Probiotic Vaccine Research Articles

Leveraging the potential of bacterial lateral gene transfer in boosting the efficacy of an edible probiotic prototype yogurt vaccine for COVID-19

Administration of coronavirus disease-2019 (COVID-19) vaccines with appropriate booster doses through painful injections under clinical supervision was challenging during the recent COVID-19 pandemic. As an alternative solution, we designed a safer, edible probiotic yogurt vaccine prototype (YoVac) that can be orally consumed by circumventing painful injections and clinical supervision. We hypothesized that YoVac prepared using Lactobacillus carrying an antigen coding gene (donor) can transfer the same to other bacteria (recipients) in the human gut microbiome (hgMb) through lateral gene transfer (LGT) for boosted antigen levels potentially triggering a robust immune response. In this study we confirmed the in vitro LGT efficiency of a plasmid (pRBD-Ampr) containing severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) spike protein-receptor binding domain (RBD) coding gene along with an ampicillin-resistance gene (selection marker) from the probiotic Lactobacillus (donor) cultured from homemade yogurt to E. coli and Helicobacter pylori (recipients). Both the donor and recipient bacteria not only exhibited ampicillin-resistance from pRBD-Ampr but also expressed RBD protein. Furthermore, Lactobacillus isolated from YoVac consistently showed the expression of RBD protein over a period of one month confirming the shelf life of our prototype stored at 4 °C. Taken together, our in vitro results provide a preliminary basis for the potential in vivo transfer of RBD coding gene from YoVac to other bacterial species in the hgMb through LGT and may potentially boost the vaccine dosage by delegating them.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Oral Lactobacillus casei expressing VP56310–500 and adjuvant flagellin C delivered by alginate-chitosan microcapsules remarkably enhances the immune protection against GCRV infection in grass carp

Due to grass carp haemorrhagic disease caused by grass carp reovirus (GCRV), aquaculture industry suffers huge economic loss. Oral Lactobacillus casei vaccine, with its excellent safety and effectiveness, is a promising strategy to prevent infectious diseases. While, the oral vaccine encapsulated with alginate–chitosan (SA/CS) can further improve its immune function. In this study, recombinant L. casei (rLc) expressing multiple fragments of VP56 was constructed, which was a key outer capsid protein of GCRV. By mixing rLcs with fodder and fed grass carp, the corresponding anti-VP56 fragment sera was produced, and VP56310–500 was screened as optimal fragment of GCRV-II by adopting enzyme linked immunosorbent assay (ELISA). Furthermore, SA/CS was used as the encapsulating material of L. casei live vaccine expressing VP56310–500, thereby preparing oral microcapsule live vaccine SCL-56310-500. With good resistance to bile (0–0.4%), NaCl (0–0.8%), simulated intestinal fluid, and simulated gastric fluid (pH = 1.5–4.5). Also, it had the ability to maintain high viability after colonizing intestine of grass carp in 7 days. In addition, Flagellin C (FlaC) was adopted as an immune adjuvant, and rLc was constructed to express VP56310–500-FlaC (56310–500C), thereby enhancing immune effect of oral live vaccine. After oral administration of encapsulated L. casei vaccine, its protective effect on GCRV-II infection was evaluated. As indicated by the results, SCL-56310-500C effectively up-regulated the activity of serum biochemical enzymes (including TSOD, lysozyme, and complement C3), and induced mRNA expression of important immune genes (including IL-1β, IFN1, MHC-IIα, IgM, and IgT in immune tissues). Simultaneously, SCL-56310-500C greatly suppressed GCRV replication in head kidney, spleen, and intestine. Correspondingly, grass carp vaccinated orally with SCL-56310-500C have distinctly higher survival rate (58% versus 24% in control) against GCRV infection. These results illustrated that an oral probiotic vaccine SCL-56310-500C effective in GCRV infection prevention was developed. Thus, this study offered an inspiring strategy for the prevention of fish viral diseases.

Oral Vaccination Using a Probiotic Vaccine Platform Combined with Prebiotics Impacts Immune Response and the Microbiome.

Unique to mucosal vaccination is the reciprocal influence of the microbiome and mucosal immune responses, where the immune system is constantly balancing between the clearance of pathogens and the tolerance of self-antigen, food, and the microbiota. Secretory IgA plays a major role in maintaining the homeostasis of a healthy gut microbiome. Natural polyreactive IgA often coats members of the commensal microbiota to aid in their colonization, while high-affinity specific IgA binds to pathogens resulting in their clearance. We developed a probiotic-based mucosal vaccination platform using the bacterium Lactobacillus acidophilus (rLA) with the potential to influence this balance in the IgA coating. In this study, we sought to determine whether repeated administration of rLA alters the host intestinal microbial community due to the immune response against the rLA vaccine. To address this, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA-bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction and that the rLA platform did not cause lasting disturbances to the microbiome.

Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-β2-ε-β1 Toxoids to Induce Protective Immunity.

Clostridium perfringens (C. perfringens) is a bacterium that commonly causes zoonotic disease. The pathogenicity of C. perfringens is a result of the combined action of α, β, and ε exotoxins. In this study, Lactobacillus crispatus (pPG-T7g10/L. crispatus) expressing the main toxoids of C. perfringens, α, ε, β1, and β2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-β2-ε-β1 toxoid was constitutively expressed for confirmation by laser confocal microscopy and western blotting, and its immunogenicity was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical assays. After booster immunization, the probiotic vaccine group showed significantly higher levels (p < 0.05) of specific secretory IgA (sIgA) and IgY antibodies in the serum and intestinal mucus. Furthermore, the levels of cytokines, including interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17, and the proliferation of spleen lymphocytes in chickens orally immunized with pPG-E-α-β2-ε-β1/L. crispatus increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-β2ε-β1/L. crispatus were significantly alleviated. These data reveal that the probiotic vaccine could stimulate mucosal, cellular, and humoral immunity and provide an active defense against the toxins of C. perfringens, suggesting a promising candidate for oral vaccines against C. perfringens.

Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase.

Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing foreign antigens. In this study, we generated and evaluated the live mucosal recombinant vaccine by integrating genes encoding influenza virus neuraminidase (NA) of the N2 subtype into the DNA of the probiotic strain Enterococcus faecium L3 (L3). We confirmed NA expression in the pili of L3 using immune electron microscopy. Mice were fed with a probiotic vaccine containing the NA gene (L3-NA) or pure L3. Oral administration of L3-NA caused detectable increase in virus-specific serum IgG and local IgA after the third feeding. Immunization with L3-NA increased the survival rate by 34% when the mice were infected using A(H1N1)pdm09 influenza virus after the third feeding. After S. pneumoniae post-influenza infection, the L3-NA-immunized mice were 50% more protected from lethality in comparison with L3-fed mice. Thus, a live probiotic vaccine candidate based on L3 induced the formation of systemic and local immunity and provide partial protection against complicated influenza.

A Live Probiotic Vaccine Prototype Based on Conserved Influenza a Virus Antigens Protect Mice against Lethal Influenza Virus Infection.

Background: Due to the highly variable nature of the antigenic properties of the influenza virus, many efforts have been made to develop broadly reactive influenza vaccines. Various vaccine platforms have been explored to deliver conserved viral antigens to the target cells to induce cross-reactive immune responses. Here, we assessed the feasibility of using Enterococcus faecium L3 as a bacterial vector for oral immunization against influenza virus. Methods: we generated two vaccine prototypes by inserting full-length HA2 (L3-HA2) protein or its long alpha helix (LAH) domain in combination with four M2e tandem repeats (L3-LAH+M2e) into genome of E.faecium L3 probiotic strain. The immunogenicity and protective potential of these oral vaccines were assessed in a lethal challenge model in BALB/c mice. Results: as expected, both vaccine prototypes induced HA stem-targeting antibodies, whereas only L3-LAH+4M2e vaccine induced M2e-specific antibody. The L3-HA2 vaccine partially protected mice against lethal challenge with two H1N1 heterologous viruses, while 100% of animals in the L3-LAH+4M2e vaccine group survived in both challenge experiments, and there was significant protection against weight loss in this group, compared to the L3 vector-immunized control mice. Conclusions: the recombinant enterococcal strain L3-LAH+4M2e can be considered as a promising live probiotic vaccine candidate for influenza prevention and warrants further evaluation in relevant pre-clinical models.

Oral Probiotic Vaccine Expressing Koi Herpesvirus (KHV) ORF81 Protein Delivered by Chitosan-Alginate Capsules Is a Promising Strategy for Mass Oral Vaccination of Carps against KHV Infection.

Koi herpesvirus (KHV) is highly contagious and lethal to cyprinid fish, causing significant economic losses to the carp aquaculture industry, particularly to koi carp breeders. Vaccines delivered through intramuscular needle injection or gene gun are not suitable for mass vaccination of carp. So, the development of cost-effective oral vaccines that are easily applicable at a farm level is highly desirable. In this study, we utilized chitosan-alginate capsules as an oral delivery system for a live probiotic (Lactobacillus rhamnosus) vaccine, pYG-KHV-ORF81/LR CIQ249, expressing KHV ORF81 protein. The tolerance of the encapsulated recombinant Lactobacillus to various digestive environments and the ability of the probiotic strain to colonize the intestine of carp was tested. The immunogenicity and the protective efficacy of the encapsulated probiotic vaccine was evaluated by determining IgM levels, lymphocyte proliferation, expression of immune-related genes, and viral challenge to vaccinated fish. It was clear that the chitosan-alginate capsules protected the probiotic vaccine effectively against extreme digestive environments, and a significant level (P < 0.01) of antigen-specific IgM with KHV-neutralizing activity was detected, which provided a protection rate of ca. 85% for koi carp against KHV challenge. The strategy of using chitosan-alginate capsules to deliver probiotic vaccines is easily applicable for mass oral vaccination of fish.IMPORTANCE An oral probiotic vaccine, pYG-KHV-ORF81/LR CIQ249, encapsulated by chitosan-alginate capsules as an oral delivery system was developed for koi carp against koi herpesvirus (KHV) infection. This encapsulated probiotic vaccine can be protected from various digestive environments and maintain effectively high viability, showing a good tolerance to digestive environments. This encapsulated probiotic vaccine has a good immunogenicity in koi carp via oral vaccination, and a significant level of antigen-specific IgM was effectively induced after oral vaccination, displaying effective KHV-neutralizing activity. This encapsulated probiotic vaccine can provide effective protection for koi carp against KHV challenge, which is handling-stress free for the fish, cost effective, and suitable for the mass oral vaccination of koi carp at a farm level, suggesting a promising vaccine strategy for fish.

Bacillus subtilis spore vaccines displaying protective antigen induce functional antibodies and protective potency

BackgroundBacillus anthracis is the causative agent of anthrax, a disease of both humans and various animal species, and can be used as a bioterror agent. Effective vaccines are available, but those could benefit from improvements, including increasing the immunity duration, reducing the shot frequency and adverse reactions. In addition, more sophisticated antigen delivery and potentiation systems are urgently required.The protective antigen (PA), one of three major virulence factors associated with anthrax was displayed on the surface of Bacillus subtilis spores, which is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administration as it is safe for human consumption and the particulate adjuvanticity. Mice were immunized orally (PO), intranasally (IN), sublingually (SL) or intraperitoneally (IP) with the PA displaying probiotic spore vaccine. Clinical observation, serological analysis and challenge experiment were conducted to investigate the safety and efficacy of the vaccine.ResultsA/J mice immunized with the PA spore vaccine via PO, IN, SL, and IP were observed to have increased levels of active antibody titer, isotype profiles and toxin neutralizing antibody in sera, and IgA in saliva. The immunized mice were demonstrated to raise protective immunity against the challenge with lethal B. anthracis spores.ConclusionsIn this study, we developed a B. subtilis spore vaccine that displays the PA on its surface and showed that the PA-displaying spore vaccine was able to confer active immunity to a murine model based on the results of antibody isotype titration, mucosal antibody identification, and a lethal challenge experiment.

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.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Protection Efficacy of Oral Bait Probiotic Vaccine Constitutively Expressing Tetravalent Toxoids against Clostridium perfringens Exotoxins in Livestock (Rabbits)

Clostridium perfringens is an opportunistic pathogen. Its main virulence factors are exotoxins, which are the etiological agents of enteritis necroticans and enterotoxemia caused in livestock (cattle, sheep, and rabbits). Here, we demonstrated effective immune protection for rabbits against α, β, and ε exotoxins of C. perfringens provided by an oral tetravalent bait probiotic vaccine delivering α, ε, β1, and β2 toxoids of C. perfringens. Results showed that the recombinant probiotic had good segregational stability and good colonization ability in the rabbit intestinal tract. Oral administration of the probiotic vaccine can effectively elicit significant levels of antigen-specific mucosa sIgA and sera IgG antibodies with exotoxin-neutralizing activity. Additionally, oral immunization with the probiotic vaccine effectively promoted lymphoproliferation and Th1/Th2-associated cytokine production. The protection rate of immunized rabbits with the probiotic vaccine was 80% after challenging rabbits with a combination of C. perfringens (toxinotypes A, C, and D) and exotoxin mixture, which was better than the 60% provided by a commercial inactivated C. perfringens A, C, and D trivalent vaccine. Moreover, obvious histopathological changes were observed in the intestinal tissues of rabbits in the commercial vaccine and PBS groups. The bait probiotic vaccine can provide effective protection against C. perfringens exotoxins, suggesting a promising C. perfringens vaccination strategy.

Protective Immunity against Canine Distemper Virus in Dogs Induced by Intranasal Immunization with a Recombinant Probiotic Expressing the Viral H Protein.

Canine distemper virus (CDV) elicits a severe contagious disease in a broad range of hosts. CDV mortality rates are 50% in domestic dogs and 100% in ferrets. Its primary infection sites are respiratory and intestinal mucosa. This study aimed to develop an effective mucosal CDV vaccine using a non-antibiotic marked probiotic pPGΔCm-T7g10-EGFP-H/L. casei 393 strain expressing the CDV H protein. Its immunogenicity in BALB/c mice was evaluated using intranasal and oral vaccinations, whereas in dogs the intranasal route was used for vaccination. Our results indicate that this probiotic vaccine can stimulate a high level of secretory immunoglobulin A (sIgA)-based mucosal and IgG-based humoral immune responses in mice. SIgA levels in the nasal lavage and lungs were significantly higher in intranasally vaccinated mice than those in orally vaccinated mice. Both antigen-specific IgG and sIgA antibodies were effectively elicited in dogs through the intranasal route and demonstrated superior immunogenicity. The immune protection efficacy of the probiotic vaccine was evaluated by challenging the immunized dogs with virulent CDV 42 days after primary immunization. Dogs of the pPGΔCm-T7g10-EGFP-H/L. casei 393 group were completely protected against CDV. The proposed probiotic vaccine could be promising for protection against CDV infection in dogs.

Recombinant Lactobacillus casei Expressing Capsid Protein VP60 can Serve as Vaccine Against Rabbit Hemorrhagic Disease Virus in Rabbits.

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD). RHD, characterized by hemorrhaging, liver necrosis, and high morbidity and mortality in rabbits and hares, causes severe economic losses in the rabbit industry worldwide. Due to the lack of an efficient in-vitro propagation system for RHDV, the current vaccine is produced via chemical inactivation of crude RHDV preparation derived from the livers of infected rabbits. Inactivated vaccines are effective for controlling RHD, but the potential problems of biosafety and animal welfare have negative effects on the application of inactivated vaccines. In this study, an oral Lactobacillus casei (L. casei) vaccine was used as an antigen delivery system to express RHDV capsid protein VP60(VP1)-eGFP fusion protein. The expression of the recombinant protein was confirmed via western blotting and immunofluorescence (IFA). Our results indicate that oral administration of this probiotic vaccine can stimulate secretory immunoglobulin A (SIgA)-based mucosal and IgG-based humoral immune responses in rabbits. The immunized rabbits were completely protected against challenge with RHDV. Our findings indicate that the L. casei expression system is a new strategy for the development of a safe and efficient vaccine against RHDV.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer’s patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiV’s immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera.

An EGFP-marked recombinant lactobacillus oral tetravalent vaccine constitutively expressing α, ε, β1, and β2 toxoids for Clostridium perfringens elicits effective anti-toxins protective immunity

ABSTRACTClostridium perfringens is a common opportunistic pathogen endangering livestock and poultry breeds. Here, using enhanced green fluorescent protein as screening marker, a recombinant lactobacillus tetravalent vaccine constitutively expressing α, ε, β1, and β2 toxoids of C. perfringens was developed, and its immunogenicity in mice was investigated via oral administration. This probiotic vaccine could effectively induce antigen-specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immune responses, and significantly high levels (p< 0.05) of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ were produced in immunized mice. Moreover, lymphoproliferation and percentage of CD4+ and CD8+ T cells significantly increased in mice of the probiotic vaccine group. Challenge experiments were performed in mice with C. perfringens toxinotypes A, C, and D crude toxins to evaluate protection efficiency of the probiotic vaccine, using a commercial inactivated C. perfringens vaccine made by C. perfringens toxinotypes A, C, and D as vaccine control. We observed 80% protection rate in the probiotic vaccine group, which was higher than commercial vaccine group, whereas all mice in control groups died and obvious histopathological changes were observed in liver, spleen, kidney, and intestines of mice. Significantly, we compared the immunogenicity and protection efficiency of lactobacillus constitutive expression system and lactobacillus inducible expression system, and results showed that lactobacillus constitutive expression system has obvious advantages. Our study clearly demonstrated that the probiotics vaccine could effectively induce mucosal, humoral, and cellular immunity, and provide effective protection against C. perfringens toxins, suggesting a promising strategy for the development of oral vaccine against C. perfringens.

Oral immunization of mice with a probiotic Lactobacillus casei constitutively expressing the α-toxoid induces protective immunity against Clostridium perfringens α-toxin

ABSTRACTClostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin.

Dietary Application of the Probiotic Lactobacillus plantarum 426951 Enhances Immune Status and Growth of Rainbow Trout (Oncorhynchus mykiss) Vaccinated Against Yersinia ruckeri.

This study was aimed to assess the effect of oral application of Lactobacillus plantarum (2×107CFUg-1 feed) as a probiotic on growth performance and immune status of vaccinated rainbow trout (29.5 ± 2g) to yersiniosis at 16 ± 2°C for 72days. Fish were randomly allocated into 12 fiber glass tanks (4100L) at a density of 80 fish per tank (240 fish per treatment). The results revealed that the activity of lysozyme and alkaline phosphatase was significantly higher in immunized fish fed with diet supplemented with probiotic (vaccine +probiotic) than that in the immunized group fed with basal diet (vaccine group) while no significant differences in levels of hematological parameters, complements, total IgM, proteins, and the intestine lactic acid bacteria (LAB) were detected. Also, significantly a better growth performance in terms of feed conversion ratio, weight gain, and thermal growth coefficient was seen in the vaccine + probiotic group than that in the vaccine group. These results indicate that feeding probiotic after vaccination can enhance the efficacy of immersion vaccination to Yersinia ruckeri.

Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences (p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.