Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.

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

Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide.

Dendritic cells (DCs) are professional antigen-presenting cells that can recognize, capture, and process antigens. Fusing molecules targeting DCs with antigens can effectively improve the efficiency with which antigens are recognized and captured by DCs. This targeting strategy can be used for vaccine development to effectively improve the efficiency of antigen recognition and capture by DCs. The targeting sequence of porcine cytotoxic T-lymphocyte associated protein 4 (CTLA4), which binds porcine DCs, was identified in this study. Recombinant Lactobacillus reuteri (L. reuteri) expressing CTLA4-6aa (LYPPPY) and CTLA4-87aa fused to the porcine epidemic diarrhea virus (PEDV) protective antigen core neutralizing epitope (COE) were used to evaluate the ability of the two targeting motifs to bind the B7 molecule on DCs. Our results demonstrate that CTLA4-6aa could bind porcine DCs, and recombinant Lactobacillus expressing the CTLA4-6aa captured by porcine DCs was more efficient than those expressing CTLA4-87aa. In addition, the expression of DC markers, toll-like receptors, and cytokines was significantly higher in the 6aa-COE/L. reuteri-stimulated porcine DCs compared to DCs treated with 87aa-COE/L. reuteri (p<0.01) and recombinant Lactobacillus expressing CTLA4-6aa enhanced the ability of porcine DCs to activate T-cell proliferation. Our analysis of the protein structure revealed that CTLA4-87aa contains intramolecular hydrogen bonds, which may have weakened the intermolecular force between the residues on porcine CTLA4 and that on B7. In conclusion, recombinant Lactobacillus expressing CTLA4-6aa were more efficiently captured by porcine DCs and had a stronger ability to promote DC maturation and enhance T-cell proliferation. The LYPPPY motif is the optimal sequence for binding to porcine DCs. Piglets immunized with recombinant Lactobacillus showed that recombinant Lactobacillus expressing CTLA4-6aa induced significant levels of anti-PEDV-specific IgG and IgA antibody responses. Our study may promote research on DC-targeting strategies to enhance the effectiveness of porcine vaccines.

Human dendritic cell targeting peptide can be targeted to porcine dendritic cells to improve antigen capture efficiency to stimulate stronger immune response.

Dendritic cells (DCs) play a key role in the natural recognition of pathogens and subsequent activation of adaptive immune responses due to their potent antigen-presenting ability. Dendritic cell-targeting peptide (DCpep) is strongly targeted to DCs, which often express antigens, to enhance the efficacy of vaccines. Our previous study showed that recombinant Lactobacillus expressing human DCpep could significantly induce stronger immune responses than recombinant Lactobacillus without DCpep, but the mechanism remains unclear. In this study, the mechanism by which DCpep enhances the immune response against recombinant Lactobacillus was explored. Fluorescence-labeled human DCpep was synthesized to evaluate the binding ability of human DCpep to porcine monocyte-derived dendritic cells (Mo-DCs) and DCs of the small intestine. The effects of Mo-DC function induced by recombinant Lactobacillus expressing human DCpep fused with the porcine epidemic diarrhea virus (PEDV) core neutralizing epitope (COE) antigen were also investigated. The results showed that human DCpep bind to porcine DCs, but not to porcine small intestinal epithelial cells. Human DCpep can also improve the capture efficiency of recombinant Lactobacillus by Mo-DCs, promote the maturation of dendritic cells, secrete more cytokines, and enhance the ability of porcine DCs to activate T-cell proliferation. Taken together, these results promote advanced understanding of the mechanism by which DCpep enhances immune responses. We found that some DCpeps are conserved between humans and pigs, which provides a theoretical basis for the development of a DC-targeted vaccine.

Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.

Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-ß2-ε-ß1 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, ß, and ε exotoxins. In this study, Lactobacillus crispatus (pPG-T7g10/L. crispatus) expressing the main toxoids of C. perfringens, α, ε, ß1, and ß2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-ß2-ε-ß1 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-ß2-ε-ß1/L. crispatus increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-ß2ε-ß1/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.

Evaluation of the Immunogenicity in Mice Orally Immunized with Recombinant Lactobacillus casei Expressing Porcine Epidemic Diarrhea Virus S1 Protein.

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.

Strategy of Developing Oral Vaccine Candidates Against Co-infection of Porcine Diarrhea Viruses Based on a Lactobacillus Delivery System.

The number of co-infections with multiple porcine diarrhea viruses has increased in recent years. Inducing mucosal immunity through oral immunization is an effective approach for controlling these pathogens. To generate a multi-pathogen vaccine against viral co-infection, we employed the Lactobacillus vector platform, which was previously used to generate potent candidate vaccines against various diseases. Two strategies were used to test the protective efficiency of recombinant Lactobacillus against multiple diarrhea viruses. First, we used a mixture of recombinant Lactobacillus separately expressing antigens of transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine rotavirus (PoRV). Next, we used a recombinant Lactobacillus expressing an antigen fusion protein of the above viruses. Twenty-four newborn piglets were divided into three groups and orally immunized with a mixture of recombinant Lactobacillus, recombinant Lactobacillus expressing the antigen fusion protein, or sterile phosphate-buffered saline daily for seven consecutive days after birth. After immunization, the piglets were randomly selected from each group for oral administration of PEDV, and these piglets were then cohabited with piglets without PEDV infection for 7 days. The protective effect against PEDV was evaluated based on clinical symptoms, viral shedding, and intestinal pathological damage. Piglets immunized with recombinant Lactobacillus showed specific mucosal and humoral immune responses to the three viruses and were protected against severe diarrhea and intestinal pathology. Our results highlight the potential of an oral multi-pathogen vaccine based on Lactobacillus to prevent transmission and limit the severity of viral co-infection.

Effect of Microencapsulation Techniques on the Stress Resistance and Biological Activity of Bovine Lactoferricin-Lactoferrampin-Encoding Lactobacillus reuteri.

Bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri (LR-LFCA) has been found to benefit its host by strengthening its intestinal barrier. However, several questions remain open concerning genetically engineered strains maintaining long-term biological activity at room temperature. In addition, probiotics are vulnerable to harsh conditions in the gut, such as acidity and alkalinity, and bile salts. Microencapsulation is a technique to entrap probiotic bacteria into gastro-resistant polymers to carry them directly to the intestine. We selected nine kinds of wall material combinations to encapsulate LR-LFCA by spray drying microencapsulation. The storage stability, microstructural morphology, biological activity, and simulated digestion in vivo or in vitro of the microencapsulated LR-LFCA were further evaluated. The results showed that LR-LFCA had the highest survival rate when microcapsules were prepared using a wall material mixture (skim milk, sodium glutamate, polyvinylpyrrolidone, maltodextrin, and gelatin). Microencapsulated LR-LFCA increased the stress resistance capacity and colonization abilities. In the present study, we have identified a suitable wall material formulation for spray-dried microencapsulation of genetically engineered probiotic products, which would facilitate their storage and transport.

His-Rich Domain of Selenoprotein P Ameliorates Neuropathology and Cognitive Deficits by Regulating TrkB Pathway and Zinc Homeostasis in an Alzheimer Model of Mice.

Selenoproteins are a family of special proteins that contain the 21st amino acid, selenocysteine (Sec), in their sequence. Selenoprotein P has 10 Sec residues and modulates selenium homeostasis and redox balance in the brain. Previously, we found that the Sec-devoid His-rich motif of selenoprotein P (Selenop-H) suppressed metal-induced aggregation and neurotoxicities of both Aß and tau in vitro. To investigate the intervening capacity of Selenop-H on the neuropathology and cognitive deficits of triple transgenic AD (3 × Tg-AD) mice, the Selenop-H gene packaged in rAAV9 was delivered into the hippocampal CA3 regions of mice via stereotaxic injection. Four months later, we demonstrated that Selenop-H (1) improved the spatial learning and memory deficits, (2) alleviated neuron damage and synaptic protein loss, (3) inhibited both tau pathology and amyloid beta protein (Aß) aggregation, (4) activated both BDNF- and Src-mediated TrkB signaling, and (5) increased MT3 and ZnT3 levels and restored Zn2+ homeostasis in the mice model of AD. The study revealed that Selenop-H is potent in ameliorating AD-related neuropathology and cognitive deficits by modulating TrkB signaling and Zn2+ homeostasis.

Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease.

Prion diseases are fatal neurodegenerative disorders of humans and animals and no effective treatments are currently available. Allogenic transplantation of immortalized human mesenchymal stem cells (MSCs) can prolong the survival of mice infected with prions. However, autologous transplantation is an appropriate model for evaluating the effects of MSCs on prion diseases. Therefore, we isolated and purified MSCs from the femur and tibia of mice as compact bone-derived MSCs (CB-MSCs). Flow cytometric analysis showed that CB-MSCs were negative for myeloid stem cell-derived cell markers CD11b and CD45, but positive for molecules such as Sca-1, CD105 and CD90.2, which are reported to be expressed on MSCs. The ability of CB-MSCs to migrate to brain extracts from prion-infected mice was confirmed by an in vitro migration assay. Intra-hippocampus transplantation of CB-MSCs at 120 days post-inoculation marginally but significantly prolonged the survival of mice infected with the Chandler prion strain. The transplantation of CB-MSCs did not influence the accumulation of disease-specific prion protein. However, the CB-MSC transplantation enhanced microglial activation, which appeared to be polarized to the M2-type activation state. These results suggest that autologous MSC transplantation is a possible treatment for prion diseases, while the modification of microglial activation may be a therapeutic target for neurodegenerative diseases.

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

Identification of the functional domain of the porcine epidemic diarrhoea virus receptor.

Porcine aminopeptidase N (pAPN) is a functional receptor for porcine epidemic diarrhoea virus (PEDV). Although PEDV is known to use the pAPN as the major receptor for cell entry, the crucial domain of the pAPN that interacts with the PEDV is still unknown. In the present study, in order to determine the crucial domain of the pAPN, the extracellular domain of pAPN was divided into three subdomains named SPA, SPB and SPC, based on its secondary structure. Recombinant plasmid pcDNA3.1 expressing SPA, SPB and SPC was constructed and introduced into Madin-Darby canine kidney (MDCK) cells by transfection. Following the detection of PEDV infection in transfected MDCK cells after PEDV challenge, we clearly demonstrated that the SPC subdomain plays a key role in cell entry of PEDV and its expression permits PEDV growth in transfected MDCK cells, while virus propagation can be inhibited by anti-SPC serum, indicating that the SPC subdomain appears to be a crucial functional domain in contributing to efficient PEDV infection.