Effects of Different Adjuvants on the Immunogenicity of PCV2-Cap Protein

The study was aimed to prepare vaccines with different adjuvants,and research its effects on immunogenicity.The PCV2 Cap gene with its signal peptide removed was connected to pET-28a vector,and then was induced to express,using sodium deoxycholate（DOC）and low concentration of urea to dissolve the inclusion body.Different adjuvants,such as alum-based adjuvants,liposome adjuvants,propolis adjuvants,white oil adjuvants and Freund adjuvant were prepared,together with protein purified to make up subunit vaccines,and commercial inactivated vaccine as positive control,immuning mice,and ELISA method were used to detect changes in concentrations of animal serum antibody and cytokines,evaluated the immune protective effect.Results showed that the expression product in the form of inclusion body,using the DOC could omit dissolving inclusion body protein renaturation steps,and the purification method was simple,the capsid protein obtained had high purity.ELISA assays showed that PCV2-Cap had good immunogenicity.And we found that the water system adjuvants had high immune activity,this could provide important previous experimental data for the commercialization of the subunit vaccine.     

不同佐剂对猪圆环病毒衣壳蛋白免疫原性的影响

试验旨在研究猪圆环病毒不同佐剂疫苗的制备及其对免疫原性的影响。将去除信号肽的PCV2（porcine circovirus type 2,PCV2）Cap蛋白基因连接在pET-28a载体上,进行诱导表达,用脱氧胆酸钠（DOC）和低浓度尿素对表达产物进行溶解,制备氢氧化铝胶体佐剂、脂质体佐剂、弗氏佐剂、白油佐剂、蜂胶佐剂,并与纯化的PCV2-Cap蛋白混合制成亚单位疫苗,以商品化的灭活疫苗作为阳性对照,免疫小鼠,并使用ELISA方法检测动物血清中抗体及细胞因子含量的变化,评估其免疫保护效果。结果显示,表达产物以包涵体的形式存在,使用DOC溶解包涵体可省略蛋白复性步骤,且纯化方法简单,获得纯度较高的衣壳蛋白;ELISA检测结果表明PCV2-Cap蛋白能诱导产生特异性较高的抗体;水系佐剂制备的亚单位疫苗具有较高的免疫活性,为亚单位疫苗的商品化提供重要的数据支持。     

利用植物生产药用蛋白的进展、局限及应对策略

以植物作为生物反应器生产药用蛋白具有廉价、安全、易于存储和运输等优点。越来越多的动物实验及临床试验研究结果显示,植物表达的外源药用蛋白,比如抗体、抗原、细胞因子等都能诱导机体产生免疫应答反应,但目前实现商业化的却极少。限制其推广应用的主要原因是外源蛋白在植物中的表达量较低,免疫原性较差。通过表达系统的选择,载体优化,使用免疫佐剂等方法可以突破这些障碍。本研究综述了近年来利用植物表达药用蛋白的进展,讨论了限制其推广应用的因素及解决策略,并对利用植物生物反应器生产药用蛋白的前景进行了展望。     

Immunology of the porcine respiratory disease complex.

PRDC is a multifactorial respiratory syndrome that includes several respiratory pathogens. As can be observed in this article, although the pathogenesis of some of the respiratory pathogens of pigs is fairly well defined, the host response and the immune response necessary to control the pathogen often remain unclear. As our ability to evaluate the porcine immune system and its ability to respond to disease improves, the knowledge of how each of these respiratory pathogens alter and evade the immune system will increase. The pathogens most commonly isolated from pigs with clinical signs of PRDC either infect the cells of the immune system or induce significant immunopathology. Thus, PRRSV and M. hyopneumoniae, the two most common pathogens associated with PRDC, alter the ability of the respiratory immune system to respond to their presence and the presence of other pathogens. By changing the respiratory immune system, these two common pathogens increase the susceptibility to the many other pathogens associated with PRDC. As we learn more about the pathogens of the respiratory system, their interactions with each other, and the mechanisms by which they modulate the immune system, our ability to develop effective control measures will improve.     

Experience gained in immunotherapy from the immunopharmacology of BCG leading to a second generation of systemic immunity adjuvants

Even after using the first arms against tumour (surgery and radiotherapy) 70% of solid tumours reappear. Chemotherapy cannot eliminate ‘the last cell’. Specific and/or non specific immunotherapy helps the organism to eliminate the last cells by stimulating its immune capacity.

The different generations of adjuvants, agents of immunotherapy were studied successively. BCG belongs to the first generation of adjuvants. We considered its association with specific immunotherapy (of irradiated tumour cells) and/or chemotherapy (cyclophosphamide), then we analysed the problems of dose and the ways of administration, advantages and disadvantages (stimulation of suppressor cells).

Second generation of adjuvant (Corynebacterium parvum, nucleotids and levamisole) allowed to specify the parameters of the efficiency of adjuvants. They have the same disadvantages as BCG.

Finally the third generation share the following characteristics: their side-effects will be minimal (they will not induce suppressor cells), their action will be limited to stimulate one population of cells only and they will be well defined chemical compounds. Thymosin, Bestatin and MDP belong to this generation. Combinations of adjuvants may also be envisaged with the objective of stimulating a particular mechanism of antitumoural activity.     

免疫佐剂研究进展

佐剂的主要作用是提高抗原(免疫原)的免疫原性和免疫反应的可持续性,它能引导机体的免疫系统对抗原产生体液免疫或细胞免疫反应.对佐剂的选择取决于免疫的目的,从用途上分,佐剂可分为试验用佐剂和疫苗用佐剂.前者主要用于特异性抗体的制备,而后者则作为疫苗的必要成分.文章主要介绍目前常用的几种佐剂包括铝盐佐剂、弗氏佐剂、免疫刺激复合物(ISCOM)、脂质体和CpG及其在科研和疫苗中的应用.     

Role of memory B-cell responses in serum and mucosal fluids of swine for protective immunity against pseudorabies virus.

We examined primary and memory isotype-specific antibody responses directed against pseudorabies virus in serum and mucosal fluids of pigs with and without passively acquired maternal antibody, and we studied the relationship between these responses and protection against virus challenge. Pigs were inoculated intranasally with the virulent NIA-3 strain or the avirulent Bartha strain, or they were inoculated IM with an inactivated vaccine containing the Phylaxia strain. Ten weeks later, all pigs were challenge-exposed intranasally with strain NIA-3. Only pigs that were without passively acquired antibody at the time they were inoculated with virulent virus appeared to have complete protective immunity against challenge exposure, as evidenced by lack of clinical signs of pseudorabies and lack of virus excretion. In contrast, pigs inoculated with strain Bartha or with the inactivated vaccine developed fever, had a period of growth arrest, and excreted virus after challenge exposure. In pigs without passively acquired antibody, intranasal inoculation with strains NIA-3 or Bartha was followed by primary IgM and IgA responses in serum and in oropharyngeal fluid as well as primary IgG1 and IgG2 responses in serum. Intramuscular inoculation with the inactivated vaccine induced primary serum IgM, IgG1, and IgG2 responses, but no mucosal responses. Challenge exposure of pigs that had been inoculated with the Bartha strain or the inactivated vaccine was followed by clear memory responses in serum and in oropharyngeal fluid. In contrast, challenge exposure of pigs that had been inoculated by the virulent NIA-3 strain was not followed by memory responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪伪狂犬病不同佐剂灭活疫苗对兔免疫原性初探

采用分离鉴定的猪伪狂犬病毒（HB—J株）以4种佐剂研制成4批灭活疫苗,以研究其对兔的免疫原性。疫苗分别免疫PRV抗体阴性兔后,用乳胶凝集试验法（LAT）和中和试验法（SNT）对试验兔进行血清抗体检测;于免疫后21、28d对试验兔分别进行攻毒,观察兔保护情况。试验结果表明,兔对不同佐剂的猪伪狂犬病疫苗均产生良好的免疫应答反应,且当兔免疫后血清凝集价≥1：32或血清抗体中和指数≥1479或中和价≥1：16时,可以抵抗10LD50剂量强毒的攻击;当兔免疫后血清凝集价≥1：64或血清抗体中和指数≥2187或中和价≥1：32时,可以抵抗100LD50剂量强毒的攻击;4种佐剂灭活疫苗均具有良好的免疫效果。     

动物肠道菌群与病原微生物感染关系的研究进展

动物肠道内寄居着大量微生物,通常被称为共生菌群。它们对动物的生长、代谢和免疫状态至关重要,还与许多疾病的发生密切相关。病毒、细菌和寄生虫感染都会使机体的肠道菌群发生紊乱,表现在益生菌丰度减少而有害菌丰度增加。其机制包括引发宿主的炎症反应和抑制机体的免疫细胞两方面。同样肠道菌群也会调控病原菌的感染,如肠道菌群对不同的病毒会产生颉颃或促进作用,对细菌和寄生虫分别产生抑制和促进作用。肠道菌群抑制病原菌的机制包括与病原菌竞争代谢产物和诱导宿主的免疫反应。肠道菌群促进病毒感染的机制包括3点,分别为提高病毒的稳定性及其与靶细胞的黏附作用、抑制机体免疫系统和刺激靶细胞的增殖。肠道菌群促进寄生虫感染的可能机制包括降低Th2细胞因子(如IL-4和IL-13)并提高调节性T细胞的表达频率。肠道菌群、病原微生物和宿主不断相互作用,形成一个动态的平衡关系,并在感染过程中不断进化。作者主要综述了病毒、细菌和寄生虫感染对动物肠道菌群的组成和丰度的影响,动物肠道菌群如何影响病毒、细菌和寄生虫的感染进程并分析相关机制,以期了解疾病的发病机理,为疫苗佐剂的研发及制定更有效的预防和治疗策略提供新视角和理论依据。     

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus

The spike 1 (S1) surface glycoprotein of infectious bronchitis virus (IBV) is the major inducer of the generation of virus neutralizing antibodies, and the administration of purified S1 has been shown to elicit a protective immune response against virulent virus challenge. On the basis of these observations, recombinant fowl poxvirus (rFPV) containing a cDNA copy of the S1 gene of IBV Mass 41 (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 in vito by indirect immunofluorescence staining and western blot analyses. Later, in vivo expression was demonstrated by the detection of IBV-specific serum immunoglobulin G and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histologic changes of the tracheas in virulent IBV Mass 41-challenged chickens previously receiving rFPV-S1 as compared with parental fowl poxvirus (FPV)-vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent virulent FPV challenge. As to a preferred method of immunization, wing web administration appeared to be superior to the subcutaneous route because a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowl pox and infectious bronchitis.     

禽流感DNA疫苗研究进展

禽流感（avian influenza,AI）是由禽流感病毒（AIV）引起的一种禽类烈性综合征,威胁动物和人类公共健康,严重影响中国养禽业发展,接种疫苗一直是控制禽流感病毒传播最有效的手段。基于基因工程技术的不断发展,各种新型疫苗相继研发并投入使用。其中,禽流感DNA疫苗具有安全性高、制备方法简单、易于储藏和运输等优点,受到了广泛关注。常见的禽流感疫苗有HA DNA疫苗、NA DNA疫苗、M DNA疫苗、NP DNA疫苗等。禽流感DNA疫苗是将含有目的基因序列的重组质粒导入动物细胞,诱导动物机体产生体液和细胞免疫应答。为了提高禽流感DNA疫苗的免疫效果,国内外学者通过添加合适的佐剂、将目的基因导入理想质粒载体、对抗原序列优化,增强DNA疫苗的转染效率和基因表达水平,取得了一定的研究成果。自DNA疫苗开始研发至今,H1、H3、H5、H7、H9等众多亚型禽流感DNA疫苗逐步研发。2018年,由中国农业科学院哈尔滨兽医研究所研制的禽流感H5亚型DNA疫苗获得国家一类新兽药证书,是中国首个获得批准的禽流感DNA疫苗,极大地推动了DNA疫苗的发展。文章主要论述了禽流感DNA疫苗的载体构建、免疫机制、佐剂和载体选择以及疫苗研发等方面的研究进展和创新,并对其应用前景进行简要分析,旨在为科研工作者研制新型禽流感疫苗提供新的思路和参考。     

Review of DNA Vaccine for Avian Influenza

Avian influenza (AI) is a kind of avian virulent syndrome caused by avian influenza virus (AIV),which threatens animal and human public health and seriously affects the development of poultry industry in China.Vaccination has always been the most effective means to control the spread of avian influenza virus.Based on the continuous development of genetic engineering technology,a variety of new vaccines have been developed and put into use.Among them,avian influenza DNA vaccine has many advantages,such as high safety,simple preparation,easy storage and transportation.Common HA DNA vaccine,NA DNA vaccine,M DNA vaccine,NP DNA vaccine.Avian influenza DNA vaccine introduces a recombinant plasmid containing the target gene sequence into animal cells to induce a humoral and cellular immune response.In order to improve the immune effect of avian influenza DNA vaccine,researchers at home and abroad have made some achievements in enhancing the transfection efficiency and gene expression level of DNA vaccine by adding appropriate adjuvants,introducing target genes into ideal plasmid vectors and optimizing antigen sequence.Since the development of DNA vaccines,many subtypes of avian influenza DNA vaccines,including H1,H3,H5,H7 and H9 subtypes,have been gradually developed.In 2018,the H5 subtype DNA vaccine developed by Harbin Veterinary Research Institute of The Chinese Academy of Agricultural Sciences obtained the National Class Ⅰ Veterinary Medicine certificate,which is the first DNA vaccine of avian influenza to be approved in China,greatly promoting the development of DNA vaccines.This review mainly discusses the development and innovation of avian influenza DNA vaccine in terms of vector construction,immune mechanism,adjuvant and vector selection,and vaccine research and development,and briefly analyzes its application prospect,in order to provide new ideas and references for researchers to develop new avian influenza vaccine.     

疫苗佐剂胞嘧啶-鸟嘌呤寡脱氧核苷酸的研究进展

胞嘧啶-鸟嘌呤寡脱氧核苷酸(cytosine phosphate guanidine oligodeoxynucleotide,CpG ODN)是指含有非甲基化的胞嘧啶和鸟嘌呤二核苷酸为核心序列的核苷酸序列,近年来,CpG ODN作为一种新型免疫佐剂的研究越来越多,可诱发机体产生多种免疫学效应,提高系统免疫和黏膜免疫水平,具有安全性高,耐受性强等特点。     

An immunologist's perspective on nutrition,immunity, and infectious diseases: Introduction and overview

The immune system is a multifaceted arrangement of membranes (skin, epithelial, and mucus), cells, and molecules whose function is to eradicate invading pathogens or cancer cells from a host. Working together, the various components of the immune system perform a balancing act of being lethal enough to kill pathogens or cancer cells yet specific so as not to cause extensive damage to “self” tissues of the host. A functional immune system is a requirement of a healthy life in modern animal production. Yet infectious diseases still represent a serious drain on the economics (reduced production, cost of therapeutics, and vaccines) and welfare of animal agriculture. The interaction involving nutrition and immunity and how the host deals with infectious agents is a strategic determinant in animal health. Almost all nutrients in the diet play a fundamental role in sustaining an optimal immune response, such that deficient and excessive intakes can have negative consequences on immune status and susceptibility to a variety of pathogens. Dietary components can regulate physiological functions of the body; interacting with the immune response is one of the most important functions of nutrients. The pertinent question to be asked and answered in the current era of poultry production is whether the level of nutrients that maximizes production in commercial diets is sufficient to maintain competence of immune status and disease resistance. This question, and how to answer it, is the basis of this overview. Clearly, a better understanding of the interactions between the immune signaling pathways and productivity signaling could provide the basis for the formulation of diets that optimize disease resistance. By understanding the mechanisms of nutritional effects on the immune system, we can study the specific interactions that occur between diet and infections. This mechanism-based framework allows for experiments to be interpreted based on immune function during an infection. Thus, these experiments would provide a “real world” assessment of nutritional modulation of immune protection separating immune changes that have little impact on resistance from those that are truly important. Therefore, a coordinated account of the temporal changes in metabolism and associated gene expression and production of downstream immune molecules during an immune response and how nutrition changes these responses should be the focus of future studies. These studies could be answered using new “-eomics” technologies to describe both the local immune environments and the host-pathogen interface.     

Strategies and hurdles using DNA vaccines to fish

DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen – and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.     

Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells

Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu–Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th₁ and CD8+ Tc₁ adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production.     

Immune evasion by pathogens of bovine respiratory disease complex

Bovine respiratory tract disease is a multi-factorial disease complex involving several viruses and bacteria. Viruses that play prominent roles in causing the bovine respiratory disease complex include bovine herpesvirus-1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parinfluenza-3 virus. Bacteria that play prominent roles in this disease complex are Mannheimia haemolytica and Mycoplasma bovis. Other bacteria that infect the bovine respiratory tract of cattle are Histophilus (Haemophilus) somni and Pasteurella multocida. Frequently, severe respiratory tract disease in cattle is associated with concurrent infections of these pathogens. Like other pathogens, the viral and bacterial pathogens of this disease complex have co-evolved with their hosts over millions of years. As much as the hosts have diversified and fine-tuned the components of their immune system, the pathogens have also evolved diverse and sophisticated strategies to evade the host immune responses. These pathogens have developed intricate mechanisms to thwart both the innate and adaptive arms of the immune responses of their hosts. This review presents an overview of the strategies by which the pathogens suppress host immune responses, as well as the strategies by which the pathogens modify themselves or their locations in the host to evade host immune responses. These immune evasion strategies likely contribute to the failure of currently-available vaccines to provide complete protection to cattle against these pathogens.     

Vaccination against chlamydial infections of man and animals

Vaccination is the best approach for controlling the spread of chlamydial infections, in animal and human populations. This review summarises the progress that has been made towards the development of effective vaccines over the last 50 years, and discusses current vaccine strategies. The ultimate goal of vaccine research is to develop efficacious vaccines that induce sterile, long-lasting, heterotypic protective immune responses. To date, the greatest success has been in developing whole organism based killed or live attenuated vaccines against the animal pathogens Chlamydophila abortus and Chlamydophila felis. However, similar approaches have proved unsuccessful in combating human chlamydial infections. More recently, emphasis has been placed on the development of subunit or multicomponent vaccines, as cheaper, safer and more stable alternatives. Central to this is a need to identify candidate vaccine antigens, which is being aided by the sequencing of representative genomes of all of the chlamydial species. In addition, it is necessary to identify suitable adjuvants and develop methods for antigen delivery that are capable of eliciting mucosal and systemic cellular and humoral immune responses. DNA vaccination in particular holds much promise, particularly in terms of safety and stability, although it has so far been less effective in humans and large animals than in mice. Thus, much research still needs to be done to improve the delivery of plasmid DNA, as well as the expression and presentation of antigens to ensure that effective immune responses are induced.