Construction of Live Attenuated Vaccine against Vib- rio alginolyticus and the Evaluation on Its Immuno- protection Effect

Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.

Evaluatingα-galactosylceramide as an adjuvant for live attenuated infuenza vaccines in pigs

Natural killer T(NKT)cells activated with the glycolipid ligandα-galactosylceramide(α-GalCer)stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses.Several studies have used this approach to adjuvant inactivated and subunit infuenza A virus(IAV)vaccines,including to enhance cross-protective infuenza immunity.However,less is known about whetherα-GalCer can enhance live attenuated infuenza virus(LAIV)vaccines,which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating infuenza vaccines.The current study used the swine infuenza challenge model to assess whetherα-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine(TX98ΔNS1)encoding a truncated NS1 protein.In one study,weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0,10,50,and 100μg/kg doses ofα-GalCer,and subsequently challenged with a heterologous H3N2 virus.All treatment groups were protected from infection.However,the addition ofα-GalCer appeared to suppress nasal shedding of the LAIV vaccine.In another experiment,pigs vaccinated with the H3N2 LAIV,with or without 50μg/kg ofα-GalCer,were challenged with the heterosubtypic pandemic H1N1 virus.Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways,and signifcantly decreased virus shedding.On the other hand,combining the vaccine withα-GalCer reduced cross-protective cellular and antibody responses,and resulted in higher virus titers in respiratory tissues.These fndings suggest that:(i)high doses ofα-GalCer impair the replication and nasal shedding of the LAIV vaccine;and(ii)α-GalCer might interfere with heterosubtypic cross-protective immune responses.This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines.

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL^(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.

Vaccination with the Live Attenuated <i>Francisella</i><i>novicida</i>Mutant <i>FTN0109</i>Protects against Pulmonary Tularemia

Francisella tularensis is considered a potential bioterrorism agent due to its low infectious dose, high mortality rate, and ability to be spread via the aerosol route. We characterized the F. tularensis subspecies novicida mutant strain FTN0109 as a potential vaccine candidate against tularemia. This strain, which lacks an outer membrane lipoprotein, is attenuated in vitro and in vivo, as it exhibits reduced replication within murine J774 macrophages and has a pulmonary LD50 in BALB/c and C57BL/6 mice of >105 CFU (compared to WT parental strain U112, LD50 FTN0109 also conferred complete protection in BALB/c mice against subsequent pulmonary challenge with 10 LD50 (60,000 CFU) of the murine virulent Francisella strain LVS. We also have demonstrated partial protection (50%) against the highly human virulent subspecies tularensis strain SCHU S4 (25 LD50, 12,500 CFU) following intratracheal vaccination in the Fischer 344 rat, a second rodent model for tularemia. Overall, our results suggest that FTN0109 serves as a potential putative vaccine candidate against pulmonary tularemia.

越南非洲猪瘟疫苗AVAC ASF LIVE(ASFV-G-ΔMGF)研究工作分析与思考

非洲猪瘟(Afcrian swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的烈性传染病。近日,越南正式将经临床试验验证的非洲猪瘟弱毒疫苗AVAC ASF LIVE(ASFV-G-ΔMGF)在其全国推广使用。本文对越南非洲猪瘟弱毒疫苗ASFV-G-ΔMGF相关研究数据进行了总结分析,分别从ASFVG-ΔMGF株攻毒保护能力评价、传代细胞培养株临床动物实验、野猪口服接种试验、毒力返强验证等方面,结合国内相关ASFV基因缺失株试验数据进行了分析。总结分析发现:越南上市的ASFV-G-ΔMGF疫苗株,二次免疫接种后攻毒保护效果更好,但在对野猪的口服接种评价中,其口鼻接种效果存在不确定性;随着疫苗株在动物体内的不断传代,其存在毒力返强和毒株变异风险,导致接种猪只临床症状明显;ASFV-G-ΔMGF株虽然有较好的攻毒保护能力和可靠的生产优势,但仍缺乏相关研究数据,特别是在垂直传播、交叉保护、基因突变等方面。后续仍需对ASFV-G-ΔMGF株的临床应用效果进一步评价。

HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN CAN BE EXPRESSED IN LIVE ATTENUATED SHIGELLA FLEXNERI 5A STRAIN SH42

Objective Attenuated strains of Shigella are attractive live vaccine candidates for eliciting mucosal immune responses which is a suitable carrier for the prophylactic human papillomaviruses (HPV) vaccine development, To examine the potential of a live Shigella based prophylactic HPV vaccine, HPV16L1should be expressed in attenuated shigella strain. Methods A Shigella large invasive plasmid (icsA/virG) based prokaryotic expression plasmid pHS3199 was constructed. HPV16L1 gene was inserted into plasmid pHS3199 to form pHS3199-HPV16 L1 construct, and pHS3199-hpv16L1 was electroporated into a live attenuated shigella strain sh42. The expression of HPV16L1 protein was demonstrated by Western blotting with monoclonal antibody to HPV16L1, The genetic stability of recombinant strain sh42-HPV16 L1 was monitored by consecutive passage culture. Invasive ability of sh42-HPV16L1 was evaluated by Hela cell infection assay. Results HPV16 L1 protein can be expressed in recombinant strain sh42-HPV16 L1, and the protein stably expressed over 140 generations. The invasive ability of sh42-HPV16L1 was diminished dramatically compared to its parent strain, but not abolished completely. Conclusion HPV16L1 protein was constitutively expressed in the attenuated strain of shigella flexneri sh42, and maintained partial invasive ability. Our strategy may represent a promising vaccine candidate against genital HPV16 infection.

Lessons from Vibrio Pathogen and the Comparative Study of Vaccines Developed

Cholera continues to be one of the most common causes of morbidity and mortality among children and adults in developing countries. Vaccine against cholera is an approach in the control of this epidemic and pandemic disease. From the development of very early oral cholera vaccine, advances in vaccine development documented due to a good illustration of the epidemiology, outbreak strategy, and pathophysiology of the disease causing pathogen. The newer-generation oral cholera vaccines are safe and guarantee a high level of protection during outbreak settings for several years. Yet infants and young children in developing countries are hyporesponsive to vaccines and show poor protection against cholera. In this review, we survey and analyse our current knowledge on the etiology of cholera, its clinical manifestation, global epidemiology and elaborate the vaccine candidates, which are effective against the pathogen and the corresponding immune responses to the available vaccines. These reviews comprehensively cover the salient features of recent discoveries related to Vibrio cholerae virulence, past and present vaccine candidates and their advantages and disadvantages with their development strategies. We believe that the advances that have been included in this review will give a comprehensive insight to the prevention and control of cholera outbreaks and development of effective cholera vaccines.

Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals

Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.

Current efforts towards safe and effective live attenuated vaccines against African swine fever: challenges and prospects

Background: African swine fever (ASF) is a fatal hemorrhagic disease in domestic pigs and wild boar caused by African swine fever virus (ASFV). Since ASF has been introduced into Europe and Asia, the major pig-raising areas, posing a huge threat to the pork industry worldwide. Currently, prevention and control of ASF are basically dependent on strict biosecurity measures and stamping-out policy once ASF occurs.Main text: The major risks of ASF spread are insufficient biosecurity measures and human behaviors. Therefore, a safe and effective vaccine seems to be a reasonable demand for the prevention and control of ASF. Due to the efficacy advantage over other types of vaccines, live attenuated vaccines (LAVs), especially virulence-associated genes deleted vaccines, are likely to be put into emergency and conditional use in restricted areas if ASF is out of control in a country with a huge pig population and pork consumption, like China. However, the safety, efficacy, and genetic stability of current candidate ASF LAVs require comprehensive clinical evaluations prior to country-wide field application. Several critical issues need to be addressed to commercialize an ideal ASF LAV, including a stable cell line for manufacturing vaccines, differentiation of infected from vaccinated animals (DIVA), and cross-protection from different genotypes.Conclusion: A safe and effective DIVA vaccine and an accompanying diagnostic assay will facilitate the prevention, control, and eradication of ASF, which is quite challenging in the near future.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Human respiratory syncytial virus(RSV)infection is the leading cause of lower respiratory tract illness(LRTI),and no vaccine against LRTI has proven to be safe and effective in infants.Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice.The constructed recombinant plasmids harbored(5′to 3′)a T7 promoter,hammerhead ribozyme,RSV Long strain antigenomic cDNA with cold-passaged(cp)mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain(A2cpts)or further combined with SH gene deletion(A2cptsΔSH),HDV ribozyme(δ),and a T7 terminator.These vectors were subsequently co-transfected with four helper plasmids encoding N,P,L,and M2-1 viral proteins into BHK/T7-9 cells,and the recovered viruses were then passaged in Vero cells.The rescued recombinant RSVs(rRSVs)were named rRSV-Long/A2cp,rRSV-Long/A2cpts,and rRSV-Long/A2cptsΔSH,respectively,and stably passaged in vitro,without reversion to wild type(wt)at sites containing introduced mutations or deletion.Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive(ts)phenotype in vitro and in vivo,all rRSVs were significantly attenuated in vivo.Furthermore,BALB/c mice immunized with rRSVs produced Th1-biased immune response,resisted wtRSV infection,and were free from enhanced respiratory disease.We showed that the combination ofΔSH with attenuation(att)mutations of cpts contributed to improving att phenotype,efficacy,and gene stability of rRSV.By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains,we have laid an important foundation for the development of RSV live attenuated vaccines.

真实世界12岁以下儿童接种麻腮风联合减毒活疫苗后30天内单纯性热性惊厥发生风险分析

目的分析甘肃省12岁以下儿童接种麻腮风联合减毒活疫苗(MMR)后30 d内发生单纯性热性惊厥(SFS)特征。方法筛选甘肃省2021年1月1日至2023年12月31日电子病历库中诊断为“热性惊厥”个案,利用病例身份信息匹配甘肃省免疫规划信息系统中该病例的接种信息,采用观察性流行病学方法分析12岁以下儿童出现SFS的流行特征及接种MMR 30 d内SFS发生风险。结果共纳入10614例SFS儿童患者,12岁以下儿童SFS总体发生率为92.42/10万,其中12~24月龄儿童发生率最高,为297.67/10万,男性儿童发生SFS风险高于女性儿童(RR值为1.61,P<0.001)。接种MMR后30 d内发生SFS风险较未接种该疫苗的高(RR值为2.66,P<0.001)。接种第1剂次的发生率(27.98/10万)较第2剂次(18.48/10万)高,12~24月龄儿童在接种第1剂次MMR 6~14 d SFS发生风险较<12月、25月~6岁组高(RR值分别为4.06和2.64,P<0.001)。结论12~24月龄儿童在接种MMR后6~14 d SFS发生风险增加,以12~24月龄儿童最为常见,应高度关注高风险人群并加强对SFS监测。

牛支原体疫苗研究进展

牛支原体(M.bovis)是牛的重要病原体,能够引起牛的乳腺炎、关节炎和肺炎等疾病。牛支原体已在全世界传播,对全球范围内的养牛业产生了重大不利影响。目前,由于牛支原体的耐药性有增强的趋势,同时缺乏有效的疫苗和治疗方法,这严重阻碍了牛支原体感染的控制。预防、控制甚至清除牛支原体最好的方法是使用有效的疫苗。虽然目前我国还没有获得新兽药证书的牛支原体疫苗,但相关的研究已取得了一定的进展。本文根据多年来牛支原体疫苗研究的相关资料,从牛支原体感染与免疫机制、灭活疫苗、弱毒活疫苗、基因工程疫苗四个方面进行综述,以期为今后牛支原体疫苗的研制提供思路及参考。

解析疫苗接种后抗体记忆的维持:进展与挑战

“首次感染可以为二次感染提供免疫保护”的生物学基本原理推动近代疫苗的诞生与使用。这种免疫保护的基础是抗体记忆,然而各种疫苗引起的有效抗体应答的持续时间各不相同。临床研究发现,接种减毒活疫苗后引起的中和抗体滴度通常水平较高且持续时间久。但减毒活疫苗存在难以回避的安全性风险,因此有必要进一步研究高水平、高质量抗体记忆的产生机制与调控因素,以开发兼具保护性与安全性的新型疫苗。抗体记忆的维持主要依赖于长寿浆细胞介导的抗体分泌,以及记忆B细胞在二次感染时介导的快速记忆性抗体应答。抗原结构与抗原效价、疫苗的抗原载量与免疫策略、病原体相关物质与疫苗佐剂等因素均可通过影响生发中心反应调控抗体记忆的持续时长。本文将梳理疫苗中各成分及因素对抗体记忆生成和维持的相关机制,并尝试讨论目前疫苗学和基础免疫学领域对此方面仍存有的问题与挑战。

流感病毒改造新策略及其应用

流感病毒有着极强的变异性和传播性,常在全球范围内引起季节性的流感爆发。流感病毒的基因组序列、蛋白结构与功能、病毒的包装机制等环节研究相对清楚,也是一种重要的模式病毒,用于条件控制基因元件的发现和确证,构建智能响应型病毒等。随着反向遗传学与合成生物学的发展,通过基因工程改造的流感病毒能更好地控制病毒复制来提高疫苗的安全性,以及诱发机体产生强烈的免疫反应,在肿瘤免疫治疗领域引发广泛关注。本文描述了蛋白质水解靶向嵌合病毒、条件复制型流感减毒活病毒和高干扰素敏感病毒等三种新型减毒流感病毒改造策略,并对编码过早终止密码子的嵌合抗原肽的流感病毒、与PD-L1或CTLA4免疫检查点重组的流感病毒、截短的NS1片段表达GM-CSF的流感病毒分别对黑色素瘤、肝癌的溶瘤作用进行评述。未来,将通过创新性地运用不同策略、不同病毒来构建减毒活疫苗和溶瘤病毒,以便在临床上获得更加安全有效的治疗手段。

副结核分枝杆菌PanCD基因缺失株的构建及毒力评价

为了开发出能有效防控副结核病的减毒疫苗,试验采用同源重组的方法构建MAP K-10ΔPanCD基因缺失菌株,以MAP K-10菌株基因组为模板,扩增PanCD基因的上下游同源臂,构建pHAE159-p0004s-PanCD重组质粒,转化入耻垢分枝杆菌(Mycobacterium smegmatis,Msg)感受态细胞中获得噬菌体,并感染MAP K-10菌株,然后挑取单克隆扩大培养,提取基因组进行PCR鉴定;绘制MAP K-10菌株与MAP K-10ΔPanCD基因缺失菌株的生长曲线;用MAP K-10ΔPanCD基因缺失菌株感染小鼠并评价其毒力,解剖取肝脏和脾脏,分析组织荷菌量和肝脏病理变化。结果表明:成功扩增出PanCD基因的上下游同源臂,经PCR扩增和酶切鉴定成功构建出pHAE159-p0004s-PanCD重组质粒,将重组质粒电转化入Msg感受态细胞中获得噬菌体,感染MAP K-10菌株后成功构建出MAP K-10ΔPanCD基因缺失菌株;与MAP K-10菌株相比,MAP K-10ΔPanCD基因缺失菌株的生长速率明显下降;攻菌2周时,MAP K-10ΔPanCD基因缺失菌株感染小鼠的肝脏和脾脏中荷菌量显著减少;小鼠肝脏病变明显减轻。说明PanCD基因是MAP的一个重要毒力因子,构建的MAP K-10ΔPanCD基因缺失菌株可作为MAP减毒活疫苗的候选菌株。

CE-EMMA法测定乙脑灭活疫苗中乙二胺四乙酸二钠残留量

目的:建立毛细管电泳(CE)结合电泳中介微分析(EMMA)测定乙脑灭活疫苗中乙二胺四乙酸二钠(EDTA-2Na)残留量的方法。方法:运行缓冲液为0.025 mol•L^(-1)磷酸氢二钠缓冲液(pH 2.5),在线络合金属离子为1.5 mg•mL Fe^(3+),孵育时间3 min,分离电压-25 kV,紫外检测波长257 nm,压力进样5000 Pa×5 s,检测温度为25.0℃。结果:EDTA-2Na在0.01~0.5 mg•mL^(-1)的浓度范围内呈现良好的线性关系,r为0.9999,最低检测限为5μg•mL^(-1),测定样品RSD小于2.87%,加样回收率为94.13%~105.56%。结论:该方法操作简便,准确度高,稳定性好,可用于乙脑灭活疫苗中EDTA-2Na残留量的测定。

毒害艾美耳球虫疫苗ENYS株TaqMan qPCR检测方法的建立

为了对E.necatrix ENYS株在生产中进行质控及对临床应用效果的监测,该研究对ENYS株和ENGD株进行全基因组重测序,结合生物信息学分析建立了一种TaqMan qPCR检测技术。结果显示,En-marker5测序结果与预期相符,最优的引物浓度和探针浓度分别为500nM和250 nM,标准曲线的斜率为-3.249,相关系数为0.999,可以通过Ct值对模板浓度定量,该方法检测下线为290个拷贝/反应和50个卵囊/反应,灵敏度是传统PCR的10倍;该方法可以特异性检测EnYS株,不能扩增其他种的疫苗株和野毒株以及肠道病原菌;重复性检测的变异系数小于0.6%,重复性良好。本研究针对E.necatrix EnYS株开发了一种灵敏度高、特异性强、稳定性好的TaqMan qPCR检测方法,为E.necatrix EnYS株的安全性评估及临床应用提供技术支持。

水痘减毒活疫苗冻干工艺的优化

目的:对冻干水痘减毒活疫苗的冻干工艺参数进行筛选和优化。方法:通过设计单因素试验和正交试验对水痘减毒活疫苗的关键冻干工艺参数进行优化,采用优化后的冻干曲线制备冻干水痘减毒活疫苗,并测定制品水分含量、外观、病毒滴度等指标。结果:最佳工艺参数为升华干燥温度-20℃,升华干燥真空度10 Pa,解析干燥温度30℃,解析干燥时间8 h;采用优化后冻干曲线制备的疫苗外观为乳白色或白色疏松体,复溶后为澄明液体,可微带乳光,无异物;各种理化性质及水分含量均符合规定,且制品病毒滴度符合标准。结论:优化后的冻干曲线适用于冻干水痘减毒活疫苗的制备。

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats

Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis.It is essential to develop an effective anti-T.gondii vaccine for the control of toxoplasmosis,and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.Methods First,theompdc anduprt genes of T.gondii were deleted through the CRISPR-Cas9 system.Then,the intracellular proliferation and virulence of this mutant strain were evaluated.Subsequently,the immune responses induced by this mutant in mice and cats were detected,including antibody titers,cytokine levels,and subsets of T lymphocytes.Finally,the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats.Furthermore,to discover the effective immune element against toxoplasmosis,passive immunizations were carried out.GraphPad Prism software was used to conduct the log-rank(Mantel–Cox)test,Student’st test and one-way ANOVA.Results The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system.Compared with the wild-type strain,the mutant notably reduced proliferation(P<0.05).In addition,the mutant exhibited virulence attenuation in both murine(BALB/c and BALB/c-nu)and cat models.Notably,limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice.Furthermore,compared with nonimmunized group,high levels of IgG(IgG1 and IgG2a)antibodies and cytokines(IFN-γ,IL-4,IL-10,IL-2 and IL-12)in mice were detected by the mutant(P<0.05).Remarkably,all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains.The immunized sera and splenocytes,especially CD8^(+)T cells,could significantly extend(P<0.05)the survival time of mice challenged with the RHΔku80 strain compared with naïve mice.In addition,compared with nonimmunized cats,cats immunized with the mutant produced high levels of antibodies and cytokines(P<0.05),and notably decreased the shedding numbers of oocysts in feces(95.3%).Conclusions The avirulent RHΔompdcΔuprt strain can provide strong anti-T.gondii immune responses,and is a promising candidate for developing a safe and effective live attenuated vaccine.

猪繁殖与呼吸综合征两种减毒活疫苗免疫后病毒载量及抗体水平分析

为了解A、B两种商用猪繁殖与呼吸综合征减毒活疫苗免疫猪后病毒血症及抗体水平的差异,在后备母猪场随机选择100头35日龄后备母猪,分为A、B两组,分组后猪混养14 d。试验猪50日龄时,对A组50头后备母猪免疫A疫苗(1头份/头),B组50头后备母猪免疫B疫苗(1头份/头),疫苗免疫后0、1、2、3、5、7、10、14、21、28、35 d各组猪随机采集血样10份,荧光定量PCR检测猪繁殖与呼吸综合征病毒载量,ELISA检测猪繁殖与呼吸综合征病毒抗体。结果显示,B疫苗免疫后,试验猪的猪繁殖与呼吸综合征病毒病毒血症期持续时间更久,持续至免疫后35 d;A疫苗免疫后,试验猪的猪繁殖与呼吸综合征病毒载量更高;B疫苗免疫后试验猪的N蛋白抗体水平变化趋势更稳定;B疫苗免疫后试验猪的PRRSV GP蛋白抗体水平更高。试验结果表明B疫苗的免疫效果优于A疫苗。