Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫     

Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens

The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% µg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.     

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.     

丙型肝炎病毒保护性抗原及其疫苗研究的现状与展望

研制实用有效的HCV疫苗是控制HCV感染的一项根本措施。本文就近年来有关HCV保护性抗原及其疫苗的研究现状进行了综述,并就HCV疫苗研制的有关问题进行了讨论。     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

Cancer testis antigen PRAME: An anti-cancer target with immunomodulatory potential

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.     

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.     

ISA 61 VG佐剂增强单核细胞增生李斯特氏菌灭活疫苗的保护性免疫应答

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是重要的人兽共患李斯特氏菌病的致病菌,疫苗免疫是预防该病原菌感染的有效手段之一。本研究研制了添加矿物油佐剂MontanideTM ISA61VG的新型灭活细菌疫苗,并对其安全性和免疫应答特性进行了研究。结果表明,ISA 61 VG佐剂疫苗具有较好的安全性;诱导小鼠产生的抗李斯特氏菌溶血素O抗体滴度以及IgG2a/IgG1比值显著高于无佐剂免疫组;在致死剂量Lm攻毒下,能对小鼠提供100%的免疫保护。因此,ISA 61VG佐剂能显著增强灭活疫苗诱导宿主产生体液免疫和细胞免疫应答的能力,从而提高灭活疫苗的保护性免疫应答作用,是预防人和动物Lm感染的潜在疫苗候选株。     

Hypoxia modulates stem cell properties and induces EMT through N-glycosylation of EpCAM in breast cancer cells

Epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein, is related to tumor progression. We demonstrated that EpCAM plays important roles in proliferation, apoptosis, and metastasis during breast cancer (BC) progression. But the role of N-glycosylation in EpCAM in tumor aggressiveness is not clear. Here, we evaluated the role of N-glycosylation of EpCAM in stemness and epithelial–mesenchymal transition (EMT) characteristics. EpCAM overexpression increases the expression of stemness markers (NANOG,SOX2, and OCT4) and EMT markers (N-cadherin and vimentin) under the condition of hypoxia in BC. Knockdown of EpCAM and mutation of N-glycosylation of EpCAM maintained in severe hypoxia lead to a significant reduction of stemness/EMT markers. In addition, we found that N-glycosylation of EpCAM is a crucial factor during this process. This demonstrates that EpCAM has a novel regulatory role in stemness/EMT dependence of hypoxia-inducible factor 1-alpha via regulating nuclear factor kappa B in BC cells. Hence, our study reveals EpCAM glycosylation modification as a new regulator of stemness/EMT under hypoxic in BC and points out EpCAM as a potential therapeutic target.     

Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice

AIMS: To determine if live recombinant Lactococcus lactis strains expressing rotavirus VP7 antigen are immunogenic in mice. METHODS AND RESULTS: Using the food-grade lactic acid bacterium L. lactis as a carrier, we expressed VP7, the major rotavirus outer shell protein and one of the main components of the infective particle, as a cytoplasmic, secreted or cell wall anchored forms. Our results showed that recombinant L. lactis strains secreting VP7 proved to be more immunogenic than strains containing the antigen in the cytoplasm or anchored to the cell wall. CONCLUSIONS: This is the first demonstration that recombinant L. lactis producing VP7 can induce the production of a neutralizing antibody response against rotavirus by the intragastric route. SIGNIFICANCE AND IMPACT OF THE STUDY: Rotaviruses are the single most important aetiological agents of severe diarrhoea of infants and young children worldwide and have been estimated to be responsible for 650 000-800 000 deaths per year of children younger than 5 years old in development countries. Thus, the development of a safe and effective vaccine has been a global public health goal. Although two of five mice orally inoculated with L. lactis strains secreting VP7 elicited a specific-antibody response, these strains could be very useful to be used as a prototype to develop a new generation of protective rotavirus vaccines.     

一种新型T细胞免疫原的原核表达及对口蹄疫疫苗的免疫增强作用

主要探讨了T细胞免疫原TI对口蹄疫疫苗的免疫增强作用。设计并原核表达产生了一种包含口蹄疫病毒VP1,VP4,3A和3D蛋白上多个T细胞表位与两个通用T细胞表位的T细胞免疫原,命名为TI;同时表达了O和Asia 1两个型口蹄疫病毒 VP1 蛋白的串联编码基因,表达产物命名为OA-VP1。将上述T细胞免疫原分别与OA-VP1和口蹄疫灭活疫苗按不同剂量组合免疫小鼠,于免疫后不同时间测定各组小鼠的体液与细胞免疫应答情况。采用微量中和试验检测小鼠O型和Asia1型中和抗体,采用流式细胞检测技术和测定γ-干扰素的水平来分析不同免疫组小鼠细胞免疫的水平。结果显示,与灭活疫苗或OA-VP1单独免疫组相比,添加TI抗原后灭活疫苗 (P＜0.01) 和OA-VP1免疫组(P＜0.05)小鼠均能产生高水平的特异性中和抗体;且CD4+ T细胞数量显著增多,IFN-γ产生水平显著升高 (P＜0.01)。说明TI抗原具有很好的诱导特异性体液与细胞免疫应答的作用,是一种很好的免疫增效剂,可作为口蹄疫蛋白亚单位疫苗和灭活疫苗中的一种有效成分,以提高疫苗的免疫效果。     

Commensal Clostridiales strains mediate effective anti-cancer immune response against solid tumors

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Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis

Abstract The molecularly cloned gene encoding the vesicular stomatitis virus (VSV) membrane glycoprotein G was modified and joined to a Bacillus subtilis secretion vector constructed from the plasmid pUB110 and containing the promoter and signal sequence regions of the α-amylase (a secretory protein) gene from Bacillus amyloliquefaciens . The regions encoding the NH₂-terminal signal peptide and the COOH-terminal hydrophobic transmembrane domains of the VSV gene were deleted to facilitate the secretion of the G protein in soluble form. The truncated G protein was found to be expressed in B. subtilis . The expression level was low, probably due to rapid proteolytic degradation of the protein and, contrary to what was expected, almost all of the protein remained cell-associated.     

Combination of CpG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been characterized as Th1-promoting immunopotentiators, an adjuvant activity desirable for vaccination against intracellular parasites like Toxoplasma gondii. In an attempt to find new antigen–adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the extent of protection in mice immunized with ROP2 and GRA4 recombinant proteins when co-administered with CpG-ODN. Both GRA4 + CpG-ODN and ROP2 + CpG-ODN formulations were shown to induce a strong humoral Th1-biased response characterized by a high IgG_2a to IgG₁ antibody ratio. Both vaccination regimens led to increased secretion of IFN-γ and IL-10, and negligible amounts of IL-4, upon specific re-stimulation of spleen cells from these groups of mice. After a non-lethal challenge with tissue cysts of a moderately virulent strain, only the brains from mice vaccinated with ROP2 or GRA4 in combination with CpG-ODN showed a significant reduction (63% and 62%, respectively) in their parasite load compared to the controls. The rate of protection obtained with GRA4 + ROP2 + CpG-ODN resulted equivalent (66%) to those achieved with the single antigens plus CpG-ODN. Taken together, these results indicate that CpG-ODN is an important candidate adjuvant for use in potential multicomponent anti-T. gondii vaccines for animals and humans.     

Development and validation of a coding framework to identify severe acute toxicity from systemic anti-cancer therapy using hospital administrative data

BackgroundThe capture of toxicities from systemic anti-cancer therapy (SACT) in real-world data will complement results from clinical trials. The aim of this study was to develop and validate a comprehensive coding framework to identify severe acute toxicity in hospital administrative data.MethodsA coding framework was developed to identify diagnostic codes representing severe acute toxicity in hospital administrative data. The coding framework was validated on a sample of 23,265 colon cancer patients treated in the English National Health Service between 1 June 2014 and 31 December 2017. This involved comparing individual toxicities according to the receipt of SACT and according to different SACT regimens, as well as assessing the associations of predictive factors and outcomes with toxicity.ResultsThe severe acute toxicities captured by the developed coding framework were shown to vary across clinical groups with an overall rate of 26.4% in the adjuvant cohort, 53.4% in the metastatic cohort, and 12.5% in the comparison group receiving no chemotherapy. Results were in line with regimen-specific findings from clinical trials. For example, patients receiving additional bevacizumab had higher rates of bleeding (12.5% vs. 2.7%), gastrointestinal perforation (5.6% vs. 2.9%) and fistulation (1.4% vs. 0.5%), and allergic drug reactions (1.4% vs. 0.5%). Severe acute toxicity was associated with pre-existing renal (p = 0.001) and cardiac disease (p = 0.038), and urgency of surgery (p = 0.004). Severe toxicity also predicted lower rates of completion of chemotherapy (p = <0.001) and an increased likelihood of altered administration route (p = <0.001).ConclusionThese results demonstrate that the developed coding framework captures severe acute toxicities from hospital administrative data of colon cancer patients. A similar approach can be used for patients with other cancer types, receiving different regimens. Toxicity captured in administrative data can be used to compare treatment outcomes, inform clinical decision making, and provide opportunities for benchmarking and provider performance monitoring.     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

Anti-idotype and recombinant antigen in immunotherapy of colorectal cancer

The CO17-1A/GA733 antigen (Ag), bound by monoclonal antibodies (MAb) CO17-1A and GA733 that define two different epitopes on the Ag, has proven a useful target in passive and active immunotherapy of colorectal carcinoma (CRC). Previous studies suggest that the antitumor effects demonstrated in MAb-treated patients may be mediated by idiotypic cascades. In approaches to active immunotherapy against the Ag, polyclonal goat and monoclonal rat anti-idiotypic antibodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with CRC (stage Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro. The humoral and cellular immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 Ag has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant Ag preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T-cells), similar to the native Ag. Antibody titers elicited in experimental animals by recombinant Ag were significantly higher than those elicited by Ab2, presumably because Ag expresses numerous epitopes, whereas Ab2 mimics a single epitope. Recombinant CO17-1A/GA733 Ag has potential as a vaccine for CRC patients.