Yigang Xu

Immunogenicity of Recombinant Classic Swine Fever Virus CD8 T Lymphocyte Epitope and Porcine Parvovirus VP2 Antigen Coexpressed by Lactobacillus casei in Swine via Oral Vaccination

ABSTRACT Classical swine fever virus (CSFV) and porcine parvovirus (PPV) are highly contagious pathogens, resulting in enormous economic losses in pig industries worldwide. Because vaccines play an important role in disease control, researchers are seeking improved vaccines that could induce antiviral immune responses against CSFV and PPV at the mucosal and systemic levels simultaneously. In this study, a genetically engineered Lactobacillus strain coexpressing the CSFV-specific cytotoxic T lymphocyte (CTL) epitope 290 and the VP2 antigen of PPV was developed, and its immunopotentiating capacity as an oral vaccine in pigs was analyzed. The data demonstrated that in the absence of any adjuvant, the recombinant Lactobacillus strain can efficiently stimulate mucosal and systemic CSFV-specific CD8+ CTL responses to protect pigs against CSFV challenge. Moreover, anti-PPV-VP2 serum IgG and mucosal IgA were induced in pigs immunized orally with the recombinant Lactobacillus strain, showing a neutralizing effect on PPV infection. The results suggest that the recombinant Lactobacillus microecological agent may be a valuable component of a strategy for development of a vaccine against CSFV and PPV.

Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants.

Novel Approach for Isolation and Identification of Porcine Epidemic Diarrhea Virus (PEDV) Strain NJ Using Porcine Intestinal Epithelial Cells

Porcine epidemic diarrhea virus (PEDV), which is the causative agent of porcine epidemic diarrhea in China and other countries, is responsible for serious economic losses in the pork industry. Inactivated PEDV vaccine plays a key role in controlling the prevalence of PEDV. However, consistently low viral titers are obtained during the propagation of PEDV in vitro; this represents a challenge to molecular analyses of the virus and vaccine development. In this study, we successfully isolated a PEDV isolate (strain NJ) from clinical samples collected during a recent outbreak of diarrhea in piglets in China, using porcine intestinal epithelial cells (IEC). We found that the isolate was better adapted to growth in IECs than in Vero cells, and the titer of the IEC cultures was 104.5 TCID50/0.1 mL at passage 45. Mutations in the S protein increased with the viral passage and the mutations tended towards attenuation. Viral challenge showed that the survival of IEC-adapted cultures was higher at the 45th passage than at the 5th passage. The use of IECs to isolate and propagate PEDV provides an effective approach for laboratory-based diagnosis of PEDV, as well as studies of the epidemiological characteristics and molecular biology of this virus.

Probiotic Lactobacillus casei expressing porcine antimicrobial peptide PR39 elevates antibacterial activity in the gastrointestinal tract.

PR39, a 4.7 kDa proline-rich antimicrobial peptide, acts as a cationic host defense peptide. In addition to killing bacteria, PR39 mediates inflammatory reactions, including cell proliferation, migration, wound healing, and angiogenesis. Here, we examined the antibacterial effects of this peptide. The synthetic gene fragment PR39 was inserted into the secretory expression vector plasmid pPG:612 of Lactobacillus casei, yielding the recombinant strain pPG:612-PR39/L. casei 393. In vitro antibacterial tests showed that expression of the PR39 peptide in recombinant L. casei resulted in antibacterial activity against Escherichia coli and Salmonella but had only minor antibacterial effects in Staphylococcus aureus. In addition, BALB/c mice fed the recombinant pPG:612-PR39/L. casei 393 grew better and had increased peripheral blood lymphocyte percentages, white blood cell numbers, and spleen indices than mice in the control group. Scanning electron microscopy showed that jejunum and duodenum villus height, crypt depth, and the ratio of villus height/crypt depth in the intestinal villi also increased. Moreover, mice fed the recombinant strain showed significantly lower mortality rates than the control group mice when challenged with the enterotoxigenic E. coli K88+. Thus, this recombinant expression system had the beneficial characteristics of both L. casei and PR39, supporting its potential as an animal feed additive.

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.

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

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.

TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells

Type II transmembrane serine proteases (TTSPs) facilitate the spread and replication of viruses such as influenza and human coronaviruses, although it remains unclear whether TTSPs play a role in the progression of animal coronavirus infections, such as that by porcine epidemic diarrhea virus (PEDV). In this study, TTSPs including TMPRSS2, HAT, DESC1, and MSPL were tested for their ability to facilitate PEDV replication in Vero cells. Our results showed that TMPRSS2 and MSPL played significant roles in the stages of cell–cell fusion and virus–cell fusion, whereas HAT and DESC1 exhibited weaker effects. This activation may be involved in the interaction between TTSPs and the PEDV S protein, as the S protein extensively co-localized with TMPRSS2 and MSPL and could be cleaved by co-expression with TMPRSS2 or MSPL. Moreover, the use of Vero cells expressing TMPRSS2 and MSPL facilitated PEDV replication in the absence of exogenous trypsin. In sum, we identified two host proteases, TMPRSS2 and MSPL, which may provide insights and a novel method for enhancing viral titers, expanding virus production, and improving the adaptability of PEDV isolates in vitro.

the role of infectious hematopoietic necrosis virus (ihnv) proteins in the modulation of Nf-κb pathway during Ihnv infection

Abstract Viral infections frequently lead to the activation of host innate immune signaling pathways involved in the defense against invading pathogens. To ensure their survival, viruses have evolved sophisticated mechanisms to overcome the host immune responses. The present study demonstrated for the first time that infectious hematopoietic necrosis virus (IHNV) activated NF‐&kgr;B pathway in fish cells. We further identified that the IHNV L protein could activate the NF‐&kgr;B signaling pathway and that IHNV NV functioned as an inhibitor of NF‐&kgr;B activation. Further results demonstrated that the NV protein blocked the degradation of the inhibitor of NF‐&kgr;B (I&kgr;B&agr;) and suppressed the SeV‐induced NF‐&kgr;B nuclear translocation. In conclusion, our study explored the functions of different IHNV proteins on NF‐&kgr;B activation, and revealed a potential mechanism by which IHNV evades innate immune responses. HighlightsOur study revealed a potential mechanism by which IHNV evades innate immune responses.Infectious hematopoietic necrosis virus (IHNV) activated NF‐&kgr;B pathway in fish cells.The IHNV L protein remarkably increased NF‐&kgr;B activation while NV protein suppressed the NF‐&kgr;B response.NV protein blocked the degradation of I&kgr;B&agr; and suppressed the SeV‐induced NF‐&kgr;B nuclear translocation.