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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. [ABSTRACT FROM AUTHOR]

Abstract 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.

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (d-integration). The recombinant SC strains containing eight ASFV antigens--KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L-induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV. [ABSTRACT FROM AUTHOR]

Infectious bursal disease (IBD), as a highly infectious immunosuppressive disease, causes severe economic losses in the poultry industry worldwide. Saccharomyces cerevisiae is an appealing vehicle used in oral vaccine formulations to safely and effectively deliver heterologous antigens. It can elicit systemic and mucosal responses. This study aims to explore the potential as oral an vaccine for S. cerevisiae expressing the capsid protein VP2 of IBDV. We constructed the recombinant S. cerevisiae, demonstrated that VP2 was displayed on the cell surface and had high immunoreactivity. By using the live ST1814G/Aga2-VP2 strain to immunize the mice, the results showed that recombinant S. cerevisiae significantly increased specific IgG and sIgA antibody titers, indicating the potential efficacy of vaccine-induced protection. These results suggested that the VP2 protein-expressing recombinant S. cerevisiae strain was a promising candidate oral subunit vaccine to prevent IBDV infection. [ABSTRACT FROM AUTHOR]

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine. [ABSTRACT FROM AUTHOR]

Abstract 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.

Oral immunization is an effective strategy for inducing protective immunity against mucosal enteric pathogens. Although live-attenuated as well as subunit approaches have been explored for vaccination against enteric pathogens, inactivated whole bacterial cells may also be effective in introducing protective immunity. Successfully accomplishing this goal with inactivated whole bacterial cells will require that a complex antigenic repertoire be presented in controlled immunogenic amounts, in a safe and relatively simple and self-contained delivery format. The benefit from immunization with whole cell vaccines can be further enhanced through genetic engineering to over-express selected antigens and also by the use of mucosal adjuvants to direct a more robust immunologic response. These steps are being taken for the development of ETVAX, the most clinically advanced vaccine candidate against the major enteric pathogen, enterotoxigenic Escherichia coli (ETEC) with significant positive impact. [ABSTRACT FROM AUTHOR]

Infectious bursal disease (IBD), as a highly infectious immunosuppressive disease, causes severe economic losses in the poultry industry worldwide. Saccharomyces cerevisiae is an appealing vehicle used in oral vaccine formulations to safely and effectively deliver heterologous antigens. It can elicit systemic and mucosal responses. This study aims to explore the potential as oral an vaccine for S. cerevisiae expressing the capsid protein VP2 of IBDV. We constructed the recombinant S. cerevisiae, demonstrated that VP2 was displayed on the cell surface and had high immunoreactivity. By using the live ST1814G/Aga2-VP2 strain to immunize the mice, the results showed that recombinant S. cerevisiae significantly increased specific IgG and sIgA antibody titers, indicating the potential efficacy of vaccine-induced protection. These results suggested that the VP2 protein-expressing recombinant S. cerevisiae strain was a promising candidate oral subunit vaccine to prevent IBDV infection.

Oral immunization is an effective strategy for inducing protective immunity against mucosal enteric pathogens. Although live-attenuated as well as subunit approaches have been explored for vaccination against enteric pathogens, inactivated whole bacterial cells may also be effective in introducing protective immunity. Successfully accomplishing this goal with inactivated whole bacterial cells will require that a complex antigenic repertoire be presented in controlled immunogenic amounts, in a safe and relatively simple and self-contained delivery format. The benefit from immunization with whole cell vaccines can be further enhanced through genetic engineering to over-express selected antigens and also by the use of mucosal adjuvants to direct a more robust immunologic response. These steps are being taken for the development of ETVAX, the most clinically advanced vaccine candidate against the major enteric pathogen, enterotoxigenic Escherichia coli (ETEC) with significant positive impact.

Introduction: Rabies is a worldwide epidemic that poses a serious threat to global public health. At present, rabies in domestic dogs, cats, and some pets can be effectively prevented and controlled by intramuscular injection of rabies vaccine. But for some inaccessible animals, especially stray dogs, and wild animals, it is difficult to prevent with intramuscular injection. Therefore, it is necessary to develop a safe and effective oral rabies vaccine. Methods: We constructed recombinant Bacillus subtilis (B. subtilis) expressing two different strains of rabies virus G protein, named CotG-E-G and CotG-C-G, immunogenicity was studied in mice. Results: The results showed that CotG-E-G and CotG-C-G could significantly increase the specific SIgA titers in feces, serum IgG titers, and neutralizing antibodies. ELISpot experiments showed that CotG-E-G and CotG-C-G could also induce Th1 and Th2 to mediate the secretion of immune-related IFN-γ and IL-4. Collectively, our results suggested that recombinant B. subtilis CotG-E-G and CotG-C-G have excellent immunogenicity and are expected to be novel oral vaccine candidates for the prevention and control of wild animal rabies. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a highly contagious, acute, febrile disease caused by the African swine fever virus (ASFV), with morbidity and mortality rates approaching 100% in domestic and wild swine, resulting in massive economic losses to the pig industry worldwide. This study aimed to express the p30, p54, and p72 proteins encoded by ASFV in vitro using the Lactobacillus lactis (L. lactis) expression system. Here, six new functional recombinant L. lactis were constructed, and the expression of the p30 protein, p54 protein, p72 protein, p30-LTB (heat-labile enterotoxin B, LTB) fusion protein, p54-LTB fusion protein, and the p72-LTB fusion protein was successfully detected by Western blot analysis. Following oral immunization of rabbits with recombinant L. lactis, serum IgG, intestinal mucosal sIgA, cytokines (IL-4 and INF-γ), and splenocyte viability were higher than in the control group via ELISA. Notably, without the LTB adjuvant group, humoral and Th1 cellular immunity were promoted, whereas, with the LTB adjuvant group, local mucosal immunity, humoral immunity, and Th2 cellular immunity were promoted, providing new insights into the design and development of an ASFV subunit vaccine. [ABSTRACT FROM AUTHOR]

VP1, a major immunogenic protein of foot-and-mouth disease virus (FMDV), facilitates viral attachment and entry into host cells. VP1 possesses critical epitope sequences responsible for inducing neutralizing antibodies but its expression using Saccharomyces cerevisiae has been hampered despite evidence that the presence of VP1 does not negatively impact the yeast's biology. In this study, we fused proteins to enhance VP1 expression using S. cerevisiae. Among short P1 chimeras containing VP1 including VP3-VP1 and VP2-VP1, VP3-VP1 fusion proteins showed higher expression levels than VP2-VP1. We subsequently designed new fusion proteins, of which 20 amino acids of N-terminal VP3 fused with VP1-Co1 (referred to 20aaVP3-VP1-Co1) showed the highest expression level. Lowering the culture temperature from 30 ⁰C to 20 ⁰C further enhanced fusion protein production. The highest expression level of 20aaVP3-VP1-Co1 was estimated to be 7.7 mg/L, which is comparable to other heterologous proteins produced using our S. cerevisiae expression system. Oral administration of the cell expressing 20aaVP3-VP1-Co1 induced VP1-specific IgG and IgA responses in mice. The S. cerevisiae -expressed 20aaVP3-VP1-Co1 fusion protein induced a significant immune response to the FMDV structural epitope protein, which opens the possibility of an oral FMDV vaccine. • High expression of the structural capsid VP1 protein of FMDV using S. cerevisiae. • Decreased proteolytic degradation of the VP1 by an extension of N-terminus of VP1. • Oral immune response of recombinant cells by fusing M cell-targeting ligands to VP1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (alr), in the genome of Lactobacillus casei strain W56 (L. casei W56) was knocked out to construct an auxotrophic L. casei strain (L. casei Δalr W56) using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/Δalr W56) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection. [ABSTRACT FROM AUTHOR]

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Background and Objectives: Hepatitis-hydropericardium syndrome (HHS) caused by Fowl adenoviruses serotype 4 (FAdV-4) leads to severe economic losses to the poultry industry. Although various vaccines are available, vaccines that effectively stimulate intestinal mucosal immunity are still deficient. In the present study, novel probiotics that surface-deliver Fiber2 protein, the major virulence determiner and efficient immunogen for FAdV-4, were explored to prevent this fecal–oral-transmitted virus, and the induced protective immunity was evaluated after oral immunization. Methods: The probiotic Enterococcus faecalis strain MDXEF-1 and Lactococcus lactis NZ9000 were used as host strains to deliver surface-anchoring Fiber2 protein of FAdV-4. Then the constructed live recombinant bacteria were orally vaccinated thrice with chickens at intervals of 2 weeks. Following each immunization, immunoglobulin G (IgG) in sera, secretory immunoglobulin A (sIgA) in jejunum lavage, immune-related cytokines, and T-cell proliferation were detected. Following challenge with the highly virulent FAdV-4, the protective effects of the probiotics surface-delivering Fiber2 protein were evaluated by verifying inflammatory factors, viral load, liver function, and survival rate. Results: The results demonstrated that probiotics surface-delivering Fiber2 protein stimulated humoral and intestinal mucosal immune responses in chickens, shown by high levels of sIgA and IgG antibodies, substantial rise in mRNA levels of cytokines, increased proliferative ability of T cells in peripheral blood, improved liver function, and reduced viral load in liver. Accordingly, adequate protection against homologous challenges and a significant increase in the overall survival rate were observed. Notably, chickens orally immunized with E. faecalis /DCpep-Fiber2-CWA were completely protected from the FAdV-4 challenge, which is better than L. lactis /DCpep-Fiber2-CWA. Conclusion: The recombinant probiotics surface-expressing Fiber2 protein could evoke remarkable humoral and cellular immune responses, relieve injury, and functionally damage target organs. The current study indicates a promising method used for preventing FAdV-4 infection in chickens. [ABSTRACT FROM AUTHOR]

Infections with Pseudomonas aeruginosa (PA) pose a major clinical threat worldwide especially to immunocompromised patients. As a novel vaccine network for many kinds of bacteria, bacterial ghosts (BGs) have recently been introduced. In the present research, using Sponge-Like Reduced Protocol, P. aeruginosa ghosts (PAGs) were prepared to maintain surface antigens and immunogenicity. This is the first study, to our knowledge, on the production of chemically induced well-structured bacterial ghosts for PA using concentrations of different chemicals. The research was carried out using diabetic rats who were orally immunized at two-week intervals with three doses of PAGs. Rats were subsequently challenged either by the oral route or by the model of ulcer infection with PA. In challenged rats, in addition to other immunological parameters, organ bioburden and wound healing were determined, respectively. Examination of the scanning and transmission electron microscope (EM) proved that PAGs with a proper three-dimensional structure were obtained. In contrast to control groups, oral PAGs promoted the generation of agglutinating antibodies, the development of IFN-γ, and the increase in phagocytic activity in vaccinated groups. Antibodies of the elicited PAGs were reactive to PA proteins and lipopolysaccharides. The defense against the PA challenge was observed in PAGs-immunized diabetic rats. The resulting PAGs in orally vaccinated diabetic rats were able to evoke unique humoral and cell-mediated immune responses and to defend them from the threat of skin wound infection. These results have positive implications for future studies on the PA vaccine. [ABSTRACT FROM AUTHOR]

Background and ObjectivesHepatitis-hydropericardium syndrome (HHS) caused by Fowl adenoviruses serotype 4 (FAdV-4) leads to severe economic losses to the poultry industry. Although various vaccines are available, vaccines that effectively stimulate intestinal mucosal immunity are still deficient. In the present study, novel probiotics that surface-deliver Fiber2 protein, the major virulence determiner and efficient immunogen for FAdV-4, were explored to prevent this fecal–oral-transmitted virus, and the induced protective immunity was evaluated after oral immunization.MethodsThe probiotic Enterococcus faecalis strain MDXEF-1 and Lactococcus lactis NZ9000 were used as host strains to deliver surface-anchoring Fiber2 protein of FAdV-4. Then the constructed live recombinant bacteria were orally vaccinated thrice with chickens at intervals of 2 weeks. Following each immunization, immunoglobulin G (IgG) in sera, secretory immunoglobulin A (sIgA) in jejunum lavage, immune-related cytokines, and T-cell proliferation were detected. Following challenge with the highly virulent FAdV-4, the protective effects of the probiotics surface-delivering Fiber2 protein were evaluated by verifying inflammatory factors, viral load, liver function, and survival rate.ResultsThe results demonstrated that probiotics surface-delivering Fiber2 protein stimulated humoral and intestinal mucosal immune responses in chickens, shown by high levels of sIgA and IgG antibodies, substantial rise in mRNA levels of cytokines, increased proliferative ability of T cells in peripheral blood, improved liver function, and reduced viral load in liver. Accordingly, adequate protection against homologous challenges and a significant increase in the overall survival rate were observed. Notably, chickens orally immunized with E. faecalis/DCpep-Fiber2-CWA were completely protected from the FAdV-4 challenge, which is better than L. lactis/DCpep-Fiber2-CWA.ConclusionThe recombinant probiotics surface-expressing Fiber2 protein could evoke remarkable humoral and cellular immune responses, relieve injury, and functionally damage target organs. The current study indicates a promising method used for preventing FAdV-4 infection in chickens.

ABSTRACT The symptoms of Clostridioides difficile infection (CDI) are largely attributed to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. Recently, we generated a novel chimeric protein (designated Tcd169), comprised of the glucosyltransferase domain (GT), the cysteine protease domain (CPD), and the receptor binding domain (RBD) of TcdB, and the RBD of TcdA. Parenteral immunizations with Tcd169 provide mice effective protection against infection with a ribotype (RT) 027 C. difficile strain. In this study, we expressed Tcd169 in a nontoxigenic C. difficile CCUG37785 strain (designated NTCD), resulting in strain NTCD_Tcd169 to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors. Oral immunizations with NTCD_Tcd169 spores induced systematic and mucosal antibody responses against, not only both toxins, but also C. difficile flagellins (FliC/FliD). Intriguingly yet importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2). Oral immunizations with NTCD_Tcd169 spores provided mice effective protection against infection with a hypervirulent RT027 C. difficile strain R20291and significantly reduced R20291spore numbers in feces compared with NTCD or PBS immunized mice. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing Tcd169 may represent a novel mucosal vaccine candidate against CDI. IMPORTANCE Clostridioides difficile is an enteric pathogen, and symptoms of C. difficile infection (CDI) are mainly by two exotoxins TcdA and TcdB. Active vaccination is cost-effective approach to prevent CDI and high rates of recurrence. Ideally, vaccines should target both C. difficile toxins and cell/spore colonization. In this study, we expressed immunodominant fragments of TcdA and TcdB (i.e., Tcd169) in a nontoxigenic C. difficile CCUG37785 strain, generating a promising oral/mucosal vaccine candidate against CDI, by targeting both toxins and colonization of pathogenic C. difficile strains. Importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2), and all of which are involved in C. difficile adhesion/colonization in vitro and in vivo.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds causing important economic losses in the poultry industry worldwide. We have previously developed a plant-based vaccine candidate for infectious bursal disease (IBD) that is able to protect against infection with IBDV when administered through intramuscular (im) route. Given that oral vaccination is non-invasive and stimulates the immunity of the mucosal gastrointestinal surface, the initial site of contact and entry of IBDV, the aim of this work was to study if our immunogen was also able to elicit a protective immune response when orally administered. We demonstrated that 85% of the animals that received two oral doses of the vaccine formulation and all animals that were orally boosted after an im prime scheme developed virus neutralizing antibodies and were protected against IBDV infection, evidenced by the bursa/body weight (BB) ratio, absence of T-cell infiltration, and low viral load in bursa. Although mild to moderate bursal damage was observed in some of these animals, these lesions were not as severe as the ones observed in challenged control groups, which also presented signs of acute inflammation, bursal atrophy, T-cell infiltration, and absence of viral clearance. These results show that two immunizations with our recombinant immunogen are able to induce a specific and protective immune response in chicken against IBDV when orally administered in a prime/boost scheme or when the oral boost follows an im prime scheme. In conclusion, our oral plant-based vaccine candidate could represent a viable alternative to conventional vaccines and is of great interest to the poultry industry. [ABSTRACT FROM AUTHOR]

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds causing important economic losses in the poultry industry worldwide. We have previously developed a plant-based vaccine candidate for infectious bursal disease (IBD) that is able to protect against infection with IBDV when administered through intramuscular (im) route. Given that oral vaccination is non-invasive and stimulates the immunity of the mucosal gastrointestinal surface, the initial site of contact and entry of IBDV, the aim of this work was to study if our immunogen was also able to elicit a protective immune response when orally administered. We demonstrated that 85% of the animals that received two oral doses of the vaccine formulation and all animals that were orally boosted after an im prime scheme developed virus neutralizing antibodies and were protected against IBDV infection, evidenced by the bursa/body weight (BB) ratio, absence of T-cell infiltration, and low viral load in bursa. Although mild to moderate bursal damage was observed in some of these animals, these lesions were not as severe as the ones observed in challenged control groups, which also presented signs of acute inflammation, bursal atrophy, T-cell infiltration, and absence of viral clearance. These results show that two immunizations with our recombinant immunogen are able to induce a specific and protective immune response in chicken against IBDV when orally administered in a prime/boost scheme or when the oral boost follows an im prime scheme. In conclusion, our oral plant-based vaccine candidate could represent a viable alternative to conventional vaccines and is of great interest to the poultry industry.

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (alr), in the genome of Lactobacillus casei strain W56 (L. casei W56) was knocked out to construct an auxotrophic L. casei strain (L. casei Δalr W56) using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/Δalr W56) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV 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. • The recombinant L. lactis expressing VP4 greatly enhanced nonspecific immune response and specific immune response of fish. • Both humoral immunity and cellular immunity could effectively induced by oral the recombinant strian. • Mucosal immunity was elicited following oral immunization with the recombinant strain to enhance the protective effect. • Compare with PBS, oral vaccine provided RPS of 42.9%. [ABSTRACT FROM AUTHOR]

Clostridium 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

Clostridium 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.

Infections with Pseudomonas aeruginosa (PA) pose a major clinical threat worldwide especially to immunocompromised patients. As a novel vaccine network for many kinds of bacteria, bacterial ghosts (BGs) have recently been introduced. In the present research, using Sponge-Like Reduced Protocol, P. aeruginosa ghosts (PAGs) were prepared to maintain surface antigens and immunogenicity. This is the first study, to our knowledge, on the production of chemically induced well-structured bacterial ghosts for PA using concentrations of different chemicals. The research was carried out using diabetic rats who were orally immunized at two-week intervals with three doses of PAGs. Rats were subsequently challenged either by the oral route or by the model of ulcer infection with PA. In challenged rats, in addition to other immunological parameters, organ bioburden and wound healing were determined, respectively. Examination of the scanning and transmission electron microscope (EM) proved that PAGs with a proper three-dimensional structure were obtained. In contrast to control groups, oral PAGs promoted the generation of agglutinating antibodies, the development of IFN-γ, and the increase in phagocytic activity in vaccinated groups. Antibodies of the elicited PAGs were reactive to PA proteins and lipopolysaccharides. The defense against the PA challenge was observed in PAGs-immunized diabetic rats. The resulting PAGs in orally vaccinated diabetic rats were able to evoke unique humoral and cell-mediated immune responses and to defend them from the threat of skin wound infection. These results have positive implications for future studies on the PA vaccine.

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low‐grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant‐based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB‐CdeM), carrying the CdeM antigen, fused to the carboxi‐terminus of the B subunit of the Escherichia coli heat‐labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB‐CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB‐CdeM antigen in the range of 52–90 μg/g dry weight leaf tissues. The antigenicity of the plant‐made LTB‐CdeM antigen was evidenced by GM1‐ELISA and immunogenicity assessment performed in test mice revealed that the LTB‐CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant‐based vaccines against C. difficile infection. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hepatitis-hydropericardium syndrome (HPS) causes severe economic losses in the global poultry industry. The present study aims to explore oral immunization of recombinant Lactococcus lactis and Enterococcus faecalis expressing Hexon protein of fowl adenovirus 4 (FAdV-4). The bacteria L. lactis NZ9000 and E. faecalis MDXEF-1 were, respectively, modified as host strain to deliver truncated Hexon protein (ΔHexon) or ΔHexon protein fusing with dendritic cell (DC) targeting peptide (DC-ΔHexon) on the surface of bacteria. The expression of target protein in L. lactis NZ9000 and E. faecalis MDXEF-1 were detected by western blot. To evaluate the immune responses and protective efficacies provided by the live recombinant bacteria, chickens were immunized with the constructed ΔHexon-expressing bacteria three times at 2-week intervals, then experimentally challenged with hypervirulent FAdV-4/GX01. The results showed that oral immunizations with the four ΔHexon-expressing bacteria (NZ9000/ΔHexon-CWA, NZ9000/DC-ΔHexon-CWA, MDXEF-1/ΔHexon-CWA, and MDXEF-1/DC-ΔHexon-CWA), especially the two bacteria carrying DC-targeting peptide, stimulated higher levels of ΔHexon-specific sera IgG and secretory IgA (sIgA) in jejunal lavage fluid, higher proliferation of peripheral blood lymphocytes (PBLs) and higher levels of Th1/Th2-type cytokines, along with significantly decreased virus loads in liver and more offered protective efficacies against FAdV infection compared with PBS and empty vector control groups (p < 0.01). For chickens in the group MDXEF-1/DC-ΔHexon-CWA, the levels of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in sera, and the virus loads in livers were significantly decreased vs. the other three ΔHexon-expressing bacteria (p < 0.01). The pathological changes in the hearts, livers, spleens and kidneys of chickens in MDXEF-1/DC-ΔHexon-CWA group were relatively slight compared to infection control group and other three ΔHexon-expressing bacteria groups. The rate of protection in MDXEF-1/DC-ΔHexon-CWA group was 90%. The present work demonstrated that cell surface-displayed target protein and immune enhancers in L. lactis and E. faecalis might be a promising approach to enhance immunity and immune efficacy against pathogen FAdV-4 infection.

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.

The reported cases of snake bite are 5.4 million per year, making the serious envenoming issue so, that WHO categorises cases of snake bite in neglected tropical diseases. Anti snake venom is produced by intramuscular injection of crude venom with different formulation of adjuvants to equine animals, but these intramuscular injections have several serious health impact in equine. Snake species categorised according to geographical locations, and there is a different composition of the snake venom at different locations. So, this research had been designed and evaluated new oral adjuvant formulation for viper family of Sindh, Pakistan. This is a animal immune based analysis consisting two Saw scale viper from Thar and costal area of Sindh because both have different habitat and morphological appearance. The venom toxin protein, combined with a mineral oil adjuvant for oral immunization. The adjuvant with venom via oral route produced of specific IgG and horse give maximum response with three doses of venom. The antibody titer was measured by ELISA showed significant results in both groups. For confirmation of specific antibodies isolated from horse serum, serum neutalization assay and immunodiffusion test were carried out. The snake venom with oral adjuvant immunization, is safe, efficient and time saving for production of equine Immunoglobulins IgG. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens-KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L- induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV., Competing Interests: Authors WZ, CK, XS, JL, YFZ, and QZ were employed by the Research Institute of Wuhan Keqian Biology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gao, Zuo, Kang, Zou, Zhang, Qiu, Shang, Li, Zhang, Zuo, Zhao and Jin.)

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

Abstract 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.

Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

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, 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

Porcine epidemic diarrhea (PED) induced by porcine epidemic diarrhea virus (PEDV) is an intestinal infectious disease in pigs that causes serious economic losses to the pig industry. To develop an effective oral vaccine against PEDV infection, we used a swine-origin Lactobacillus johnsonii (L. johnsonii) as an antigen delivery carrier. A recombinant strain pPG-T7g10-COE/L. johnsonii (L. johnsonii-COE) expressing COE protein (a neutralizing epitope of the viral spike protein) was generated. The immunomodulatory effect on dendritic cell in vitro and immunogenicity in pregnant sows was evaluated following oral administration. L. johnsonii-COE could activate monocyte-derived dendritic cell (MoDC) maturation and triggered cell immune responses. After oral vaccination with L. johnsonii-COE, levels of anti-PEDV-specific serum IgG, IgA, and IgM antibodies as well as mucosal secretory immunoglobulin A (SIgA) antibody were induced in pregnant sows. High levels of PEDV-specific SIgA and IgG antibodies were detected in the maternal milk, which provide effective protection for the piglets against PEDV infection. In summary, oral L. johnsonii-COE was able to efficiently activate anti-PEDV humoral and cellular immune responses, demonstrating potential as a vaccine for use in sows to provide protection of their piglets against PEDV.

The hepatitis E virus (HEV) genotype 3 (GT3) is an emergent pathogen in industrialized countries. It is transmitted zoonotically and may lead to chronic hepatitis in immunocompromised individuals. We evaluated if the major antigen of HEV, the capsid protein, can be used in combination with immunobiotic bacterium-like particles (IBLP) for oral vaccination in a mouse model. We have cloned and expressed the RGS-His5-tagged HEV GT3 capsid protein (ORF2) in E. coli and purified it by NiNTA. IBLP were obtained from two immunobiotic Lactobacillus rhamnosus strains acid- and heat-treated. ORF2 and the IBLP were orally administered to Balb/c mice. After three oral immunizations (14-day intervals), blood, intestinal fluid, Peyer´s patches, and spleen samples were drawn. IgA- and IgG-specific antibodies were determined by ELISA. Mononuclear cell populations from Peyer's patches and spleen were analyzed by flow cytometry, and the cytokine profiles were determined by ELISA to study cellular immunity. Orally administered recombinant ORF2 and IBLP from two L. rhamnosus strains (CRL1505 and IBL027) induced both antigen-specific humoral and cellular immune responses in mice. IBLP027 was more effective in inducing specific secretory IgA in the gut. IFN-γ, TNF-α, and IL-4 were produced by Peyer's plaques lymphocytes stimulated with ORF2 ex vivo suggesting a mixed Th1/Th2-type adaptive immune response in immunized mice. Oral vaccines are not invasive, do not need to be administered by specialized personal, and elicit both systemic and local immune responses at the port of entry. Here, we present an experimental oral vaccine for HEV GT3, which could be further developed for human and/or veterinary use. [ABSTRACT FROM AUTHOR]

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium -mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) has proven to be a major problem for the porcine industry worldwide. Conventional injectable vaccines induce effective systemic immune responses but are less effective in preventing PEDV at mucosal invasion sites, including the nasal or oral mucosa. Additionally, antigens delivered orally are easily degraded. Nasal immunization induces intestinal mucosal immune responses, which can aid in blocking viral invasion, and requires fewer antigen inoculation doses. Therefore, nasal immunizations are considered to be a potential approach to overcome viral infections. In the present study, nasal immunization of piglets was performed using inactivated PEDV combined with Bacillus subtilis as an immunopotentiator and the efficacy of nasal immunization was assessed. The results demonstrated that compared with oral immunization, piglets from the nasal immunization group exhibited higher levels of neutralizing antibodies (P<0.01) in the intestine, PEDV-specific immunoglobulin (Ig)G (P<0.01) in serum and PEDV-specific secretory IgA (SIgA) in saliva (P<0.01) and nasal secretions (P<0.01). An increased number of intestinal CD3+ T cells, IgA-secreting cells and intraepithelial lymphocytes (P<0.05) were also observed. Furthermore, the protein expression levels of interleukin-6 and interferon-γ, relative to the control PEDV infection, were also significantly elevated (P<0.05). The results of the present study indicate that nasal immunization is more effective at inducing the intestinal mucosal immune response, and provide new insights into a novel vaccination strategy that may be used to decrease the incidence of PEDV infections. [ABSTRACT FROM AUTHOR]

• Fusion protein of C. perfringens Cpa247-370, Cpb108-305 and EtxH118P toxin was generated. • B. subtilis expressing C. perfringens recombinant toxin protein rCpa-b-x was constructed. • Immunization with recombinants can elicit cellular and humoral immune responses. • Immunization with recombinants protected mice from C. perfringens toxins. The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD 100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens. [ABSTRACT FROM AUTHOR]

Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that resembles a disease-causing microorganism and is often made from weak or killed forms of the microbe. Vaccines are agents that stimulate the body's immune system to recognize the antigen. Now, a new form of vaccine was introduced which will have the power to mask the risk side of conventional vaccines. This type of vaccine was produced from plants which are genetically modified. In the production of edible vaccines, the gene-encoding bacterial or viral disease-causing agent can be incorporated in plants without losing its immunogenic property. The main mechanism of action of edible vaccines is to activate the systemic and mucosal immunity responses against a foreign disease-causing organism. Edible vaccines can be produced by incorporating transgene in to the selected plant cell. At present edible vaccine are developed for veterinary and human use. But the main challenge faced by edible vaccine is its acceptance by the population so that it is necessary to make aware the society about its use and benefits. When compared to other traditional vaccines, edible vaccines are cost effective, efficient and safe. It promises a better prevention option from diseases. [ABSTRACT FROM AUTHOR]

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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. • A recombinant Saccharomyces cerevisiae strain with the simultaneous quadrivalent expression of MG antigen protein was constructed innovatively. • Oral administration of ST1814G-MG could strongly stimulate the immune response in chickens, improve the composition of the gut microbiota. • Oral ST1814G-MG had better protection against MG infection than existing inactivated vaccines, and co-administration was even more effective. [ABSTRACT FROM AUTHOR]

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.