomyces cerevisiae (ST1814G-MG) displaying on MGC2, MGC3, VLH5, and VLH3, proteins was innovatively constructed, and its protective efficiency was evaluated in birds. The results showed that oral immunization with ST1814G-MG stimulates specific antibodies in chickens, reshapes the composition of the gut microbiota, reduces the Mycoplasma loading and pulmonary disease injury in the lungs. In addition, we found that oral ST1814G-MG had better protection against MG infection than an inactivated vaccine, and co-administration with the inactivated vaccine was even more effective. The results suggest that ST1814G-MG is a potentially safer and effective agent for controlling MG infection.

Oral immunization with recombinant L. lactis expressing GCRV-II VP4 produces protection against grass carp reovirus infection.

The hemorrhagic disease causing by grass carp reovirus (GCRV) infection, is associated with major economic losses and significant impact on aquaculture worldwide. VP4 of GCRV is one of the major outer capsid proteins which can induce an immune response in the host. In this study, pNZ8148-VP4/L. lactis was constructed to express recombinant VP4 protein of GCRV, which was confirmed by the Western-Blot and enzyme-linked immunosorbent assay. Then we performed the oral immunization for rare minnow model and the challenge with GCRV-II. After oral administration, pNZ8148-VP4/L. lactis can continuously reside in the intestinal tract to achieve antigen presentation. The intestinal and spleen samples were collected at different time intervals after immunization, and the expression of immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that VP4 recombinant L. lactis could induce complete cellular and humoral immune responses in the intestinal mucosal system, and effectively regulate the immunological effect of the spleen. The immunogenicity and the protective efficacy of the oral vaccine was evaluated by determining IgM levels and viral challenge to vaccinated fish, a significant level (P < 0.01) of antigen-specific IgM with GCRV-II neutralizing activity was able to be detected, which provided a effective protection in the challenge experiment. These results indicated that an oral probiotic vaccine with VP4 expression can provide effective protection for grass carp against GCRV-II challenge, suggesting a promising vaccine strategy for fish.

Oral administration of Lactobacillus plantarum expressing aCD11c modulates cellular immunity alleviating inflammatory injury due to Klebsiella pneumoniae infection.

BACKGROUND: Klebsiella pneumoniae (KP), responsible for acute lung injury (ALI) and inflammation of the gastrointestinal tract, is a zoonotic pathogen that poses a threat to livestock farming worldwide. Nevertheless, there is currently no validated vaccine to prevent KP infection. The development of mucosal vaccines against KP using Lactobacillus plantarum (L. plantarum) is an effective strategy. RESULTS: Firstly, the L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c were constructed via homologous recombination to express the aCD11c protein either inducibly or constitutively. Both NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c strains could enhance the adhesion and invasion of L. plantarum on bone marrow-derived dendritic cells (BMDCs), and stimulate the activation of BMDCs compared to the control strain NC8-pSIP409 in vitro. Following oral immunization of mice with NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c, the cellular, humoral, and mucosal immunity were significantly improved, as evidenced by the increased expression of CD4+ IL-4+ T cells in the spleen, IgG in serum, and secretory IgA (sIgA) in the intestinal lavage fluid (ILF). Furthermore, the protective effects of L. plantarum against inflammatory damage caused by KP infection were confirmed by assessing the bacterial loads in various tissues, lung wet/dry ratio (W/D), levels of inflammatory cytokines, and histological evaluation, which influenced T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood and lung. CONCLUSIONS: Both the inducible and constitutive L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c have been found to stimulate cellular and humoral immunity levels and alleviate the inflammatory response caused by KP infection. These findings have provided a basis for the development of a novel vaccine against KP.

Safety profile of intravenous administration of live Pichia pastoris cells in mice.

Pichia pastoris has been widely used to produce antigenic proteins aimed to integrate subunit vaccines. Moreover, increasing interest in large-scale vaccine production at the lowest cost is rapidly focusing in the development of yeast surface display (YSD) systems for delivery of antigens. In this scenario, the safety of live yeast administration must be warranted, however, such information is very scarce. Here, we assess the intravenous administration (i.v.) of live P. pastoris cells in order to trace dissemination in BALB/c mice and to evaluate the immune response raised against the yeast compared to the well-defined pathogen Candida albicans. Our results demonstrate dissemination of P. pastoris to the heart, kidney, and spleen, but it is quickly eliminated during the first 48 h postinfection (hpi), with persistence in the liver along with mild mononuclear (MN) and polymorphonuclear (PMN) infiltrate, which was resolved at 144 hpi. In vivo delayed-type hypersensitivity test (DTH) or in vitro antigenic stimulation of mice splenocytes demonstrate that transient infection of P. pastoris did not induce a cell-mediated immune response nor increase the level of circulating IgG or IgM. These results demonstrate the innocuous profile of P. pastoris and support its use as a safe delivery system for vaccine development.

Oral or intranasal immunization with recombinant Lactobacillus plantarum displaying head domain of Swine Influenza A virus hemagglutinin protects mice from H1N1 virus.

BACKGROUND: Swine influenza A virus (swIAV) is a major concern for the swine industry owing to its highly contagious nature and acute viral disease. Currently, most commercial swIAV vaccines are traditional inactivated virus vaccines. The Lactobacillus plantarum-based vaccine platform is a promising approach for mucosal vaccine development. Oral and intranasal immunisations have the potential to induce a mucosal immune response, which confers protective immunity. The aim of this study was to evaluate the probiotic potential and adhesion ability of three L. plantarum strains. Furthermore, a recombinant L. plantarum strain expressing the head domain of swIAV antigen HA1 was constructed and evaluated for its ability to prevent swIAV infection. RESULTS: The three L. plantarum strains isolated from healthy pig faecal samples maintained the highest survival rate when incubated at pH 3 and at bile salt concentration of 0.3%. They also showed high adherence to intestinal cells. All three L. plantarum strains were monitored in live mice, and no major differences in transit time were observed. Recombinant L. plantarum expressed swIAV HA1 protein (pSIP401-HA1-ZN-3) and conferred effective mucosal, cellular and systemic immune responses in the intestine as well as in the upper respiratory airways of mice. In conclusion, the oral and intranasal administration of L. plantarum strain pSIP401-HA1-ZN-3 in mice induced mucosal immunity and most importantly, provided protection against lethal influenza virus challenge. CONCLUSION: In summary, these findings suggest that the engineered L. plantarum strain pSIP401-HA1-ZN-3 can be considered as an alternative approach for developing a novel vaccine during an swine influenza A pandemic.

Distinct roles for Peyer's patch B cells for induction of antigen-specific IgA antibody responses in mice administered oral recombinant Salmonella.

Our previous study demonstrated an indispensable role of Peyer's patches (PPs) for the induction of antigen-specific secretory (S)IgA antibody responses after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella-Tox C). In this study, we defined the PP lymphoid structures and immune cells required for the induction of mucosal SIgA antibody responses. Adoptive transfer of mononuclear cells (MNCs) from PPs into PP-deficient (PP-null) mice failed to elicit tetanus toxoid (TT)-specific mucosal immunity. However, when the same PP MNCs were transferred into lethally irradiated PP-normal recipient mice, PP MNCs preferentially emigrated to recipient PPs, leading to PP lymphoid structures and TT-specific SIgA antibody responses. Significantly reduced numbers of TT-specific IgA antibody-forming cells were detected in the mesenteric lymph nodes (MLNs) and intestinal lamina propria of mice when surface expression of the sphingosine 1-phosphate receptor on lymphocytes was inhibited by its agonist FTY720. However, FTY720 treatment did not alter dendritic cell migration or Salmonella dissemination into these tissues. When rSalmonella-Tox C-stimulated CD4+ T cells isolated from PPs, MLNs and the spleen were co-cultured with B cells from these tissues, significantly increased levels of TT-specific IgA antibody responses were exclusively induced in cultures containing PP B cells. Furthermore, surface IgA+ PP B cells produced TT-specific IgA antibody responses in vitro. These findings suggest that PP lymphoid structures and surface IgA+ PP B cells are essential elements for the induction of antigen-specific intestinal SIgA antibody responses to oral Salmonella.

Oral immunization of carps with chitosan-alginate microcapsule containing probiotic expressing spring viremia of carp virus (SVCV) G protein provides effective protection against SVCV infection.

Spring viremia of carp (SVC) is highly contagious and lethal disease in cyprinid fish, in particular common carps (Cyprinus carpio), causing numerous economic losses to the aquaculture industry. SVC is presently endemic disease in Europe, America, and several countries in Asia and its causative agent is spring viremia of carp virus (SVCV). In this study, a chitosan-alginate microcapsule probiotic vaccine expressing G protein of SVCV was prepared, and the immunogenicity in carps of orally administrated with the microcapsule probiotic vaccine was evaluated. Our results showed that the microcapsule probiotic vaccine can induce potent antigen-specific immune responses in carps via oral vaccination, and provide effective anti-SVCV protection for carps. Significantly, the microcapsule probiotic vaccine is suitable for mass fish immunization, suggesting a promising vaccine strategy for fish.

Oral Immunization of Chickens with Lactococcus lactis Expressing cjaA Temporarily Reduces Campylobacter jejuni Colonization.

Campylobacter jejuni is the leading cause of human foodborne enteritis worldwide. Poultry products are regarded as the main source of human campylobacteriosis. Strategies are being developed to reduce colonization of poultry by Campylobacter. The membrane transport protein CjaA was reported to stimulate mucosal immune responses, which can reduce the C. jejuni load in chickens. In this study, oral immunization of broilers with food-grade Lactococcus lactis NZ3900/pNZ8149 carrying the C. jejuni cjaA gene was examined for the ability to reduce colonization of broilers by Campylobacter. The Usp45 signal peptide and the Escherichia coli heat-labile enterotoxin B subunit (LTB) gene fragments were inserted into the upstream and downstream of the cjaA gene for secretory expression and immune enhancement, respectively. The cjaA gene and the fusion cjaA-ltb gene were both expressed in recombinant L. lactis, and the single cjaA gene was secretory expressed in the recombinant strain. Oral administration of two recombinant L. lactis strains expressing the cjaA gene and the fusion cjaA-ltb gene both stimulated specific anti-CjaA serum IgY responses significantly. While the average intestinal sIgA responses in these groups were higher compared with the control groups, they were not significantly different. Chicken challenge experiments showed that the colonization levels of C. jejuni in the groups provided oral immunization with two recombinant L. lactis-delivered CjaA strains were significantly lower than that of the control group at 5 d postinoculation, but there was no significant difference in C. jejuni colonization among all groups at 9 d. These results indicated that recombinant L. lactis with secretory expression of CjaA is a promising live vector vaccine against C. jejuni colonization of chickens. The immunization regimen requires further optimization to ideally stimulate detectable levels of intestinal sIgA to enhance the level of inhibition of C. jejuni colonization.

A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice.

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.

Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein.

Recombinant strains of Lactococcus lactis NZ9000 that express native and codon-optimized E6 protein (fused to the SPusp45 secretion signal) were successfully constructed by using the nisin-controlled gene expression (NICE) system. Expression of the recombinant strains was evaluated by Western blot analysis. Female mice of strain C57BL/6 were immunized orally with recombinant lactococci expressing inducible E6 oncoprotein and the antigen-specific antibody production (IgA and IgG) and cytokines were measured by ELISA and ELISPOT assay, respectively. Our outcomes indicate that the HPV-16 E6 specific IL-2- and IFN-γ-secreting lymphocytes in the antigen-stimulated intestinal mucosal lymphocytes, splenocytes and vaginal lymphocytes were significantly higher than the control groups. We showed that L. lactis having codon-optimized E6 oncogene had better inhibitory effect on tumor growth, better treatment effects on progression of tumor size, and better survival rate in comparison with L. lactis having native E6 oncogene, (P < 0.0001). In conclusion, the rE6 protein displayed by L. lactis can induce humoral and cellular immunity. Taken together, these preclinical results represent a promising step towards the development of recombinant L. lactis as a live oral vector vaccine to treat the HPV-16 associated with cervical cancer.

Effective oral delivery of gp100 plasmid vaccine against metastatic melanoma through multi-faceted blending-by-blending nanogels.

Plasmid DNA gp100 is able to act as an available vaccine against metastatic melanoma, but its administration is usually limited to parenteral route. Since oral delivery of plasmid DNA is intervened by various physical obstacles, here we constructed a nanogel (Alg-Tat-gp100) with multi-faceted functions through blending-by-blending method. Due to the cooperation of alginate and Tat peptide, Alg-Tat-gp100 demonstrated the significant improvement of stability in the stomach, mucus penetration ability in intestine, and transport across mucus layer and MDCK cells. Moreover, the bone marrow-derived cells were activated with an enhanced co-stimulatory molecule expression. Following immunization using Alg-Tat-gp100 nanogels in C57BL/6 mice, the secretion of IFN-γ and the activation of cytotoxic T cells were significantly improved. Benefiting from those cases, the B16F10 tumor inhibition rate achieved 42.5% by this oral DNA vaccine, suggesting that this multi-faceted nanogel prepared by simple blending-by-blending method may provide a new strategy for oral DNA vaccine delivery.

Surface Display of Antigen Protein VP8* of Porcine Rotavirus on Bacillus Subtilis Spores Using CotB as a Fusion Partner.

Porcine rotavirus is a major cause of acute viral gastroenteritis in suckling piglets, and vaccination is considered to be an effective measure to control these infections. The development of a live mucosal vaccine using Bacillus subtilis spores as an antigen delivery vehicle is a convenient and attractive vaccination strategy against porcine rotavirus. In this study, a shuttle vector was constructed for the spore surface display of the spike protein VP8* from porcine rotavirus (the genotype was G5P[7]). A successful display of the CotB-VP8* fusion protein on the spore surface was confirmed by Western blot and immunofluorescence microscopy analysis. The capacity for immune response generated after immunization with the recombinant strain was evaluated in a mouse model. The intestinal fecal IgA and serum IgG were detected by enzyme-linked-immunosorbent serologic assay (ELISA). Importantly, recombinant strain spores could elicit strong specific mucosal and humoral immune responses. These encouraging results suggest that recombinant B. subtilis BV could provide a strategy for a potential novel application approach to the development of a new and safe mucosal subunit vaccine against porcine rotavirus.

Oral Immunization with Nontoxigenic Clostridium difficile Strains Expressing Chimeric Fragments of TcdA and TcdB Elicits Protective Immunity against C. difficile Infection in Both Mice and Hamsters.

The symptoms of Clostridium difficile infection (CDI) are attributed largely to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenic C. difficile (NTCD) strain, resulting in strain NTCD_mTcd138. Oral immunization with spores of NTCD_mTcd138 provided mice full protection against infection with a hypervirulent C. difficile strain, UK6 (ribotype 027). The protective strength and efficacy of NTCD_mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD_mTcd138 also provided hamsters significant protection against infection with 2 × 104 UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI.

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

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 be used 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 enzyme-linked immunosorbent assay and immunoblotting, 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 after 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 toward the establishment of B. subtilis as an enteric vaccine agent.

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

BACKGROUND: We previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model. RESULTS: In order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin. CONCLUSIONS: The reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus.

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, significantly higher levels of anti-PEDV specific 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 significant difference 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 expressing the COE antigen of PEDV can efficiently induce anti-PEDV mucosal, humoral, and cellular immune responses via oral administration, suggesting a promising vaccine strategy against PEDV infection.

M cell-targeting strategy enhances systemic and mucosal immune responses induced by oral administration of nuclease-producing L. lactis.

Efficient delivery of antigens to the gut-associated lymphoid tissue (GALT) is the most critical step for the induction of mucosal immunity by oral vaccines. As M cells are the main portal for luminal antigens into the GALT, the M cell-targeting of antigens affords a promising strategy toward the development of effective oral vaccines. Lactococcus lactis is a fascinating recombinant host for oral vaccines, as they survive and produce antigens in the gut and have a particularly safe profile for human use. In this study, we developed and evaluated an M cell-targeting oral immunization system using recombinant L. lactis strains. For the purpose, we generated an L. lactis strain that secretes a model antigen fused with the OmpH ß1α1 domain of Yersinia enterocolitica, which has been shown to bind to a complement C5a receptor on the M cell surface. As the model antigen, Staphylococcus aureus nuclease was used for fusion, resulting in L. lactis-expressing Nuc-OmpH (LL/Nuc-OmpH). Ex vivo intestinal loop assays showed that the amount of Nuc-OmpH taken up into Peyer's patches was more than that of the unfused nuclease (Nuc). In addition, oral administration of the recombinant L. lactis strains to mice demonstrated that LL/Nuc-OmpH-induced nuclease-specific fecal IgA and serum IgG titers were significantly higher than those induced by LL/Nuc. These results indicate that OmpH works as an M cell-targeting molecule when fused with antigens secreted from L. lactis and that the M cell-targeting strategy affords a promising platform for L. lactis-based mucosal immunization.

Immune responses induced by recombinant Lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets.

Transmissible gastroenteritis coronavirus (TGEV) is one of the most severe threats to the swine industry. In this study, we constructed a suite of recombinant Lactobacillus plantarum with surface displaying the spike (S) protein coming from TGEV and fused with DC cells targeting peptides (DCpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. Our research results found that the recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA-S-DCpep) group expressing S fused with DCpep could not only significantly increase the percentages of MHC-II+CD80+ B cells and CD3+CD4+ T cells but also the number of IgA+ B cells and CD3+CD4+ T cells of ileum lamina propria, which elevated the specific secretory immunoglobulin A (SIgA) titers in feces and IgG titers in serum. Taken together, these results suggest that NC8-pSIP409-pgsA-S-DCpep expressing the S of TGEV fused with DCpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of TGEV vaccines.

Plasmacytoid dendritic cells are dispensable for noninfectious intestinal IgA responses in vivo.

Intestinal DCs orchestrate gut immune homeostasis by dampening proinflammatory T-cell responses and inducing anti-inflammatory IgA responses. Although no specific DC subset has been strictly assigned so far to govern IgA response, some candidate subsets emerge. In particular, plasmacytoid DCs (pDCs), which notoriously promote anti-viral immunity and T-cell tolerance to innocuous antigens (Ags), contribute to IgA induction in response to intestinal viral infection and promote T-cell-independent IgA responses in vitro. Here, using two transgenic mouse models, we show that neither short-term nor long-term pDC depletion alters IgA class switch recombination in Peyer's patches and frequency of IgA plasma cells in intestinal mucosa at steady state, even in the absence of T-cell help. In addition, pDCs are dispensable for induction of intestinal IgA plasma cells in response to oral immunization with T-cell-dependent or T-cell-independent Ags, and are not required for proliferation and IgA switch of Ag-specific B cells in GALT. These results show that pDCs are dispensable for noninfectious IgA responses, and suggest that various DC subsets may play redundant roles in the control of intestinal IgA responses.