CD4+CD25+调节性T细胞治疗反复性流产的动物实验研究

目的:通过动物实验,探讨CD4~+CD25~+调节性T细胞用于反复性流产免疫治疗的效果。方法:密度梯度离心法从正常昆明白新生鼠的血液和能正常生育的BALB/c雄鼠血液中分别分离单核淋巴细胞,再用免疫磁珠法筛选出CD4~+CD25~+调节性T细胞;以CBA/J雌鼠×DBA/2J雄鼠交配组合作为反复自然流产的动物模型,于孕期给予鼠尾静脉注射不同种类的淋巴细胞;采用独立样本t检验对各组得到的数据进行分析和处理。结果:注射CD4~+CD25~+T细胞组小鼠CD4~+CD25~+T细胞在CD4~+T细胞中的占比为(17.49±0.60)%,高于注射单核淋巴细胞组的(14.68±0.83)%、无菌PBS组的(9.54±0.85)%和不注射组的(9.28±0.68)%(t=4.754,P0.05;t=13.242,P0.05;t=15.621,P0.05)。CD4~+CD25~+调节T细胞中的Foxp3蛋白相对表达量(5.85±0.45)高于注射单核淋巴细胞组(2.86±0.54)、无菌PBS组(1.08±0.16)和不注射组(1.00±0.00)(t=7.276,P0.05;t=17.227,P0.05;t=18.635,P0.05)。在第4天和第8天分别注射调节T细胞得到了较好的效果,胚胎吸收率为(4.92±0.08)%,低于注射单核淋巴细胞组的(13.07±0.06)%、注射无菌PBS组的(23.11±0.12)%和不注射组的(25.47±0.11)%(t=-2.603,P0.05;t=-4.012,P0.05;t=-4.700,P0.05)。结论:给反复性流产小鼠于孕期进行多次CD4+CD25+T细胞注射,免疫治疗效果好于传统的注射单核淋巴细胞。     

HPV18-E2与IL-12联合基因疫苗免疫效果研究

目的 研究人乳头瘤病毒18型E2基因(HPV18-E2)疫苗的免疫效果及白细胞介素-12(IL-12)对其免疫作用的影响. 方法 选6～8周龄Balb/c雌性小鼠35只,随机分为5组,即PBS组、pcDNA3.1(+)空质粒组、pcD-NA3.1(+)/E2组、pcDNA3.1(+)/IL-12组和pcDNA3.1(+)/E2+pcDNA3.1(+)/IL-12组,每组7只.小鼠肌肉注射DNA疫苗,200μg/次.隔2周免疫1次,共免疫4次.于0、2、4、6周剪尾取血,第8周摘眼球放血.ELISA测血清中的特异性IgG抗体及小鼠脾淋巴细胞培养上清IFN-γ、IL-4,MTT比色法检测淋巴细胞的增殖. 结果免疫8周pcD-NA3.1(+)/E2及pcDNA3.1(+)/E2+pcDNA3.1(+)/IL-12疫苗组抗体IgG A值显著升高,与对照组比较差异有统计学意义(P＜0.01),pcDNA3.1(+)/E2及pcDNA3.1(+)/E2+pcDNA3.1(+)/IL-12组小鼠脾淋巴细胞培养上清IFN-γ、IL-4水平明显高于其他对照组,差异有统计学意义(P＜0.01),pcDNA3.1(+)/E2+pcDNA3.1(+)/IL-12组小鼠脾淋巴细胞增殖活性明显增强,与pcDNA3.1(+)/E2组比较,差异无统计学意义(P＞0.05).与其他对照组比较差异有统计学意义(P＜0.01). 结论 pcDNA3.1(+)/E2+pcDNA3.1(+)/IL-12核酸疫苗联合免疫小鼠能够有效的诱导细胞免疫和体液免疫应答,且免疫效应比pcDNA3.1(+)/E2基因疫苗强.     

微小隐孢子虫pcDNA3.0-23重组质粒免疫效果评价

目的 比较微小隐孢子虫表面抗原CP23真核表达载体pcDNA3.0-23经不同免疫途径产生的免疫效果.方法 提取微小隐孢子虫基因组DNA,PCR扩增CP23基因片段,克隆至真核表达载体pcDNA.3.0,构建pcDNA3.0-23重组质粒,分别通过肌肉注射和滴鼻(粘膜)免疫2种途径免疫BALB/c小鼠,免疫3次,2周后检测抗CP23特异性抗体IgG滴度、小鼠脾脏、血清中CD4+和CD8+T细胞、细胞因子γ干扰素(IFN-γ);用微小隐孢子虫攻击感染被免疫小鼠,收集小鼠粪便,计算小鼠排出的卵囊量.结果 肌注组与滴鼻组小鼠血清抗CP23特异性抗体IgG滴度随免疫次数增加均明显升高,高于对照组及空质粒组(P＜0.05),肌注组高于滴鼻组(P＜0.05);肌注组与滴鼻组小鼠的CD4+T细胞、CD4+/CD8+比值均高于磷酸缓冲液(PBS)对照组及pcDNA3.0空质粒组(P＜0.05),但2种免疫途径的差异无统计学意义(P＞0.05);肌注组和滴鼻组脾细胞培养上清中IFN-γ明显高于对照组及空质粒组(P＜0.05);微小隐孢子虫攻击小鼠后,2种免疫途径的小鼠排出卵囊量明显少于对照组,且排出时间缩短,2种途径的差异无统计学意义.结论 pcDNA3.0-23重组质粒作为基因疫苗,可产生较好的细胞及体液免疫反应;不同的免疫途径可产生不同的免疫反应.     

结核病鼠T细胞γ-干扰素及白介素-2表达

目的 探讨结核病鼠T细胞亚群中CD4~+、CD8~+及CD4~+CD8~+(DP)T细胞γ-干扰素(IFN-γ)和白介素-2(IL-2)的表达水平.方法 将30只实验小鼠随机分成对照组和结核病组,饲养6周后取血,以流式细胞仪检测分析CD4~+、CD8~+及(DP)T细胞内表达IFN-γ、IL-2的阳性细胞计数和各自占淋巴细胞百分率.结果 表达IFN-γ的CD4~+T细胞占同亚群细胞总数的比值(t=1.83±0.59)高于对照组(t=1.04±0.22),差异有统计学意义(P＜0.05);表达IL-2的CD4~+T细胞、CD4~+CD8~+(DP)T细胞占同亚群细胞总数的比值(t=2.13±0.93,t=0.06±0.04)低于对照组(t=2.86±1.78,t=0.26±0.79),差异有统计学意义(P＜0.05).结论 结核病鼠CD4~+T细胞表达IFN-γ的水平升高;结核病鼠CD4~+、DPT细胞表达IL-2的水平降低;结核病进展期早期T细胞亚群表达IFN-γ与IL-2呈矛盾现象,提示该阶段虽然保护性免疫已经启动,但仍为非特异性免疫占优势.     

卡氏肺孢子虫p55基因片段DNA疫苗的免疫原性研究

[目的]构建卡氏肺孢子虫抗原p55基因片段DNA疫苗,并研究该疫苗的体外表达及在小鼠体内的免疫原性.[方法]构建真核表达质粒pcDNA3.1(+)-582,用免疫印渍技术检测目的蛋白的免疫原性.将雌性BALB/c小鼠分为3组,以肌肉注射方式分别注射重组表达质粒pcDNA3.1(+)-582(免疫组)、pcDNA3.1(+)及PBS(对照组).ELISA法检测小鼠血清中特异性抗体水平,MTT法检测免疫小鼠的淋巴细胞增殖能力.[结果]成功构建了重组真核表达质粒pcDNA3.1(+)-582,免疫印渍技术证实目的蛋白与免疫后的小鼠阳性血清能发生特异性反应;ELISA结果显示免疫后pcDNA3.1(+)-582组小鼠抗体水平呈增高的趋势,pcDNA3.1(+)-582组小鼠淋巴细胞的增殖指数明显高于pcDNA3.1(+)组及PBS组.免疫组与对照组相比较差异显著,差异有统计学意义(P<0.05).[结论]卡氏肺孢子虫抗原p55基因片段DNA疫苗构建成功,免疫小鼠后能诱导产生一定的体液免疫和细胞免疫.     

MUC1基因疫苗对乳腺肿瘤抑制的实验研究

目的:研究人粘蛋白MUC1基因DNA疫苗诱导小鼠体内免疫应答及对乳腺肿瘤的特异性抑制作用。方法:采用股四头肌注射法接种PcDNA3.1(+)-MUC1质粒,通过ELISA法检测小鼠血清MUC1抗体、脾细胞产生IL-2和IFN-γ的量及血清中TNF水平,通过乳酸脱氢酶释放法测定CTL对MCF-7细胞的杀伤活性。结果:经PcDNA3.1(+)-MUC1免疫小鼠后脾细胞分泌IL-2、IFN-γ及血清TNF水平较对照组明显增高,差异有统计学意义;在效靶比为100∶1、50∶1、25∶1、12.5∶1时,MUC1基因疫苗免疫组小鼠CTL对人乳腺癌细胞系MCF-7杀伤率分别为63.8%、51.2%、43.5%、22.5%,较空载体PcDNA3.1(+)组小鼠和灭菌生理盐水组小鼠均明显升高,差异有统计学意义(P<0.05)。结论:MUC1基因DNA疫苗能够激活小鼠Th1细胞,诱导小鼠产生抗MUCl特异性抗体,诱导产生杀伤高表达MUC1基因的乳腺癌细胞的CTL,为MUC1基因疫苗用于乳腺肿瘤生物治疗提供了一定的实验依据。     

弓形虫复合疫苗滴鼻免疫小鼠诱导肠淋巴细胞变化

目的观察弓形虫可溶性速殖子抗原(STAg)联合蜂胶和γ干扰素(IFN-γ)滴鼻免疫BALB/c小鼠后诱导肠上皮内淋巴细胞及其亚群的动态变化。方法将96只5～6周龄雌性BALB/c小鼠随机分为免疫组和对照组,免疫组以弓形虫复合疫苗(20μg STAg+40μg蜂胶+1 000 U IFN-γ)滴鼻免疫,对照组以磷酸盐缓冲液滴鼻;分别于滴鼻2次(间隔2周)后第1、2、3、4、6、8、10和12周处死小鼠,分离肠上皮内淋巴细胞(IEL),计数并涂片;免疫细胞化学法检测CD4+、CD8+T细胞亚群水平。结果免疫组小鼠IEL的T淋巴细胞增生第2周达高峰(2.27×105),之后逐渐降低;在第1、2、3和4周对照组T淋巴细胞数分别为(0.81×105)、(0.74×105)、(0.87×105)、(0.74×105)个,免疫组IEL中T淋巴细胞数分别为(1.79×105)、(2.27×105)、(2.07×105)、(1.69×105)个,2组差异均有统计学意义(F=8.42,P<0.01);IEL中以CD8+T淋巴细胞增生为主,对照组CD8+T细胞水平无明显变化,免疫组CD8+T细胞明显增生,第2周达峰值(50.5%),随后逐渐下降,第1、2、3、4和第6周分别为43.60%、50.51%、44.70%、40.45%、39.73%,与对照组的25.71%、26.37%、29.31%、27.54%、26.35%比较,差异均有统计学意义(F=6.38,P<0.05);第1、2周免疫组CD4+/CD8+比值低于对照组,差异有统计学意义(F=5.74,P<0.05)。结论 STAg联合蜂胶和IFN-γ滴鼻免疫BALB/c小鼠,可诱导肠上皮内淋巴细胞持续性免疫应答。     

人巨细胞病毒IE1核酸疫苗的免疫效果研究

[目的]用人巨细胞病毒(HCMV)IE1核酸疫苗pcDNA3.1(-)-IE1免疫BALB/c小鼠,初步研究其产生的体液免疫和细胞免疫应答水平。[方法]将pcDNA3.1(-)-IE1通过肌肉注射免疫BALB/c小鼠,通过PCR测定和免疫组化检测其在肌细胞中的表达,细胞因子测定、淋巴细胞转化实验检测免疫效果。[结果]PCR检测到与IE1大小一致的片段,免疫组化结果显示IE1基因在小鼠肌细胞中表达IE1目的蛋白。小鼠脾淋巴细胞经PHA刺激后,实验组IL-4、IL-2、IFN-γ含量以及淋巴细胞转化率显著增高,与对照组比较差异有统计学意义(P﹤0.05)。[结论]pcDNA3.1(-)-IE1核酸疫苗能在BALB/c小鼠肌细胞中稳定存在并能表达HCMV IE1蛋白,pcDNA3.1(-)-IE1核酸疫苗可诱导BALB/c小鼠脾细胞分泌IL-4、IL-2、IFN-r并刺激BALB/c小鼠脾细胞增殖。     

手足口病患儿外周血淋巴细胞亚群变化研究

目的探讨临床不同分型手足口病患儿外周血T淋巴细胞亚群水平变化研究。方法选择2012年6月至2015年11月诊断手足口病患儿150例,依据2010年版手足口病诊疗指南,按照患儿入院病情严重程度分为普通组120例与重症组30例。入院后第24h采用流式细胞术检测外周血T淋巴细胞亚群(CD3~+T细胞、CD4~+T细胞、CD4~+/CD8~+T细胞比及CD4~+CD25~+调节性T淋巴细胞比例变化),采用酶联免疫吸附法检测血清中IL-10以及TGF-β1水平变化,分析2组患者血清IL-10、TGF-β1水平以及外周血CD3~+T细胞、CD4~+T细胞、CD4~+/CD8~+T细胞比及CD4~+CD25~+调节性T淋巴细胞比例变化差异。结果重症组与普通组患儿外周血CD3~+T细胞[(50.61±5.83)%比(59.08±6.21)%,t=3.017]、CD4~+T细胞[(23.87±3.91)%比(32.90±4.06)%,t=2.874]、CD4~+/CD8~+T细胞比[(1.05±0.47)%比(1.43±0.51)%,t=2.478]明显减少,外周血CD4~+CD25~+调节性T淋巴细胞比例[(17.42±3.85)%比(11.09±3.26)%,t=2.804]明显减少,,2组比较差异有统计学意义(P0.05)。血清IL-10[(37.90±6.17)ug/L比(11.09±3.28)ug/L,t=2.873],TGF-β1[(41.09±5.83)ug/L比(13.80±4.27)ug/L,t=2.905]水平明显升高,2组间点比较差异有统计学意义(P0.05)。结论重症手足口病患儿发病后免疫调节机制紊乱,表现为抑制性调节性T细胞水平升高,炎性细胞因子分泌增多,水平升高,机体处于免疫抑制状态。     

血清CD3~+、CD4~+、CD8~+T细胞诊断造血干细胞恶性克隆性疾病(白血病)的价值

目的:探讨外周血CD3~+、CD4~+、CD8~+T细胞在白血病中的诊断价值。方法:分析67例白血病患者的临床资料,同期选择30例正常人为对照组,采用流式细胞仪检测两组患者的CD3~+T细胞、CD4~+T细胞、CD8~+T细胞的表达率,同时将观察组患者分为急性白血病(A组)和慢性白血病(B组),比较两组CD3~+T细胞、CD4~+T细胞、CD8~+T细胞表达情况,分析白血病轻重与免疫功能的关系。结果:观察组血清CD3~+T细胞、CD4~+T细胞表达率依次为(61.72±15.43)%、(32.64±7.51)%,均显著低于对照组(68.95±16.24)%、(37.54±7.43)%,CD8~+T细胞表达率(33.45±6.36)%显著高于对照组(26.51±7.27)%(P0.05);A组患者的CD3~+T细胞、CD4~+T细胞表达水平为(57.59±10.43)%、(62.93±11.08)%,均显著低于B组(62.93±11.08)%、(33.42±5.19)%,CD8~+T细胞表达率(34.85±6.27)则高于B组(31.67±5.58)%(P0.05);且rsCD3~+T=0.623,rsCD4~+T=0.714,rsCD8~+T=-0.755,P均0.05。结论:白血病患者的外周血CD3~+、CD4~+、CD8~+T细胞表达紊乱,临床可借助上述T细胞表达水平进行疾病诊断及病情判断。     

Comparison of intranasal and transcutaneous immunization for induction of protective immunity against Chlamydia muridarum respiratory tract infection

Chlamydia pneumoniae causes a range of respiratory infections including bronchitis, pharyngitis and pneumonia. Infection has also been implicated in exacerbation/initiation of asthma and chronic obstructive pulmonary disease (COPD) and may play a role in atherosclerosis and Alzheimer's disease. We have used a mouse model of Chlamydia respiratory infection to determine the effectiveness of intranasal (IN) and transcutaneous immunization (TCI) to prevent Chlamydia lung infection. Female BALB/c mice were immunized with chlamydial major outer membrane protein (MOMP) mixed with cholera toxin and CpG oligodeoxynucleotide adjuvants by either the IN or TCI routes. Serum and bronchoalveolar lavage (BAL) were collected for antibody analysis. Mononuclear cells from lung-draining lymph nodes were stimulated in vitro with MOMP and cytokine mRNA production determined by real time PCR. Animals were challenged with live Chlamydia and weighed daily following challenge. At day 10 (the peak of infection) animals were sacrificed and the numbers of recoverable Chlamydia in lungs determined by real time PCR. MOMP-specific antibody-secreting cells in lung tissues were also determined at day 10 post-infection. Both IN and TCI protected animals against weight loss compared to non-immunized controls with both immunized groups gaining weight by day 10-post challenge while controls had lost 6% of body weight. Both immunization protocols induced MOMP-specific IgG in serum and BAL while only IN immunization induced MOMP-specific IgA in BAL. Both immunization routes resulted in high numbers of MOMP-specific antibody-secreting cells in lung tissues (IN>TCI). Following in vitro re-stimulation of lung-draining lymph node cells with MOMP; IFNgamma mRNA increased 20-fold in cells from IN immunized animals (compared to non-immunized controls) while IFNgamma levels increased 6- to 7-fold in TCI animals. Ten days post challenge non-immunized animals had >7,000 IFU in their lungs, IN immunized animals <50 IFU and TCI immunized animals <1,500 IFU. Thus, both intranasal and transcutaneous immunization protected mice against respiratory challenge with Chlamydia. The best protection was obtained following IN immunization and correlated with IFNgamma production by mononuclear cells in lung-draining LN and MOMP-specific IgA in BAL.     

Influence of maternal antibodies on Chlamydophila psittaci-specific immune responses in turkeys elicited by naked DNA

Plasmid DNA (pcDNA1::MOMP D) expressing the major outer membrane protein (MOMP) of an avian Chlamydophila psittaci serovar D strain was tested for its ability to induce protective immunity against C. psittaci challenge in the presence of maternal antibodies. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was used. Following pcDNA1::MOMP vaccination, both T helper and B cell memory were primed. However, high maternal antibodies titres affected the induction of vaccine-specific antibody responses as assessed by MOMP-specific antibody levels in enzyme-linked immunosorbent assay (ELISA). Cell-mediated immunity was unaltered as demonstrated by the significantly heightened proliferative responses of peripheral blood lymphocytes (PBL) following vaccination. DNA vaccination could significantly reduce clinical symptoms, pharyngeal and cloacal excretion as well as Chlamydophila replication, even in the presence of maternal antibodies.     

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4⁺IFN-γ⁺ T cells and CD8⁺IFN-γ⁺ T cells among total CD3⁺ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.     

Oral immunization with a novel lipid-based adjuvant protects against genital Chlamydia infection

Oral immunization is attractive as a delivery route because it is needle-free and useful for rapid mass vaccination programs to target pandemics or bioterrorism. This potential has not been realized for human vaccination, due to the requirement of large antigen doses and toxic (to humans) adjuvants to overcome the induction of oral tolerance and potential degradation of antigens in the stomach. To date, only oral vaccines based on live attenuated organisms have been approved for human use. In this study we describe the use of a lipid-based delivery system/adjuvant, Lipid C, for oral immunization to protect mice against genital tract chlamydial infection. Lipid C is formulated from food-grade purified and fractionated triglycerides. Bacterial shedding following vaginal challenge with Chlamydia muridarum was reduced by 50% in female mice orally immunized with the chlamydial major outer membrane protein (MOMP) formulated in Lipid C, protection equivalent to that seen in animals immunized with MOMP admixed with both cholera toxin (CT) and CpG oligodeoxynucleotides (CpG-ODN). Protection was further enhanced when MOMP, CT and CpG were all combined in the Lipid C matrix. Protection correlated with production of gamma interferon (IFN) by splenic T cells, a serum MOMP-specific IgG response and low but detectable levels of MOMP-specific IgA in vaginal lavage.     

A DNA vaccine expressing the E2 protein of classical swine fever virus elicits T cell responses that can prime for rapid antibody production and confer total protection upon viral challenge

Immunization of domestic pigs with a DNA vaccine expressing the complete E2 protein of classical swine fever virus (CSFV) conferred total protection against a severe viral challenge. Immunization with three doses of plasmid pcDNA3.1/E2 elicited a consistent and specific, MHC class II restricted T cell response in the three domestic pigs analyzed, in the absence of detectable anti-CSFV antibodies in serum. Upon challenge specific T cell responses were boosted in the three vaccinated pigs, and a rapid rise in the titers of CSFV neutralizing antibodies was noticed in two of them, which correlated with a total protection. In these two pigs, neither disease symptoms were observed nor was virus detected at any time after CSFV infection. Neutralizing antibody titers were lower in the third vaccine, which developed a mild and transient peak of pyrexia. As expected, similar analyses in three control pigs (injected with the empty vector or PBS) did not reveal the induction of specific T cells or viral antibodies and, upon challenge, animals developed severe symptoms of the disease, including high titers of viremia, hyperthermia and virus spread to different organs. Control pigs developed, also, a marked leucopenia, resulting in SWC3+ (myelomonocytic cells) being the major PBMC population, and a drastic decrease CD3+ T cells. This T cell depletion was prevented in animals immunized with pcDNA3.1/E2. The total protection achieved, in the absence of CSFV antibodies before challenge, supports the relevance in the antiviral response observed of specific T cell responses primed by pcDNA3.1/E2 vaccine, which, upon challenge, led to a rapid induction of neutralizing antibodies. The observation that CSFV antibodies could only be detected in protected animals after viral challenge opens the possibility of exploring the potential of the DNA vaccine approach used to develop marker vaccines against CSF.     

CD4 T cells reduce the tissue burden of Chlamydia muridarum in male BALB/c mice

Male chlamydial infections are becoming more recognised as an aetiological agent in infertility. An IFN-γ response is required for protection against Chlamydia in females, but may have the potential to induce pathology in the immune-privileged male reproductive tract. We examined the induction of immunity following intranasal immunisation with major outer membrane protein (MOMP) of Chlamydia muridarum in male BALB/c mice, and the role of MOMP-specific CD4⁺ T cells in clearance of an intrapenile infection. Here we report that adoptive transfer of MOMP-specific CD4⁺ T cells into naïve mice confers partial protective immunity, which significantly reduces the tissue burden of Chlamydia.     

DNA vaccination based on pyolysin co-immunized with IL-1β enhances host antibacterial immunity against Trueperella pyogenes infection

Trueperella pyogenes is a commensal and opportunistic pathogen normally causes mastitis, liver abscesses and pneumonia of economically important livestock. To date, no specific control measure was reported to prevent T. pyogenes infections. In this study, we first constructed a recombinant plasmid pVAX1-PLO based on the main virulent factor pyolysin gene as DNA vaccine against T. pyogenes infection. Subsequently, transient expression of pVAX1-PLO and pcDNA3.1/V5-fIL-1β were identified in Human embryonic kidney cells (HEK293T) by immunofluorescence assay. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity between different immunized groups. The results showed that the successful expression of PLO or fIL-1β protein was detected by confocal microscopy for cells transfected with plasmid pVAX1-PLO and/or pcDNA3.1/V5-fIL-1β. The mice immunized with pVAX1-PLO elicited a higher titer of PLO-specific antibody than the control group. The levels of IFN-γ and IL-2 were significantly increased in the pVAX1-PLO immunized mice, while the expression level of IL-4 was slightly increased but not significant. These findings suggested that the DNA vaccine pVAX1-PLO can primarily induce Th1 immune response. The residual Colony-Forming Units (CFUs) from the liver and peritoneal fluid were decreased obviously in the pVAX1-PLO treated mice compared with the control. Importantly, co-immunization with pcDNA3.1/V5-fIL-1β and pVAX1-PLO could enhance host humoral and cellular immune responses and significantly protect mouse from T. pyogenes infection. In conclusion, our study provides a promising strategy against T. pyogenes infections and implies the potential clinical application of combined DNA vaccines in diseases control.     

Humoral immunoreaction induced by TCR DNA vaccine for beta chain of T cell lymphoma

We exploited the humoral immunoreaction of mice induced by TCR DNA vaccine of beta chain of T cell lymphoma. The plasmids of pcDNA3.1/TCR V beta 8 was constructed. The BALB/c mice were randomly divided into four groups which were pcDNA3.1, pcDNA3.1/TCR V beta 8, pcDNA3.1/TCR V beta 8 + CpG + liposome and phosphorothioate CpG groups with six mice in each group. Vaccines were injected in bilateral musculus quadriceps femoris of mice in the 0, second, and fourth week, respectively. The antibody formation was tested by indirect immuofluorescence in the 0, second, fourth, sixth and eighth weeks, respectively, before and after immunization. Production of antibody against TCR V beta 8 antigen was observed in the groups of pcDNA3.1/TCR V beta 8 and pcDNA3.1/TCR V beta 8 + CpG + liposome. The antibody titer began to rise in the fourth week and attain the maximal value in the sixth week. The antibody titer in the group of pcDNA3.1/TCR V beta 8 + CpG + liposome was higher than that in the group of pcDNA3.1/TCR V beta 8 in the fourth and eighth weeks (both P<0.01); the antibody titer in the group of pcDNA3.1/TCR V beta 8 + CpG + liposome was markedly higher than that in the group of pcDNA3.1/TCR V beta 8 in the sixth week (P<0.001). The result indicate that TCR V beta 8 antigen can induce formation of special antibody in mice. CpG and liposome can improve the humoral immunoreaction induced by TCR V beta 8 gene vaccine.