Construction and Evaluation of an Efficient Live Attenuated Salmonella Choleraesuis Vaccine and Its Ability as a Vaccine Carrier to Deliver Heterologous Antigens.

The gram-negative facultative intracellular pathogen Salmonella enterica serotype Choleraesuis, also known as S. Choleraesuis, is a major financial loss for the pig business. C500 is a vaccine strain that has been used for preventing S. Choleraesuis infection in pigs for many years in China. Although it possessed good immunogenicity and protection efficacy, it still showed severe side effects. The truncation of the key gene rpoS in C500 was believed to take the major responsibility for its attenuation. To achieve a good balance between attenuation and immunogenicity, rpoS was restored to an active state, and other essential virulent genes of crp, fur, phoP, and aroA were evaluated for their effects of deletion on safety and immunogenicity. Animal experiments demonstrated that C5001 (C500 rpoS+ Δcrp10) and C5002 (C500 rpoS+ Δfur9) showed an excellent ability to induce an immune response. To further decrease the endotoxic activity, the combination mutations of ΔpagL7 ΔpagP81::PlpplpxE ΔlpxR9 were introduced into the mutant strains to generate 1'-dephosphorylated lipid A. Animal experiments showed that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9) induced higher levels of IgG and secreted IgA antibodies and provided a higher protection rate than SC1 (C500 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9) and SC2 (C500 rpoS+ Δcrp10 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9). We also evaluated the ability of SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9) as a vaccine carrier to deliver heterologous protein antigens and polysaccharide antigens. The results indicated that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9) showed an excellent ability to deliver heterologous antigens and induce the host to produce high levels of antibodies. Together, these results indicate that we constructed a safe and efficient attenuated strain of the S. Choleraesuis vaccine, which demonstrated strong resistance to infection with wild-type S. Choleraesuis and can be employed as a universal vector for the delivery of recombinant antigens.

Construction and characterization of Aeromonas hydrophila crp and fur deletion mutants and evaluation of its potential as live-attenuated vaccines in crucian carp.

Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur, a Fe2+ regulatory protein, and the Crp, a cAMP receptor protein, play important roles in bacterial virulence in many bacteria, but no research has been investigated on A. hydrophila. In this study, the Δfur and Δcrp mutant strains were constructed by the suicide plasmid method. These two mutant strains exhibited a slightly diminished bacterial growth and also were observed some alterations in the number of outer membrane proteins, and the disappearance of hemolysis in the Δcrp strain. Animal experiments of crucian carp showed that the Δfur and Δcrp mutant strains significantly decreased virulence compared to the wild-type strain, and both mutant strains were able to induce good immune responses by two kinds of administration routes of intraperitoneal immunization (i.p) and immersion immunization, and the protection rates through intraperitoneal injection of Δfur and Δcrp to crucian carp were as high as 83.3 % and 73.3 %, respectively, and immersion immunization route of Δfur and Δcrp to crucian carp provided protection as high as 40 % and 20 %, respectively. These two mutant strains showed abilities to induce changes in enzymatic activities of the non-specific enzymes SOD, LZM, AKP, and ACP in crucian carp. Together, these results indicated the Δfur and Δcrp mutants were safe and effective candidate vaccine strains, showing good protection against the wild-type A. hydrophila challenge.

Attenuated Salmonella Typhimurium with truncated LPS and outer membrane-displayed RGD peptide for cancer therapy.

Gram-negative, facultatively anaerobic bacteria Salmonella Typhimurium is a candidate agent or delivery vector for cancer therapy. Effective targeted therapies in addition to radiotherapy, chemotherapy and surgery have been urgently needed as an alternative or supplement. This study expected to further improve the tumor-targeting ability of Salmonella bacteria through genetic modifications. Based on an auxotrophic Salmonella bacterial strain (D2), we constructed Salmonella mutants with altered LPS length to facilitate displaying the RGD4C targeting peptide on the outer membrane surface of Salmonella. The expression of RGD4C peptide in fusion with OmpA was identified by outer membrane protein extraction and WB detection in different mutant strains. However, flow cytometry analysis following immunofluorescence staining demonstrated that the extracellular length of Salmonella LPS did affect the surface display of RGD4C peptide. The strain D2-RGD4C that synthesized intact LPS including lipid A, core oligosaccharides and O antigen polysaccharides could hardly display RGD4C peptide, showing the same fluorescence signal intensity as the strains not expressing RGD4C peptide. Among different strains, D2 ∆rfaJ-RGD4C that synthesized truncated LPS including lipid A and partial core oligosaccharides was capable of displaying RGD4C peptide most efficiently and showed the highest ability to target HUVECs expressing αV integrin and tumor tissue with abundant neovascularization. Animal experiments also demonstrated that this tumor-targeting attenuated Salmonella strain to simultaneously deliver endostatin and TRAIL, two agents with different anti-tumor activities, could significantly inhibit tumor growth and prolong mouse survival. Thus, our studies revealed that Salmonella could be genetically engineered to improve its tumor targeting via the truncation of LPS and surface display of targeting peptides, thereby eliciting superior anti-tumor effects through targeted delivery of drug molecules.

Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines.

Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

Effect of deletion of gene cluster involved in synthesis of Enterobacterial common antigen on virulence and immunogenicity of live attenuated Salmonella vaccine when delivering heterologous Streptococcus pneumoniae antigen PspA.

BACKGROUND: Enterobacterial common antigen (ECA) is a family-specific surface antigen shared by all members of the Enterobacteriaceae family. Previous studies showed that the loss of ECA results in Salmonella attenuation, indicating its usefulness as a vaccine candidate for Salmonella infection, but no studies have shown whether the mutation resulting from the deletion of the ECA operon in conjunction with other mutations could be used as an antigen vehicle for heterologous protein antigen delivery. RESULTS: In this study, we introduced a nonpolar, defined ECA operon deletion into wild-type S. Typhimurium χ3761 and an attenuated vaccine strain χ9241, obtaining two isogenic ECA operon mutants, namely, χ12357 and χ12358, respectively. A number of in vitro and in vivo properties of the mutants were analyzed. We found that the loss of ECA did not affect the growth, lipopolysaccharide (LPS) production and motility of S. Typhimurium wild type strain χ3761 and its attenuated vaccine strain χ9241 but significantly affected the virulence when administered orally to BALB/c mice. Furthermore, the effects of the ECA mutation on the immunogenicity of a recombinant S. Typhimurium vaccine strain χ9241 when delivering the pneumococcal antigen PspA were determined. The result showed that the total anti-PspA IgG level of χ12358 (pYA4088) was slightly lower than that of χ9241 (pYA4088), but the protection rate was not compromised. CONCLUSIONS: ECA affects virulence and benefits the Th2 immunity of Salmonella Typhimurium, therefore, it is feasible to use a reversible ECA mutant mode to design future Salmonella vaccine strains for heterologous protective antigens.

Endostatin gene therapy delivered by attenuated Salmonella typhimurium in murine tumor models.

Salmonella typhimurium (hereafter S. typhimurium), as Gram-negative facultative anaerobic bacteria, are good candidates for cancer therapy and delivering therapeutic antitumor agents. However, it is necessary to reduce the virulence of such bacteria and enhance their tumor-targeting ability, and their immunostimulatory ability to induce tumor cell apoptosis. In this study, we constructed a S. typhimurium mutant named S634 harboring aroA mutation and additional mutations involved in modifications of lipid A. Upon intraperitoneal infection in mice, the aroA-deficient strain S634 showed greatly attenuated virulence and preferential accumulation within tumor tissue. We next investigated the ability of S636, the asd mutant derivative of S634, to deliver the anti-angiogenic agent "endostatin" (S636/pES) and to inhibit tumor growth in mouse CT26 colon carcinoma and B16F10 melanoma models. S636/pES-treated tumor-bearing mice showed suppressed tumor growth and prolonged survival, compared to mice treated with either the bacteria carrying empty plasmids or PBS intraperitoneally. Immunohistochemical studies demonstrated that, when tumor-bearing mice were infected with S636/pES, Salmonella colonization and endostatin expression were accompanied by the increase of apoptosis level and suppression of tumor angiogenesis within tumor tissues. Our findings showed that endostatin gene therapy delivered by attenuated S. typhimurium displays therapeutic antitumor effects in murine tumor models.

Fuji Intelligent Chromo Endoscopy and staining technique for the diagnosis of colon tumor.

BACKGROUND: Colon cancer is a common malignant tumor in the clinic with an incidence rate that is increasing in recent years. The key point for improving the survival rate is the diagnosis and treatment at an early stage. The purpose of this study was to compare the difference of the Fuji Intelligent Chromo Endoscopy (FICE) and staining technique for the diagnosis of colon tumors and non-tumor lesions. METHODS: From March to November 2007, 654 patients were examined with ordinary colonoscopy. Among them 223 patients with colon neoplasm or polypoid lesion were included. The patients were examined with a magnifying ordinary colonoscopy, a magnifying FICE technique and magnifying staining technique. The pit pattern and blood capillary form of the lesion were examined, an endoscopic diagnosis was made and it was compared with the pathologic diagnosis. RESULTS: Four hundred and fifty-one neoplasms were detected in the 223 patients, among those 91.1% (411/451) were detected with the magnifying ordinary endoscopy while 99.1% (447/451) were detected with the FICE technique; there was a significant difference between the two methods. FICE could clearly show the structure and form of mucosal blood capillaries (P < 0.01) but there was no significant difference between the two methods for showing the pit pattern. The coincident rate of FICE for the diagnosis of tumor and non-tumor lesions was 91.6% (413/451), that of the magnifying staining technique was 82.0% (370/451) (P < 0.05). CONCLUSIONS: Magnifying FICE could show the mucosal microstructure and blood capillary form and it had a superiority of high coincident rate, high sensitivity and specificity when compared with ordinary magnifying colonoscopy and magnifying staining endoscopy. In addition, it was easy to operate and a biopsy could be taken from the target, so it has a satisfactory clinical practical value.