Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r =  - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer\'s patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1β, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Avian influenza virus subtype H9N2 has the ability to infect birds and humans, further causing significant losses to the poultry industry and even posing a great threat to human health. Oral vaccine received particular interest for preventing majority infection due to its ability to elicit both mucosal and systemic immune responses, but their development is limited by the bad gastrointestinal (GI) environment, compact epithelium and mucus barrier, and the lack of effective mucosal adjuvants. Herein, we developed the dendritic fibrous nano-silica (DFNS) grafted with Cistanche deserticola polysaccharide (CDP) nanoparticles (CDP-DFNS) as an adjuvant for H9N2 vaccine. Encouragingly, CDP-DFNS facilitated the proliferation of T and B cells, and further induced the activation of T lymphocytes in vitro. Moreover, CDP-DFNS/H9N2 significantly promoted the antigen-specific antibodies levels in serum and intestinal mucosal of chickens, indicating the good ability to elicit both systemic and mucosal immunity. Additional, CDP-DFNS facilitate the activation of CD4 + and CD8 + T cells both in spleen and intestinal mucosal, and the indexes of immune organs. This study suggested that CDP-DFNS may be a new avenue for development of oral vaccine against pathogens that are transmitted via mucosal route.

Oral delivery of cells, such as probiotics and vaccines, has proved to be inefficient since cells are generally damaged in an acidic stomach prior to arrival at the intestine to exert their health benefits. In addition, short retention in the intestine is another obstacle which affects inefficiency. To overcome these obstacles, a cell-in-shell structure was designed with pH-responsive and mucoadhesive properties. The pH-responsive shell consisting of three cationic layers of chitosan and three anionic layers of trans-cinnamic acid (t-CA) was made via layer-by-layer (LbL) assembly. t-CA layers are hydrophobic and impermeable to protons in acid, thus enhancing cell gastric resistance in the stomach, while chitosan layers endow strong interaction between the cell surface and the mucosal wall which facilitates cell mucoadhesion in the intestine. Two model cells, probiotic L. rhamnosus GG and dead Streptococcus iniae, which serve as inactivated whole-cell vaccine were chosen to test the design. Increased survival and retention during oral administration were observed for coated cells as compared with naked cells. Partial removal of the coating (20-60% removal) after acid treatment indicates that the coated vaccine can expose its surface immunogenic protein after passage through the stomach, thus facilitating vaccine immune stimulation in the intestine. As a smart oral delivery platform, this design can be extended to various macromolecules, thus providing a promising strategy to formulate oral macromolecules in the prevention and treatment of diseases at a cellular level.

We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by Agrobacterium-mediated co-transformation and conducted a double-blind, randomized, placebo-controlled phase I trial in Japan and the United States. Although MucoRice-CTB 51A was acceptably safe and well tolerated by healthy Japanese and U.S. subjects and induced CTB-specific antibodies neutralizing cholera toxin secreted by Vibrio cholerae, we were limited to a 6-g cohort in the U.S. trial because of insufficient production of MucoRice-CTB. Since MucoRice-CTB 51A did not grow in sunlight, we re-examined the previously established marker-free lines and selected MucoRice-CTB line 19A. Southern blot analysis of line 19A showed a single copy of the CTB gene. We resequenced the whole genome and detected the transgene in an intergenic region in chromosome 1. After establishing a master seed bank of MucoRice-CTB line 19A, we established a hydroponic production facility with LED lighting to reduce electricity consumption and to increase production capacity for clinical trials. Shotgun MS/MS proteomics analysis of MucoRice-CTB 19A showed low levels of α-amylase/trypsin inhibitor-like proteins (major rice allergens), which was consistent with the data for line 51A. We also demonstrated that MucoRice-CTB 19A had high oral immunogenicity and induced protective immunity against cholera toxin challenge in mice. These results indicate that MucoRice-CTB 19A is a suitable oral cholera vaccine candidate for Phase I and II clinical trials in humans, including a V. cholerae challenge study.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Oral vaccines are a safe and convenient alternative to injected vaccines and have great potential to prevent major infectious diseases. However, the harsh gastrointestinal (GI) environment, mucus barriers, low immunogenicity, and lack of effective and safe mucosal adjuvants are the major challenges for oral vaccine delivery. In recent years, nanoparticle-based strategies have become attractive for improving oral vaccine delivery. Here, the dendritic fibrous nano-silica (DFNS) grafted with Cistanche deserticola polysaccharide (CDP) nanoparticles (CDP-DFNS) were prepared and investigated how to impact the immune responses. CDP-DFNS facilitated the antigen uptake in mouse bone marrow-derived dendritic cells (BMDCs), and induce the activation of DCs in vitro. Furthermore, in vivo experiments, the result showed that the uptake efficiency by Peyer\'s patches (PPs) of CDP-DFNS/BSA was the best. And CDP-DFNS/BSA then significantly activated the DCs in lamina propria (LP), and T/B cells in PPs and mesenteric lymph nodes (MLNs). Moreover, the memory T cell responses in later period of vaccination was stronger than other groups. In addition, CDP-DFNS/BSA enhanced BSA-specific antibody IgG, IgA production, and SIgA secretion, was effective at inducing a strong mixed Th1/Th2 response and mucosal antibody responses. These results indicated that CDP-DFNS deserves further consideration as an oral vaccine adjuvant delivery system.

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of Johne\'s disease (JD) in ruminants, establishes a prolonged and often lifelong enteric infection. The implementation of control measures for bovine JD has faced obstacles due to the considerable expenses involved in disease surveillance and hindered by unreliable and inadequate diagnostic tests, emphasizing the need for an effective vaccine that can stimulate mucosal immunity in the gastrointestinal tract. Previous investigations have demonstrated that deletion of the BacA gene in MAP produces an attenuated strain that can transiently colonize the calf small intestine while retaining its capacity to stimulate systemic immune responses similar to wildtype MAP strains. This study assessed the efficacy of the BacA gene deletion MAP strain, referred to as the BacA vaccine, when administered orally to young calves. The research aimed to evaluate its effectiveness in controlling MAP intestinal infection and to investigate the immune responses elicited by mucosal vaccination. The study represents the first evaluation of an enteric modified live MAP vaccine in the context of an oral MAP challenge in young calves. Oral immunization with BacA reduced MAP colonization specifically in the ileum and ileocecal valve. This partially protective immune response was associated with an increased frequency of CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFNγ+/TNFα+) in vaccinated animals. Moreover, re-stimulated PBMCs from vaccinated animals showed increased expression of IFNγ, IP-10, IL-2, and IL-17 at 10- and 12-weeks post challenge. Furthermore, immunophenotyping of blood leukocytes revealed that vaccinated calves had increased levels of T cells expressing cell-surface markers consistent with long-term central memory. Overall, our findings provide new insights into the development and immunogenicity of a modified live MAP vaccine against bovine JD, demonstrating oral vaccination can stimulate host immune responses that can be protective against enteric MAP infection.

Micropterus salmoides rhabdovirus (MSRV) is one of the main pathogens of largemouth bass, leading to serious economic losses. The G protein, as the only envelope protein present on the surface of MSRV virion, contains immune-related antigenic determinants, thereby becoming the primary target for the design of MSRV vaccines. Here, we displayed the G protein on the surface of yeast cells (named EBY100/pYD1-G) and conducted a preliminary assessment of the protective efficacy of the recombinant yeast vaccine. Upon oral vaccination, a robust immune response was observed in systemic and mucosal tissue. Remarkably, following the MSRV challenge, the relative percent survival of EBY100/pYD1-G treated largemouth bass significantly increased to 66.7 %. In addition, oral administration inhibited viral replication and alleviated the pathological symptoms of MSRV-infected largemouth bass. These results suggest that EBY100/pYD1-G could be used as a potential oral vaccine against MSRV infection.