Xinyuan Qiao

Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice

BackgroundPorcine rotavirus infection is a significant cause of morbidity and mortality in the swine industry necessitating the development of effective vaccines for the prevention of infection. Immune responses associated with protection are primarily mucosal in nature and induction of mucosal immunity is important for preventing porcine rotavirus infection.ResultsLactobacillus casei expressing the major protective antigen VP4 of porcine rotavirus (pPG612.1-VP4) or VP4-LTB (heat-labile toxin B subunit from Echerichia coli) (pPG612.1-VP4-LTB) fusion protein was used to immunize mice orally. The expression of recombinant pPG612.1-VP4 and pPG612.1-VP4-LTB was confirmed by SDS-PAGE and Western blot analysis and surface-displayed expression on L. casei was verified by immunofluorescence. Mice orally immunized with recombinant protein-expressing L. casei produced high levels of serum immunoglobulin G (IgG) and mucosal IgA. The IgA titters from mice immunized with pPG612.1-VP4-LTB were higher than titters from pPG612.1-VP4-immunized mice. The induced antibodies demonstrated neutralizing effects on RV infection.ConclusionThese results demonstrated that VP4 administered in the context of an L. casei expression system is an effective method for stimulating mucosal immunity and that LTB served to further stimulate mucosal immunity suggesting that this strategy can be adapted for use in pigs.

Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production.

The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier.

Virulence and serological studies of recombinant infectious hematopoietic necrosis virus (IHNV) in rainbow trout

Infectious hematopoietic necrosis virus is a highly contagious disease of juvenile salmonid species. From the IHNV HLJ-09 isolated in China, two recombinant viruses were generated by reverse genetics using the RNA polymerase II transcription system. The recombinant viruses were confirmed by RT-PCR, indirect immunofluorescence assay and electron microscopy. They were referred to as rIHNV HLJ-09 and rIHNV-EGFP. rIHNV HLJ-09 and rIHNV-EGFP could stably replicate in EPC cell lines and had the same cellular tropism as wtIHNV HLJ-09. But the titer of rIHNV-EGFP was significantly lower than rIHNV HLJ-09 and wtIHNV HLJ-09. rIHNV-EGFP strain could express EGFP stably at least in 20 passages, and the fluorescence could be observed clearly. To assess the virulence and pathogenicity of the recombinant viruses in vivo, juvenile rainbow trout were challenged by intraperitoneal injection with 20μl of rIHNV HLJ-09, rIHNV-EGFP or wtIHNV HLJ-09 (1×10(6)pfuml(-1)). Fish challenged with rIHNV HLJ-09 and wtIHNV HLJ-09 exhibited clinical signs typical of IHN disease and both produced 90% cumulative percent mortality, whlie rIHNV-EGFP produced only 5%. Pathological sectioning results showed that the tissues (liver, kidney, heart muscle, back muscle) of the fish infected with rIHNV HLJ-09 exhibited pathological changes, with the exception of cerebral neurons and the cheek. However, no lesions of liver, kidney, heart, muscle, brain in rainbow trout of rIHNV-EGFP or the control group were observed. Indirect ELISA results showed that a high level of serum antibody was detected in the experimental fish challenged with rIHNV HLJ-09, just as the same as wtIHNV HLJ-09, while a lower titer was detecred in the fish infected with rIHNV-EGFP. This indicated that the recombinant viruses could induce humoral immune response in the experimental fish. The recombinant viruses had unique genetic tags and could be used for genetic engineering, laying new ground for further investigation of IHNV pathopoiesis molecular mechanism, host tropism and the development of novel vaccines against IHN.

Development of a loop-mediated isothermal amplification assay for rapid detection of bovine parvovirus.

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8 mM Mg²⁺, 1.2 mM betaine, and an incubation at 63°C for 45 min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. To develop an effective bivalent oral vaccine against TGEV and PEDV infection, the D antigenic site of the TGEV spike (S) protein and the major antigen site (core neutralizing epitope—COE) of the PEDV S protein were used as immunogens, and the enhanced green fluorescent protein (eGFP) gene was used as a reporter to construct genetically engineered Lactobacillus casei rLpPGF-T7g10-eGFP-6D-COE. The expression of proteins of interest by the recombinant L. casei was confirmed by confocal laser scanning microscopy and a Western blot assay, and the immunogenicity of rLpPGF-T7g10-eGFP-6D-COE in orally immunized mice was evaluated. The results showed that levels of anti-PEDV and anti-TGEV serum immunoglobulin G (IgG) and mucosal secreted immunoglobulin A (sIgA) antibodies obtained from the mice immunized with rLpPGF-T7g10-eGFP-6D-COE, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (PBS) or rLpPG-T7g10. Moreover, the serum IgG antibodies showed neutralizing effects against PEDV and TGEV. Our data suggest that the antibiotic resistance-free genetically engineered L. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against PEDV and TGEV.

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

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

Recombinant Lactobacillus casei expressing Clostridium perfringens toxoids α, β2, ε and β1 gives protection against Clostridium perfringens in rabbits

The present study used Lactobacillus casei ATCC 393 as antigen delivery system to express C. perfringens toxoids α-β2-ε-β1 to construct the recombination Lactobacillus casei pPG-2-α-β2-ε-β1/L. casei 393. After being induced by 1% xylose, the specificity and integrity of recombinant strain were determined by Western-blotting. Rabbits as native animal model were immunized orally with pPG-2-α-β2-ε-β1/L. casei 393 and the titers of specific IgG and sIgA were determined by ELISA. The result showed that oral administration with the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in rabbits immunized with pPG-2-α-β2-ε-β1/L. casei 393 was observed. Levels of IL-4 and IFN-γ produced were significantly higher in lymphocytes isolated from the vaccine group than those from the control groups. Flow cytometry assay showed that both the percentages of CD4+T cells and CD8+T cells from the vaccine group were significantly increased than the control groups. All these results showed that immunizing with recombinants can elicit both humoral immunity and cellular immunity. Besides, in order to determine the effectiveness of oral immunization with pPG-2-α-β2-ε-β1/L. casei 393, rabbits of vaccine group and control groups were challenged with 1×LD100 unit of culture filtrate of C. perfringens type C and type D toxins respectively. After challenge, 100% of the immunized rabbits survived, while the rabbits of the control group were killed within 48h. Observation on histopathology showed that histopathological changes were obviously found in heart, liver, spleen, lung, kidney, intestine and brain of rabbits from the control groups, while no apparent histopathological change was observed in the vaccine group. All the results show that pPG-2-α-β2-ε-β1/L. casei 393 can eliciteffective immunoprotection against C. perfringens. All of these suggest that the use of pPG-2-α-β2-ε-β1/L. casei 393 can be regarded as candidate for the development of a vaccine against C. perfringens.

Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets

Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets. In our previous study, a genetically engineered Lactobacillus casei oral vaccine (pPG-COE-DCpep/L393) expressing a dendritic cell (DC)-targeting peptide fused with porcine epidemic diarrhea virus (PEDV) COE antigen was developed. This vaccine induced significant levels of anti-PEDV specific IgG and IgA antibody responses in mice, indicating a potential strategy against PEDV infection. In this study, pPG-COE-DCpep/L393 was used for oral vaccination of newborn piglets against PEDV. We then assessed the immune responses and protection efficacy of pPG-COE-DCpep/L393. An indirect enzyme-linked immunosorbent assay (ELISA) showed that the recombinant Lactobacillus vaccine elicits a specific systemic and mucosal immune response. The T-helper cells mediated by pPG-COE-DCpep/L393 and PEDV infection display a Th1 phenotype. The histopathological results showed that pPG-COE-DCpep/L393 promotes lymphocyte proliferation and effectively protects piglets against PEDV infection. The transforming growth factor-β level indicated that the recombinant Lactobacillus vaccine plays a role in anti-inflammatory responses in mesenteric lymph nodes during PEDV infection. These results show that pPG-COE-DCpep/L393 is a potential vaccine against PEDV infection.