The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

Protectivity conferred by immunization with intranasal recombinant outer membrane protein H from Pasteurella multocida serovar A:1 in chickens

Recombinant outer membrane protein H (rOmpH) is a potential fowl cholera vaccine candidate. The present study was aimed at developing rOmpH formulations for intranasal administration. The rOmpH was purified and formulated with either Escherichia coli enterotoxin B (LTB) or CpG oligodeoxynucleotides (ODN) as an adjuvant. Antibody responses in chickens intranasally immunized with rOmpH in combination with 2 different adjuvants were significantly increased (P<0.05) post immunization. Chicken survival rates showed that rOmpH formulated with ODN and LTB elicited 90% and 70% protection, respectively. Our findings indicated that rOmpH formulated with ODN elicited protection better than that formulated with LTB. Therefore, the vaccines formulations in the present study can be considered new intranasal vaccine formulations for fowl cholera in chickens.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定

为了筛选生长快、毒力强、免疫原性好、副反应小的牛源荚膜A型多杀性巴氏杆菌（Pasteurella multocida，Pm）灭活疫苗菌株，本试验选取6株来自不同地区致犊牛肺炎死亡的牛源荚膜A型多杀性巴氏杆菌分离株，测定了培养基生长曲线、小鼠毒力、菌体脂多糖（LPS）含量及各菌株灭活菌苗免疫小鼠和家兔后的抗体效价，并进行了攻毒保护试验。结果显示，分离株Pm2、Pm3、Pm5生长速度较快、毒力较强、LPS含量较多，均含有与毒力和免疫相关的ptfA和fimA基因；免疫小鼠及家兔未发现明显不良反应，在二免后14 d血清抗体达1：64~1：128，强毒攻毒后全部存活，而PBS对照组全部死亡。本试验结果表明，Pm2、Pm3、Pm5均可作为多杀性巴氏杆菌灭活菌苗的候选菌株，其中Pm3作为首选株。     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

Immunogenicity of multiple-epitope antigen gene of HCV carried by novel biodegradable polymers

In order to develop a promising vaccine candidate utilizing a combined approach to induce both antibody production and T-cell activity, the DNA fragment containing MA of HCV with five conserved epitopes was synthesized. Two types of HCV vaccine candidates (the DNA type and DNA/polymers) were constructed using MA. PLA-PEG-PLA and PLGA-PEG-PLGA were synthesized and used as micelles with encapsulated plasmid pcDNA3.1(+)-MA. The preparation of copolymers, the cloning and analysis of recombinant plasmid DNA, in vitro expression, and immunogenicity in transgenic mice were evaluated in detail. The results indicated that even single immunization and oral immunization with DNA/polymers achieved satisfying immune responses in vivo tests. As biodegradable and nontoxic triblock copolymers, the novel copolymers demonstrated a great advantage, as they made long-term and single-immunizing vaccines possible; in addition, the copolymers showed a better adjuvant effect and scarcely any side effects.     

细菌鞭毛蛋白作为佐剂的研究进展

细菌鞭毛蛋白作为Toll样受体5(TLR5)或NOD样受体C4(NLRC4)的配体,其结构决定了其既有抗原性又具有佐剂效应。将细菌鞭毛蛋白与外源抗原混合或融合表达,已获得多种有效的候选疫苗。细菌鞭毛蛋白佐剂效应主要是通过TLR5和NLRC4信号途径协同实现的。TLR5位于细胞表面,可触发炎性因子、趋化因子和Ⅰ型干扰素的分泌,启动天然免疫应答。NLRC4是细胞质中的模式识别受体,可识别细胞质中的多种配体并诱导相应免疫应答。另外,细菌鞭毛蛋白能募集T淋巴细胞和B淋巴细胞至次级淋巴器官,促进DCs和T淋巴细胞的活化。细菌鞭毛蛋白的可塑性将会使得以细菌鞭毛蛋白为基础的疫苗成为当前和未来研究的热点。     

Preparation of Adjuvant Diluent for Live Vaccine against Classical Swine Fever and Evaluation of its Immune Effects

The aim of this study was to develop a novel oil-in-water (O/W) emulsion as adjuvant diluents (AD) for live vaccine against classical swine fever (CSF) that could effectively enhance the immune effect of vaccine.The AD was prepared by high-pressure homogenization technique.Formulations and preparation parameters were optimized with response surface design.Its stability, particle size, polydispersity (PDI) and Zeta potential were characterized.The humoral immune response and cellular immune response of the AD were evaluated with BALB/c mice by intramuscular injection.The particle size of the AD prepared by optimized formulation and parameters was 100.4 nm, PDI was 0.147, and Zeta potential was —28.7 mV.The experiment results showed that the AD had good stability.The AD was inoculated combined with live vaccine against CSF into BALB/c mice by intramuscular injection.The results showed that the live vaccine against CSF specific immune responses could be evoked in mice by co-inoculation with AD and vaccine.The cellular immune response levels in co-inoculated groups were significantly higher than control group (P<0.05), with obvious phenomena of higher levels of IFN-γ, IL-6 and IL-4 in serum.The result revealed that cellular immune capability significantly improved with the AD.The results strongly revealed that cellular immune capability significantly improved by introducing AD for effective immune-adjuvant for live vaccine against CSF.     

血吸虫疫苗研究现状和发展方向

血吸虫疫苗的研究工作已经进行了很长一段时间,发现了一批有价值的候选疫苗分子。同时通过改进和优化免疫途径,血吸虫疫苗的免疫保护力正在逐步提高。随着研究的日益深入,对血吸虫疫苗的免疫机制等了解更加透彻,距离研制出能诱导高的、稳定的免疫保护力的疫苗分子的目标将越来越近。     

A型塞尼卡病毒检测方法及疫苗研究进展

A型塞尼卡病毒（Senecavirus A,SVA）也称为塞尼卡谷病毒（Seneca Valley virus,SVV）,属于小RNA病毒科塞尼卡病毒属成员。SVA主要引起猪的水泡性疾病,与口蹄疫、水泡性口炎、猪水泡病等临床症状相似,可导致新生仔猪急性死亡,严重影响养猪业发展。自2015年广东省发生SVA感染以来,中国多省份陆续有该病发生的报道。当前,中国因猪群缺乏针对SVA的免疫屏障,加之该病传染性较强,存在大范围暴发的潜在风险。如何有效防控SVA感染是迫切需要解决的问题。目前已开发出多种SVA诊断方法用于进行实验室及现场条件下早期的鉴别诊断。SVA的分离鉴定、原位杂交和免疫组化可用于检测病原体的存在及其与组织内形态学变化关系;血清学诊断方法包括基于不同结构蛋白的间接ELISA方法、竞争ELISA方法、均相光激化学发光免疫技术和病毒中和试验,用免疫学方法检测抗体有助于了解SVA感染进程,是临床诊断的主要手段;病毒核酸检测方法主要有PCR技术、等温扩增技术、基因组测序等分子生物学技术,在病毒感染的早期快速检测及检测新发病毒中具有重要作用。目前仍无商品化疫苗预防SVA感染,但科研人员已研发出了灭活疫苗、弱毒疫苗、核酸疫苗和亚单位疫苗等多种具有潜力的候选疫苗。笔者系统总结了SVA检测方法及疫苗研发的最新进展,以期为SVA感染的防控提供参考依据。