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.

Reducing the endotoxic activity or enhancing the vaccine immunogenicity by altering the length of lipid A acyl chain in Salmonella.

The balance of the attenuation and reactogenicity is an issue in the development of recombinant attenuated Salmonella vaccines (RASV). Some reactogenic strains produced side effects are partially induced by lipid A. As reported, the number of lipid A acyl chains influence the strength and outcome of immune responses. However, there is rarely any study to investigate the modifications of acyl chain length on the effect of the toxicity and immunogenicity in Salmonella. In this study, foreign acyltransferase genes lpxA and lpxD were introduced into S. Typhimurium, which produced the S006 (ΔaraBAD::PlppCtlpxAC10) or S007 (ΔproBA::PlppSslpxDC16) strains with C10 or C16 acyl chains respectively. The results showed that the increased polymyxin B susceptibility, reduced swimming and invasion capabilities were observed in the S006. In addition, it also exhibited a lower endotoxicity and colonization ability compared to the parent strain. The result indicated the introduction of C10 acyl chains could be as a candidate choice for lipid A detoxifying strategy in engineering bacteria. However, the longer acyl chain modification didn't obviously change these abilities. Parallelly, these modifications were introduced into a Salmonella vaccine strain to determine their influences on the immune responses against Pneumonia. After inoculation by the strain V003 (ΔaraBAD ΔproBA::PlppSslpxDC16 χ9241), the mice produced robust levels of anti-PspA IgG, and a balanced Th1/Th2 immunity, which resulted in a significant survival improvement of mice with challenging against Streptococcus pneumonia. Therefore, the combination of lipid A modification with C16 acyl chain may be a better strategy for the development of ideal RASVs.

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.

[Advances in the research of enterobacterial common antigen].

The enterobacterial common antigen (ECA) is a polysaccharide composed of polysaccharide repeats that are located in the outer membrane of almost all Enterobacteriaceae bacteria and has diverse biological functions. ECA is synthesized by the synergistic action of multiple genes that are present in clusters on the genome of Enterobacteriaceae bacteria, forming the ECA antigen gene cluster, an important virulence factor that plays a role in host invasion and survival of Enterobacteriaceae in vivo. ECA also plays an important role in the maintenance of the bacterial outer membrane permeability barrier, flagella gene expression, swarming motility, and bile salts resistance. In addition, ECALPS, anchored in the core region of bacterial lipopolysaccharide, is an important surface antigen for bacteria, stimulating high levels of antibody production in the host and could be a target for vaccine research. This review summarizes ECA purification, genes involved in ECA biosynthesis, its immunological characteristics, biological functions and clinical applications.

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.

Optimized Attenuated Salmonella Typhimurium Suppressed Tumor Growth and Improved Survival in Mice.

The gram-negative facultative anaerobic bacteria Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) has always been considered as one candidate of anti-tumor agents or vectors for delivering drug molecules. In this study, we compared several widely studied S. Typhimurium strains in their anti-tumor properties aiming to screen out the best one for further optimization and use in cancer therapy. In terms of the motility, virulence and anti-tumor efficacy, the three strains 14028, SL1344, and UK-1 were similar and obviously better than LT-2, and UK-1 showed the best phenotypes among them. Therefore, the strain UK-1 (D) was selected for the following studies. Its auxotrophic mutant strain (D1) harboring ∆aroA and ∆purM mutations was further optimized through the modification of lipid A structure, generating a new strain named D2 with stronger immunostimulatory activity. Finally, the ∆asd derivative of D2 was utilized as one live vector to deliver anti-tumor molecules including the angiogenesis inhibitor endostatin and apoptosis inducer TRAIL and the therapeutic and toxic-side effects were evaluated in mouse models of colon carcinoma and melanoma. After intraperitoneal infection, engineered Salmonella bacteria equipped with endostatin and/or TRAIL significantly suppressed the tumor growth and prolonged survival of tumor-bearing mice compared to PBS or bacteria carrying the empty plasmid. Consistently, immunohistochemical studies confirmed the colonization of Salmonella bacteria and the expression of anti-tumor molecules inside tumor tissue, which were accompanied by the increase of cell apoptosis and suppression of tumor angiogenesis. These results demonstrated that the beneficial anti-tumor efficacy of attenuated S. Typhimurium bacteria could be improved through delivery of drug molecules with powerful anti-tumor activities.

New technologies in developing recombinant-attenuated bacteria for cancer therapy.

Cancer has always been a global problem, with more cases of cancer patients being diagnosed every year. Conventional cancer treatments, including radiotherapy, chemotherapy, and surgery, are still unable to bypass their obvious limitations, and developing effective targeted therapies is still required. More than one century ago, the doctor William B. Coley discovered that cancer patients had tumor regression by injection of Streptococcus bacteria. The studies of cancer therapy using bacterial microorganisms are now very widespread. In particular, the facultative anaerobic bacteria Salmonella typhimurium is widely investigated as it can selectively colonize different types of tumors, locally deliver various antitumor drugs, and inhibit tumor growth. The exciting antitumor efficacy and safety observed in animal tumor models prompted the well-known attenuated Salmonella bacterial strain VNP20009 to be tested in human clinical trials in the early 21st century. Regrettably, no patients showed significant therapeutic effects and even bacterial colonization in tumor tissue was undetectable in most patients. Salmonella bacteria are still considered as a promising agent or vehicle for cancer therapy. Recent efforts have been focused on the generation of attenuated bacterial strains with higher targeting for tumor tissue, and optimization of the delivery of therapeutic antitumor cargoes into the tumor microenvironment. This review will summarize new technologies or approaches that may improve bacteria-mediated cancer therapy.

Bacteria-derived minicells for cancer therapy.

Bacteria are always a considerable tool for the cancer therapy. The bacteria-derived minicells are re-emerged as a promising drug delivery system for cancer therapy. The minicells are nano-sized, anucleated, non-dividing, and metabolically active cells produced by abnormal bacterial cell division that are able to transcribe and translate the gene of interest. Minicells encapsulate a wide range of chemotherapeutic and molecular drugs, si/shRNA, antigens and therapeutic toxins to precisely deliver them to the cancer cells through the easy modification of minicell surface with bi-specific antibodies against receptor-targeted cancer cells. Minicell-mediated chemotherapy may inhibit the growth of drug-resistant tumors and exhibits the potential to successfully deliver the chemotherapeutics into hypoxic and necrotic region in solid tumors. This novel approach significantly overcomes drug leakage and severe toxicity by enhancing targeting specificity and therapeutic index of drugs in cancer therapy. Many antibody-conjugated drug-loaded minicells are being investigated in clinical trials for cancer therapy. This review summarizes the advantages of bacteria-derived minicells as delivery systems for anti-cancer drugs or agents and the recent advances and emerging future in cancer therapy.

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.

Attenuated Salmonella Typhimurium expressing Salmonella Paratyphoid A O-antigen induces protective immune responses against two Salmonella strains.

Salmonella enterica serovar Paratyphi A is the main causative agent of paratyphoid fever in many Asian countries. As paratyphoid is spread by the fecal-oral route, the most effective means of controlling S. Paratyphi A infection is through the availability of clean water supplies and working sanitation services. Because sanitation facilities improve slowly in these poor areas and antibiotic resistance is severe, the development of a safe and effective vaccine remains a priority for controlling the spread of paratyphoid disease. In this study, we investigated the strategy of heterologous O-antigenic O2 serotype (S. Paratyphi A characterized) conversion in S. Typhimurium to prevent paratyphoid infections. A series of S. Typhimurium mutants were constructed with replacement of abe, wzxB1 and wbaVB1 genes with respective prt-tyvA1, wzxA1 and wbaVA1, and the results showed that only three genes including prt, wbaVA1 and wzxA1 from S. Paratyphi A presence enable S. Typhimurium to sufficiently express O2 antigen polysaccharide. We also constructed a series of live attenuated S. Typhimurium vaccine candidates expressing heterologous O2 O-antigens, and a mouse model was used to evaluate the immunogenicity of live vaccines. ELISA data showed that vaccine candidates could induce a comparatively high level of S. Paratyphi A and/or S. Typhimurium LPS-specific IgG and IgA responses in murine model, and IgG2a levels were consistently higher than IgG1 levels. Moreover, the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays. Our work highlights the potential for developing S. Typhimurium live vaccines against S. Paratyphi A.

Immunization with outer membrane vesicles of avian pathogenic Escherichia coli O78 induces protective immunity in chickens.

Avian pathogenic Escherichia coli (APEC) typically causes colibacillosis and is a major concern for the poultry industry and public health. As a vaccine platform, the outer membrane vesicles (OMVs) derived from various gram-negative bacteria and even some gram-positive bacteria have been reported to be immunogenic in laboratories or upon commercial usage worldwide. Here, we purified OMVs from APEC serotype O78 strain by ultracentrifugation and gradient isolation. By SDS-PAGE and LC-MS/MS analysis, the 20 most abundant proteins located on OMVs were identified and analyzed; the lipopolysaccharide (LPS) profiles of OMVs were not different from those of the bacteria. Moreover, three groups of chickens were immunized with OMV-, outer membrane protein (OMP)- and PBS, with the latter two serving as positive and negative controls, respectively. By analyzing the anti-OMP and anti-LPS IgG titers stimulated by the tested vaccine candidates, the macrophage opsonophagocytic activity and the bactericidal activity mediated by serum antibodies in vaccinated chickens, we found that the OMV-vaccinated chicken group was superior to the two other groups. These findings were confirmed by additional chicken challenge tests, in which all OMV-vaccinated group chickens obtained complete protection but those of the other two groups were barely protected. Our data demonstrate that native APEC O78 OMVs can induce protective immunity in chickens and therefore be used as a candidate vaccine for APEC serotype O78 strain infections.

Regulated Delayed Shigella flexneri 2a O-antigen Synthesis in Live Recombinant Salmonella enterica Serovar Typhimurium Induces Comparable Levels of Protective Immune Responses with Constitutive Antigen Synthesis System.

Shigella flexneri (S. flexneri), a leading cause of bacillary dysentery, is a major public health concern particularly affecting children in developing nations. We have constructed a novel attenuated Salmonella vaccine system based on the regulated delayed antigen synthesis (RDAS) and regulated delayed expression of attenuating phenotype (RDEAP) systems for delivering the S. flexneri 2a (Sf2a) O-antigen. Methods: The new Salmonella vaccine platform was constructed through chromosomal integration of the araC PBADlacI and araC PBADwbaP cassettes, resulting in a gradual depletion of WbaP enzyme. An expression vector, encoding Sf2a O-antigen biosynthesis under the control of the LacI-repressible Ptrc promoter, was maintained in the Salmonella vaccine strain through antibiotic-independent selection. Mice immunized with the vaccine candidates were evaluated for cell-mediate and humoral immune responses. Results: In the presence of exogenous arabinose, the Salmonella vaccine strain synthesized native Salmonella LPS as a consequence of WbaP expression. Moreover, arabinose supported LacI expression, thereby repressing Sf2a O-antigen production. In the absence of arabinose in vivo, native Salmonella LPS synthesis is repressed whilst the synthesis of the Sf2a O-antigen is induced. Murine immunization with the Salmonella vaccine strain elicited robust Sf2a-specific protective immune responses together with long term immunity. Conclusion: These findings demonstrate the protective efficacy of recombinant Sf2a O-antigen delivered by a Salmonella vaccine platform.

[Role of bacterial minicells in cancer therapy].

Cancer is one of the most important diseases threatening human health. Frequently-used traditional cancer treatment methods, like radiotherapy, chemotherapy and surgery, have serious toxic side effects and limitations. The widely-used drug delivery carriers (liposomes, nanoparticles, etc.) have also possessed many issues such as drug leakage and incomplete loading in the late clinical stage. Currently, using tumor-targeting vectors to deliver anti-tumor drugs or small molecules is one of the promising strategies for mediating safe and effective tumor therapy. In recent years, bacterial-derived non-replicating minicells, which are nanoscale non-nucleated cells produced during abnormal bacterial division, have got more and more attention. With a diameter of 200-400 nm, minicells have a large drug loading capacity. Meanwhile, the surface of minicells are able to be modified to load the assembly of antibodies/ligands that bind to tumor cell surface specific antigens or receptors, which can significantly improve tumor targeting of minicells. This tumor-targeting nanomaterials of minicells not only are used to deliver anti-tumor chemotherapeutic drugs, functional nucleic acids or plasmids encoding functional small molecules to mammalian cells, but also greatly increase drug loading and reduce drug penetration. Thus, the use of minicells combining with chemical therapy could help reduce the toxicity and maximize the effectiveness of the drug in the body. This paper summarizes the research and development of production purification, drug loading, tumor cells targeting, and internalization process of minicells, as well as its use in the delivery of anti-tumor drugs, to provide some information for the development and utilization of minicell carriers.

[Application of lysis system in bacterial vector vaccines].

Recombinant bacterial vector vaccines have been widely used as carriers for the delivery of protective antigens and nucleic acid vaccines to prevent certain infectious diseases because of their ability to induce mucosal immunity, humoral immunity and cellular immunity. However, protective antigens and nucleic acids recombined into bacterial vector vaccines are difficult to be released into host cells because of the presence of bacterial cell wall. Vaccine strains that are residual in animals or livestock products may also cause environmental contamination and spread of the vaccine strains. The effective solution for these problems is to construct an auto-lysis system that can regulate the vaccine strains to grow normally in vitro while lysis in vivo. The lysis systems that have been applied in germs mainly include: the lysis system based on regulated delayed peptidoglycan synthesis, the lysis system based on the regulation of bacteriophage lysis protein and the lysis system based on the toxin-antitoxin system. In addition, a potential lysis system based on bacterial Type Ⅵ Secretion System (T6SS) is also expected to be a new method for the construction of auto-lysis strains. This review will focus on the regulatory mechanisms of these bacterial lysis systems.

Identification of Fur in Pasteurella multocida and the Potential of Its Mutant as an Attenuated Live Vaccine.

Pasteurella multocida is a pathogenic microorganism that causes a variety of serious diseases in humans and animals worldwide. The global regulator gene, fur, plays an important role in pathogenesis and regulates the virulence of many bacteria. Here, we identified a fur gene in P. multocida by complementing a Salmonella Choleraesuis Δfur mutant, and characterized a fur mutant strain of P. multocida. The P. multocida Δfur mutant strain exhibited no significant differences in growth and outer membrane protein (OMP) profiles when the complemented strain was compared to the parent. Ducks were used as the model organism to determine the virulence and protection efficacy induced by Δfur mutant strain. Animal experiments showed that colonization by the mutant was decreased by oral infection of live Δfur mutant strain. The LD50 of the ducks infected with the Δfur mutant was 146-fold higher than that of the ducks infected with the wild-type strain when administered through the oral route. Evaluation of the immunogenicity and protective efficacy of the Δfur mutant of P. multocida revealed strong serum IgY and bile IgA immune responses following oral inoculation with the Δfur strain. Ducks that were orally inoculated with the Δfur mutant strain demonstrated 62% protection efficacy against severe lethal challenge with the wild-type P. multocida. This study provides new insights into P. multocida virulence and the potential use of an attenuated vaccine against P. multocida.

Genetically engineered Salmonella Typhimurium: Recent advances in cancer therapy.

Bacteria have been investigated as anti-tumor therapeutic agents for more than a century, since Coley first observed successful curing of a patient with inoperable cancer by injection of streptococcal organisms. Previous studies have demonstrated that some obligate or facultative anaerobes can selectively accumulate and proliferate within tumors and suppress their growth. Developments in molecular biology as well as the complete genome sequencing of many bacterial species have increased the applicability of bacterial organisms for cancer treatment. In particular, the facultative anaerobe Salmonella Typhimurium has been widely studied and genetically engineered to improve its tumor-targeting ability as well as to reduce bacterial virulence. Moreover, the effectiveness of engineered attenuated S. Typhimurium strains employed as live delivery vectors of various anti-tumor therapeutic agents or combined with other therapies has been evaluated in a large number of animal experiments. The well-known S. Typhimurium mutant VNP20009 and its derivative strain TAPET-CD have even been applied in human clinical trials. However, Salmonella-mediated cancer therapies have not achieved the expected success, except in animal experiments. Many problems remain to be solved to exploit more promising strategies for combatting cancer with Salmonella bacteria. Here, we summarize the promising studies regarding cancer therapy mediated by Salmonella bacteria and highlight the main mechanisms of Salmonella anti-tumor activities.

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.

Bi-valent polysaccharides of Vi capsular and O9 O-antigen in attenuated Salmonella Typhimurium induce strong immune responses against these two antigens.

Salmonella Typhi is the causative agent of typhoid fever in humans, responsible for approximately 21 million infections and 222,000 deaths globally each year. The current licensed vaccines provide moderate protection to recipients aged >2 years. Prior work on typhoid vaccines has focused on injectable Vi capsular polysaccharide or Vi-protein conjugates and live, oral attenuated S. Typhi vaccines to induce humoral anti-Vi antibodies, while the value and importance of anti-O9 antibodies is less well established. In this study, we constructed a S. Typhimurium strain that synthesizes Vi capsular antigen in vivo and produces the immunodominant O9-antigen polysaccharide instead of its native O4-antigen. The live recombinant attenuated S. Typhimurium mutants were effective in stimulating anti-Vi and anti-O9 antibodies in a mouse model, and the surface Vi capsular expression did not affect the immune responses against the O9 O-antigen polysaccharide. Moreover, the resulting anti-Vi and anti-O9 antibodies were effective at killing S. Typhi and other Salmonella spp. expressing Vi or O9 antigen polysaccharides and provided efficient protection against lethal challenge by S. Typhimurium and S. Enteritidis. Our work highlights the strategy of developing live attenuated S. Typhimurium vaccines to prevent typhoid fever by targeting the both Vi capsular and O9 O-polysaccharide antigens simultaneously.