Conventional cancer therapies, such as radiotherapy, chemotherapy, and surgery, exhibit notable limitations in the treatment of solid tumors. Bacteria-mediated tumor therapy has been proven to be effective in clinical studies and is regarded as a promising strategy for tumor treatment. Salmonella, as a facultative anaerobic bacterium, can preferentially colonize tumors and remodel the tumor microenvironment to activate the antitumor immune response of the host. Genetically engineered Salmonella possess excellent tumor-targeting capacity, controllability, and adaptability to diverse therapeutic demands, making them ideal carriers for drug delivery. In this study, RGD4C short peptide was displayed on the constitutively expressed flagella of attenuated Salmonella enterica serovar Typhimurium to develop a vector with enhanced motility, tumor-targeting ability, and capacity for deep colonization in hypoxic tumor regions. Meanwhile, to enable Salmonella to exert antitumor activity safely and effectively, a regulated lysis system based on the novel lysing protein M4lys encoded by phage was applied to lyse Salmonella peptidoglycan to release endogenous and exogenous antitumor molecules, and a balanced lethal vector-host system was used to deliver exogenous antitumor molecules (e.g., l-asparaginase, L-ASNase). Collectively, a novel genetically engineered live Salmonella was successfully constructed, which could deliver exogenous antitumor molecules to exert antitumor effects for tumor therapy.

Live attenuated Salmonella vectors hold great promise for antigen delivery due to their ability to elicit broad mucosal and systemic immunity, but their clinical translation is hindered by safety concerns (e.g., virulence reversion, persistent colonization) and suboptimal antigen release kinetics. Here, we devised and constructed a novel, programmed lysis system in Salmonella based on regulated delayed gene expression and integrated it with self-assembled nanoparticles to create a versatile vaccine platform. This programmed lysis system involved a tse1 gene from Pseudomonas aeruginosa (P. aeruginosa) encoding a peptidoglycan-cleaving lytic effector and a controllable promoter PpagC regulated by Mg²⁺ concentration, and Streptococcus pneumoniae (S. pneumoniae) antigen PspA was used to evaluate this system, which was fused with ferritin (FR) or dihydrolipoyl acetyltransferase (E2p) to self-assembled nanoparticles. The results confirmed Mg²⁺-responsive programmed lysis both in vitro and in vivo and the successful synthesis and assembly of antigen-displaying nanoparticles in S. Typhimurium. Animal studies demonstrated that all engineered autonomously lysing Salmonella strains colonized gut-associated lymphoid tissues equivalently to the control. Notably, this Salmonella vaccine platform showed no significant histopathological signs of virulence in liver or spleen tissues relative to PBS-treated controls. Immunization with the control and this Salmonella vaccine platform elicited robust anti-PspA IgG responses and conferred significant protection against challenge with wild-type S. pneumoniae. Moreover, the nanoparticle-fused constructs (FR and E2p) also induced significant mucosal IgA responses, and the anti-PspA-specific IgG response induced by this Salmonella vaccine platform was significantly higher than that elicited by the non-nanoparticle controls in long-term immune responses. Collectively, our findings establish a programmable lysis-based Salmonella vaccine platform that achieves an optimal balance between the biosafety of attenuated Salmonella and the efficacy of antigen delivery, providing a versatile strategy for next-generation live vector vaccines.

Conventional cancer therapies such as radiotherapy, chemotherapy, and surgery have obvious limitations in treating solid tumors. Bacteria-mediated tumor therapy has been proven effective in clinical studies and considered to be a promising strategy for tumor treatment. Salmonella, as a facultative anaerobic bacterium, can preferentially colonize tumors and activate the anti-tumor immune response of the host by remodeling the tumor microenvironment. Genetically engineered Salmonella have good tumor targeting ability and controllability, and are highly adaptable to different therapeutic requirements, thus serving as ideal carriers for drug delivery. In this study, RGD4C short peptide was displayed on the constitutively expressed flagellum of attenuated Salmonella Typhimurium to develop a vector suitable for fast moving and targeting and deep colonizing within hypoxic region in tumor. At the same time, in order to safely and effectively exert the anti-tumor activity in Salmonella, a regulated lysis system based on the new lysing protein M4lys encoded by phage was applied to lyse Salmonella peptidoglycan to release endogenous and exogenous anti-tumor molecules, and balanced lethal vector-host system was used to deliver exogenous anti-tumor molecules. In all, a novel genetically engineered live Salmonella was successfuly constructed and able to deliver exogenous anti-tumor molecules to exert the anti-tumor activity for tumor therapy.

Shiga toxin-producing Escherichia coli (STEC) is one of the major pathogens responsible for severe foodborne infections, and the common serotypes include E. coli O157, O26, O45, O103, O111, O121, and O145. Vaccination has the potential to prevent STEC infections, but no licensed vaccines are available to provide protection against multiple STEC infections. In this study, we constructed an engineered S. Typhimurium to rapidly produce the outer membrane vesicle (OMV) with low endotoxic activity to deliver the O-antigen of E. coli. S. Typhimurium OMV (STmOMV), which displays mixed heterologous O-antigens, was systematically investigated in mice for immunogenicity and the ability to prevent wild-type STEC infection. Animal experiments demonstrated that STmOMV displaying both E. coli O111 and O157 O-antigens by intraperitoneal injection not only induced robust humoral immunity but also provided effective protection against wild-type E. coli O111 and O157 infection in mice, as well as long-lasting immunity. Meanwhile, the O-antigen polysaccharides of E. coli O26 and O45, and O145 and O103 were also mixedly exhibited on STmOMV as O-antigens of the O111 and O157 did. Three mixed STmOMVs were inoculated intraperitoneally to mice, and confer effective protection against six E. coli infections. The STmOMV developed in this study to display mixed heterologous O-antigens provides an innovative and improved strategy for the prevention of multiple STEC infections.

Non-typhoidal Salmonella enterica (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines are available for protecting against infection of multiple Salmonella serotypes, therefore, the development of Salmonella vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live Salmonella in vivo for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns in vitro to keep their original ability to invade host cells for inducing cross-protection against infection of multiple Salmonella serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit in vivo regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9 Δfur9) containing ∆pagP81::Plpp lpxE mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 (waaI), SW116 (waaJ), SW118 (waaL), and SW120 (wbaP) induced a significant production of IgG and IgA against OMPs isolated from S. Typhimurium, S. Enteritidis, and S. Choleraesuis. SW114 (waaI), SW116 (waaJ), and SW118 (waaL) were capable of conferring significant protection against infection of wild-type S. Enteritidis and S. Choleraesuis, with SW118 (waaL) triggering significant CD4+ T-cell responses as well as the B220low IgG+ BM cell. In conclusion, regulated delayed attenuated Salmonella vaccines with the whole core oligosaccharides of LPS showed a goo.d ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous Salmonella infections. These results give new insight into the development of the Salmonella vaccine against multiple serotypes of Salmonella.

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.

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::Plpp lpxE ΔlpxR9 were introduced into the mutant strains to generate 1′-dephosphorylated lipid A. Animal experiments showed that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) induced higher levels of IgG and secreted IgA antibodies and provided a higher protection rate than SC1 (C500 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) and SC2 (C500 rpoS+ Δcrp10 ΔpagL7 ΔpagP81:: PlpplpxE ΔlpxR9). We also evaluated the ability of SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) as a vaccine carrier to deliver heterologous protein antigens and polysaccharide antigens. The results indicated that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE 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.

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.

根据大肠杆菌W3110菌株的waaL基因序列设计CRISPR/Cas9的作用靶点,构建sgRNA表达质粒,然后设计同源修复供体DNA序列,通过电转化法导入宿主菌内,从而构建完整的大肠杆菌CRISPR/Cas9基因编辑系统.结果显示,应用该系统成功地构建了大肠杆菌waaL基因缺失株.将该系统继续用于大肠杆菌W3110菌株wecA基因的缺失,结果表明,该系统可快速高效地用于大肠杆菌基因缺失,这为构建大肠杆菌生物工程菌奠定基础.

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.

SignificancePneumococcal infection-caused diseases are responsible for substantial morbidity and mortality worldwide. Traditional pneumococcal vaccines are developed based on purified capsular polysaccharides (CPS) or CPS conjugated to a protein carrier. Production processes of the traditional vaccines are laborious, and thereby increase the vaccine cost and limit their use in developing nations. A cost-effective pneumococcal vaccine using the recombinant attenuatedSalmonellavaccine (RASV) was developed in this study. We cloned and expressed genes for seven serotypes of CPSs in the RASV strain. The RASV-delivered CPSs induced robust humoral and cell-mediated responses and mediated efficient protection of mice against pneumococcal infection. Our work provides an innovative strategy for mass producing low-cost bioconjugated polysaccharide vaccines for needle-free mucosal delivery against pneumococcal infections.

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.

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.

旨在鉴定鸭疫里默氏杆菌(Riemerella anatipestifer)中的crp基因,并探究该基因与菌株毒力的关系,了解crp基因突变株的免疫保护效力,为开发新型R.anatipestifer减毒活疫苗奠定基础.采用P-BLAST在R.anatipesti er 蛋白质组中搜寻与沙门菌crp基因相似的蛋白基因,发现R.anatipesti fer CH-1株的B739-0373基因与沙门菌crp 基因相似度为45％;将B739-0373基因克隆到表达载体上,并回补到沙门菌△crp突变株中,利用麦芽糖-麦康凯培养基颜色反应对其糖代谢功能进行鉴定,结果发现该基因可完全回补沙门菌自身crp的缺失,推测R.anati pesti er CH-1株的B739-0373基因具有类似沙门菌crp基因的功能;利用同源重组和自杀质粒,构建R.anatipesti fer CH-1△crp突变株,比较野生及突变株的生长情况和毒力(包括黏附率、侵染率、LD50、在雏鸭肝脑中的定殖能力),结果表明,crp基因的缺失使菌株生长变得缓慢,黏附力(P＜0.01)和侵染力(P＜0.001)显著下降,对雏鸭的半数致死量(LD50)显著提高,在雏鸭肝脏、脑中定殖能力也显著下降(P＜0.01);在CH-1△crp突变株免疫雏鸭后7,14,21 d,检测雏鸭血清中IgG水平,并以R.anatipestifer野生株对免疫后的雏鸭进行肌注攻毒,观察其临床症状和免疫保护率,发现△crp突变株能刺激雏鸭产生高水平的IgG抗体(P＜0.01),对雏鸭的免疫保护率为100％.综上表明,B739-0373基因鉴定为R.anatipesti fer CH-1株的类似crp基因,可全局调控该菌株的生长和毒力;安全剂量的CH-1△crp突变株可诱导雏鸭产生较强的免疫保护效力,可作为新型减毒活疫苗的候选菌株.

近年来,应用减毒沙门菌载体递呈异源抗原开发口服活疫苗受到广泛的重视.沙门菌作为活的疫苗载体,其优点有:(1)沙门菌疫苗通过常规的细菌培养方法即可大量获得,而且可以通过食物或饮水口服给药,是大规模养殖场接种疫苗的经济、方便选择;(2)经口服接种的减毒沙门菌能够通过粘膜途径感染宿主,诱发特异性粘膜、体液和细胞免疫应答[1];(3)沙门菌可以在活细胞中生物合成多糖,并且这些多糖可以连接至类脂A (Lipid A)的核心寡糖部分,从而诱导针对异源多糖抗原的系统性免疫应答[2-31.此外,使用延迟减毒系统能够兼顾减毒沙门菌疫苗的安全性和有效性.该系统的主要原理是,用可调控启动子(如阿拉伯糖调控子)将某些基因的启动子替换,在体外诱导条件下该基因可以正常表达,沙门菌可以如野生菌一样感染宿主,而一旦沙门菌到达缺乏诱导条件的体内环境,该基因不再表达,从而引起细菌毒力弱化[4].外膜多糖是存在于大多数致病菌的一种毒力因子,经修饰后可以被用来预防相关致病菌引发的疾病.因此,使用减毒沙门菌载体开发多糖疫苗也是相关研究的热点之一.本文将对减毒沙门菌载体递呈异源多糖的研究进展及其潜在的免疫机制进行综述.

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.