ESAT-6 and CFP-10 can be combined to reduce the cost of testing for Mycobacterium tuberculosis infection, but CFP-10 responses associate with active disease

Commercial tests measuring IFN-gamma responses to ESAT-6 and CFP-10 are available for diagnosing Mycobacterium tuberculosis infection. Measures that minimize cost and complexity will facilitate their application in less-developed countries. We investigated whether overlapping peptides representing both ESAT-6 and CFP-10 are required to detect M. tuberculosis infection in a high TB-burden country, and whether they can be combined in a single pool. ESAT-6 and CFP-10 peptides were compared in IFN-gamma enzyme-linked immunospot (ELISPOT) in 183 HIV-negative smear-positive TB cases and 1673 HIV-negative household contacts. Separate peptide pools for each antigen were compared with a combined pool in 498 contacts. Forty per cent of responsive contacts recognized both antigens, 51% only ESAT-6 and 10% only CFP-10, whereas 56% of responsive cases recognized both antigens, 30% only ESAT-6 and 13% only CFP-10. Accordingly, CFP-10 response rates were higher for TB cases (odds ratio 2.409, P<0.001). Low purified protein derivative response rates indicated that responses to CFP-10 only were non-specific in contacts. Agreement between peptides in separate versus combined pools was good (kappa=0.758, r=0.840). Therefore a combined ESAT-6/CFP-10 peptide pool provided maximum sensitivity and efficiency, but CFP-10 was mainly required to detect active disease.     

ESAT-6/CFP-10融合蛋白的抗原性分析及其对诊断结核临床应用价值

目的 分析ESAT-6/CFP-10融合蛋白作为结核抗原的特性,并探讨以其作为抗原的酶联免疫斑点实验(ELISPOT)在结核诊断中的应用价值.方法 以ESAT-6/CFP- 10融合蛋白为刺激抗原的EIISPOT法(ESAT-6/CFP-10-ELISPOT)检测经结核特异性抗原刺激后分泌γ干扰素的效应T淋巴细胞数量方法,测定65例单纯结核病患者,16例HIV/结核双重感染患者,20例HIV感染患者,32例非结核呼吸道疾病患者,30名健康体检者外周血单个核细胞中结核菌抗原特异的T淋巴细胞的频率.结果 ESAT-6/CFP- 10-ELISPOT与结核菌素皮肤试验(TST)在所有81例结核患者中的比较,ESAT-6/CFP- 10-ELISPOT阳性率98.8％,TST阳性率为56.8％;ESAT-6/CFP-10-ELISPOT在涂阳结核组、涂阴结核组、HIV/结核双重感染组阳性率分别为100.0％、97.1％、100.0％,其敏感性远高于痰涂片检查,且各组结果差异无统计学意义;20例HIV感染组有1例阳性,非结核呼吸道疾病患者组与健康对照组均为阴性,提示ESAT-6/CFP- 10-ELISPOT方法的特异度为98.8％.结论 ESAT-6/CFP-10融合蛋白可以很好的刺激效应T淋巴细胞分泌γ干扰素,适合作为结核诊断中的特异性抗原,因而可以用于ELISPOT的检测,ESAT-6/CFP- 10-ELISPOT对结核诊断有应用价值.     

Screening for tuberculosis among 2381 household contacts of sputum-smear-positive cases in The Gambia

Contact investigation is a key component of tuberculosis (TB) control in developed, but not developing, countries. We aimed to measure the prevalence of TB among household contacts of sputum-smear-positive TB cases in The Gambia and to assess the sensitivity of an enzyme-linked immunospot (ELISPOT) assay in this regard. Household contacts of adult smear-positive TB patients were assessed by questionnaire, purified protein derivative (PPD) skin test, ELISPOT assay, physical examination, chest X-ray and sputum/gastric aspirate. Thirty-three TB cases were identified from 2174 of 2381 contacts of 317 adult smear-positive pulmonary TB patients, giving a prevalence of 1518/100000. The cases identified tended to have milder disease than those passively detected. The sensitivity of ESAT-6/CFP-10 ELISPOT test as a screening test for TB disease was estimated as 71%. Fifty-six per cent of contacts with a PPD skin test result >or=10mm induration had detectable responses to ESAT-6/CFP-10 by ELISPOT; 11% with a negative PPD skin test (<10mm) had a positive ESAT-6/CFP-10 response. Active screening for TB among contacts of TB patients may have a role in TB control in The Gambia. These individuals are a high-risk group, and the disease identified is less advanced than that found through passive case detection. An ELISPOT assay was relatively insensitive as a screening test for TB.     

优化T-SPOT. TB在区分脊柱结核与其他脊柱感染中的诊断效能

目的 探讨结核感染T细胞斑点试验(T-SPOT.TB)在脊柱结核(STB)鉴别诊断中的效能,并通过受试者工作特征(ROC)曲线最佳截断值优化诊断效能。方法 收集2010年1月—2019年5月某院脊柱感染患者的临床资料,包括术前T-SPOT.TB检测结果、白细胞计数、C-反应蛋白、血沉、降钙素原和结核抗体等相关数据,根据诊断标准进行临床诊断,分析T-SPOT.TB在术前诊断STB与其他脊柱感染中的灵敏度和特异度,评价优化后的T-SPOT.TB指标的诊断效能。结果 共纳入132例患者,其中78例(59.09%)为STB,54例(40.91%)为非结核脊柱感染。T-SPOT.TB在鉴别诊断STB方面的灵敏度为67.68%,特异度为66.67%。单因素logistic回归分析显示,与非结核脊柱感染比较,T-SPOT.TB检测诊断STB的OR值为4.188(95%CI:1.847～9.974,P<0.001)。优化T-SPOT.TB评价指标,通过绘制ROC曲线,确定ESAT-6、CFP-10、CFP-10+ESAT-6在STB和非结核脊柱感染鉴别诊断中的最佳截断值,分别为12.5、19.5...     

Evaluation of cell-mediated immune responses to two BCG vaccination regimes in young children in South Korea

Children in South Korea are vaccinated with either BCG Pasteur vaccine intradermally (ID), or with BCG Tokyo vaccine given by multipuncture device (MP). Data from a recent national survey indicated that in children under 6 years old, 31.1% had received the ID vaccine and 64.5% the MP vaccine. To compare the T cell responses induced by the two vaccines, children aged 3-7 were recruited and tested for tuberculin skin test reactivity and for in vitro IFN-γ responses to mycobacterial antigens. DTH responses were not significantly different in children vaccinated by either the ID or MP vaccines. PPD-induced IFN-γ was measured in supernatants of 6-day diluted whole blood cultures. IFN-γ production to PPD was not significantly different in the two vaccine groups, although there is a trend that the MP group gives a higher proportion of IFN-γ positivity than the ID group. In addition, when IFN-γ responses to the antigens ESAT-6 and CFP-10 were assessed in the 6-7 year old group, there was no significant difference between the two vaccine groups. Thus, there was no evidence that the increasing use of MP vaccination has reduced protection against M. tuberculosis in young children in South Korea, based on immunogenicity as assessed by DTH and IFN-γ responses to PPD, and also equivalent frequency of responses to ESAT-6 and CFP-10.     

活动性肺结核病患者外周血ESAT6、Ag85B水平与细胞因子相关性分析

目的 检测结核病患者外周血血清中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的水平,探讨分析ESAT-6、Ag85B水平与Treg细胞的增殖之间的相关性。 方法 ELISA方法检测活动性结核病患者和健康志愿者外周血中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的表达水平,并用统计学方法分析血清中特异性分泌蛋白ESAT-6、Ag85B的浓度与Treg细胞刺激分泌的细胞因子IL-10、TGF-β1、IL-35的水平的相关性。 结果 活动性结核病患者血清中特异性分泌蛋白ESAT-6、Ag85B和细胞因子IL-10、TGF-β1的浓度与正常对照组相比均显著升高(P<0.001),但IL-35的浓度与正常对照组相比却显著降低,差异有统计学意义(P<0.001)。活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平与Treg细胞刺激活化分泌的细胞因子IL-10、TGF-β1、IL-35之间的相关性分析结果显示差异无统计学意义(P>0.05)。 结论 活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平均明显高于健康对照组,提示活动性结核病患者Treg细胞的高水平表达可能与结核分枝杆菌毒力系统有关。     

Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p < 0.001) and IL-12 (191 pg/mL, p < 0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10 pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB.     

结核感染T淋巴细胞γ干扰素释放试验在骨关节结核诊断中的应用

目的探讨结核感染T淋巴细胞γ干扰素释放试验(斑点试验,T-SPOT.TB)在骨关节结核中的应用价值。 方法回顾性分析2016年1月至2018年3月在我院骨外科行T-SPOT.TB检测的127疑似骨关节结核感染病例,分析T-SPOT.TB在骨关节结核中的诊断性能;比较在不同部位骨关节结核的阳性率差异;同时比较以早期分泌抗原靶6 (early secreted antigenic target 6,ESAT-6)和培养滤液蛋白10 (culture filtrate protein 10,CFP-10)诊断骨关节结核的阳性率差异。 结果T-SPOT.TB检测的正确率、灵敏度、特异度、阳性预测值、阴性预测值分别为86.61%(110/127)、83.33%(35/42)、88.24%(75/85)、77.78%(35/45)、91.46%(75/82);且在不同类型骨关节结核的阳性率差别无统计学意义(χ²=1.72,P>0.05);单独以ESAT-6和CFP-10抗原的点数诊断骨关节结核阳性率为69.04%、61.90%,差异无统计学意义(χ²=0.27,P>0.05),两者联合诊断阳性率(83.33%)均高于ESAT-6和CFP-10抗原单独诊断的阳性率,差异有统计学意义(χ²=4.16、7.11,P<0.05)。 结论T-SPOT.TB在骨关节结核诊断中具有重要的应用价值,值得临床推广。     

Analysis of complex formation and immune response of CFP-10 and ESAT-6 mutants

ESAT-6 and CFP-10 form a 1:1 heterodimeric complex which contributes to the virulence of Mycobacterium tuberculosis H37Rv. Based on the structure of CFP-10-ESAT-6 complex, we have selected four point mutations each of CFP-10 and ESAT-6 and have analyzed complex formation for the 25 possible combinations between wild-type and mutant CFP-10 and ESAT-6 proteins. We observed that the mutations L25R or F58R of CFP-10 and L29D or L65D of ESAT-6 lead to disruption of complex formation. We have evaluated the immunogenic responses of the wild-type and mutant CFP-10 and ESAT-6 proteins, the wild-type CFP-10-ESAT-6 complex, six complex-forming and two non-complex-forming combinations of wild-type/mutant CFP-10 and ESAT-6 proteins. CFP-10 mutants I21R, L25R, and W43R were found to have better immunogenic potential than wt-CFP-10, while none of the ESAT-6 mutants were better than wt-ESAT-6. Very interestingly, we have discovered that the non-complex-forming mixture of CFP-10-I21R and ESAT-6-L29D gives a strong immunogenic response.     

Epitope based recombinant BCG vaccine elicits specific Th1 polarized immune responses in BALB/c mice

Developing an efficacious vaccine is one of the highest priorities in tuberculosis research. A vaccine based on T cell epitopes representing multiple antigens is an ideal approach to generate effective cellular immunity against the disease. In the present study, we have selected four T cell epitopes from four well defined Mycobacterium tuberculosis antigens, Ag85C (Rv2903c), 10-kDa culture filtrate protein (CFP-10) (Rv3874), PPE68 (Rv3873) and INV (Rv1478). The epitope encoding genes were grafted into a Cpn 10 based epitope delivery system. The cpn 10-epitope chimeras were further cloned and expressed in BCG to obtain four rBCGs (BCG::CFP, BCG::FBP, BCG::PPE and BCG::INV). Both cellular and humoral immune responses induced by these r-BCG strains were evaluated in BALB/c mice after subcutaneous injection of a single dose of 1×10(6)CFU of the individual rBCGs. Compared to the parent BCG immunized animals the splenocytes derived from rBCG vaccinated groups showed greater antigen specific proliferation, characterized with higher IFN-γ response and reduced IL-4 secretion. Also rBCG vaccination was able to induce specific humoral immune response with an enhanced IgG2a/IgG1 ratio. The rBCGs therefore favor an epitope specific Th1 type response, which is known to be important for mycobacterial immunity. Further when two of the rBCGs (BCG::CFP and BCG::FBP) were tested for their protective efficacy both the rBCGs were comparable to BCG in a H37Rv challenge study performed in guinea pigs.     

7种结核分枝杆菌重组蛋白的纯化及血清学特性

目的分离纯化结核分枝杆菌重组蛋白38-kDa、16-kDa、Mtb81、ESAT-6、CFP10、Rv3425、Rv3391,并比较7种蛋白的免疫学特性,评价其在结核血清学诊断中的应用价值。方法利用间接酶联免疫吸附试验评价确诊结核病人、非结核病人、健康人血清中7种重组蛋白的抗原性。结果成功分离纯化了7种重组蛋白;酶联免疫吸附试验结果表明,7种抗原均能有效区分确诊结核病人、非结核病人和健康人,其中阳性率最高的是ESAT-6为47.3%,特异性最高的是Rv3391为98.3%。联合抗原中阳性率和特异性最高的是CFP10+ESAT-6+Rv3391抗原组合。结论单一抗原中ESAT-6具有较高的免疫原性,联合抗原中CFP10+ESAT-6+Rv3391抗原具有很高的阳性率和特异性,在结核诊断具有很高的诊断潜力。     

A novel DNA vaccine containing multiple TB-specific epitopes casted in a natural structure (ECANS) confers protective immunity against pulmonary mycobacterial challenge

Epitope-based DNA vaccines designed to induce T cell responses specific for Mycobacterium tuberculosis (M. tb) are being developed as a means of addressing vaccine potency. In this study, we predicted 4 T cell epitopes from ESAT-6, Ag85A/B and CFP-10 antigens and constructed an ECANS (epitopes casted in a natural structure) DNA vaccine by inserting the epitope DNA segments separately into the gene backbone of M. tb-derived HSP65 (heat shock protein 65) carrier. The immunogenicity and protective efficacy of pECANS DNA vaccine were assessed in BALB/c mice after intramuscular immunization with 4 doses of 50 μg ECANS DNA and followed by mycobaterial challenge 4 weeks after the last immunization. Compared to plasmid encoding HSP65, pECANS DNA immunization elicited remarkably higher levels of IFN-γ production by both CD4⁺ and CD8⁺ T cells, which were coupled with higher frequencies of antigen-specific T cells and higher CTL activity. Significantly enhanced levels of Th1 cytokines (IFN-γ and IL-12) and increased serum IgG2a/IgG1 ratio were also noted, indicating a predominant Th1 immune response achieved by pECANS DNA immunization. In the consequence, a better protection against Mycobacterium bovis BCG challenge was achieved which was evidenced by reduced bacterial loads in lungs and spleens and profound attenuation of lung inflammation and injury. Our results suggested that multi-T cell-epitope based ECANS gene vaccine induced T cell response to multiple T cell epitopes and led to enhanced protection against mycobacterial challenge. This strategy might be a useful platform to design multi-T cell epitope-based vaccine against M. tb infection.     

ESX-5-targeted export of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine tuberculosis with low virulence and reduced persistence

Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette–Guérin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The incorporation of the Mycobacterium tuberculosis (Mtb) secretion system ESX-1 into BCG improves immunogenicity and protection against TB in animal models, which is associated with the secretion of the ESX-1-dependent protein ESAT-6. However, the resulting strain, BCG::ESX1^Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice. In this study, we describe a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. The strain was constructed by fusing the ESAT-6-encoding gene esxA to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing revealed that ESX-5-targeted ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice. Additionally, compared to BCG::ESX1^Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results warrant further studies to evaluate BCG::ESAT6-PE25SS as a potential live attenuated vaccine candidate for TB.     

应用酶联免疫斑点试验检测入伍新兵结核潜伏感染

目的应用酶联免疫斑点试验（ELISPOT）检测入伍新兵结核潜伏感染情况,评价ELISPOT在检测结核潜伏感染中的价值。方法以结核菌素纯蛋白衍生物 (PPD) 皮肤试验为对照,应用ELISPOT试剂盒检测366例2009年驻京部队入伍新兵外周血中分泌结核菌抗原特异性γ干扰素（IFN γ）的T淋巴细胞数。对PPD和ELISPOT均为阴性的入伍新兵接种卡介苗,10个月后再做PPD 皮肤试验和ELISPOT,比较结果。结果366例入伍新兵中,PPD皮肤试验和ELISPOT阳性率分别为44.81%和31.69%。202例PPD皮肤试验阴性和164例PPD皮肤试验阳性者中,分别有53例 （26.24%）和63例（38.41%）ELISPOT阳性,两者的一致率为57.92%（212/366）, 两者的检测结果差异具有统计学意义（χ2=14.34,P＜0.001）。在接种过卡介苗者中,PPD皮肤试验阳性率为58.53%（127/217）,ELISPOT阳性率为29.03%（63/217）,斑点形成细胞数为32.44±26.52 ;在未接种卡介苗者中,PPD皮肤试验阳性率为24.83%（37/149）,ELISPOT阳性率为35.57%（53/149）,斑点形成细胞数为41.81±30.48。110例PPD和ELISPOT均为阴性的入伍新兵接种卡介苗10个月后, PPD 皮肤试验阳转率为78.18%,而ELISPOT检测均为阴性。结论ELISPOT具有较高的特异性和敏感性,能真实反映入伍新兵的结核潜伏感染情况,可推广应用。     

Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates

Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4⁺ and CD8⁺ T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4⁺ and CD8⁺ T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4⁺ T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.     

Elicitation of efficient, protective immune responses by using DNA vaccines against tuberculosis

DNA vaccination is an effective method for elicitation of strong humoral as well as cellular immune responses. DNA vaccines expressing mycobacterial antigens ESAT-6 (Rv3875), alpha-crystallin (Rv2031c) and superoxide dismutase A (Rv3846) were evaluated for their immune responses in Balb/c mice and protective efficacy in guinea pigs. Immunization of mice with the DNA vaccines expressing superoxide dismutase A and alpha-crystallin resulted in markedly higher levels of IFN-gamma as compared to the levels of IL-10. The DNA vaccine expressing ESAT-6 elicited a mixed Th1/Th2 response. Immunization of guinea pigs with these DNA vaccines and subsequent challenge of animals with Mycobacterium tuberculosis H(37)Rv, showed that DNA vaccine expressing superoxide dismutase imparted the maximum protection as observed by a 50 and 10 folds reduction in bacillary load in spleens and lungs, respectively, in comparison to immunization with vector control.     

Immunogenicity of a recombinant Mycobacterium bovis bacille Calmette-Guèrin expressing malarial and tuberculosis epitopes

Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.     

结核分枝杆菌模拟抗原的筛选及初步应用

目的 通过对甲期分泌抗原-6(ESAT-6)及培养基滤过蛋白-10(CFP-10)肽库的结核分枝杆菌模拟抗原的筛选,旨在建立一个临床鉴别诊断结核分枝杆菌感染的新方法.方法 基于蛋白ESAT-6和CFP 10的序列,随机设计合成一组1 9肽库,通过γ-干扰素释放试验筛选特异性结核分枝杆菌抗原模拟多肽,并研究确立其工作浓度.结果 经过多轮筛选获得3条特异性模拟多肽,分别是P1MG、P8NV、P11LD,灵敏度分别为93.3％、90.0％、80.0％,特异性均＞90.0％;工作浓度均为2 μg/ml;将3种模拟多肽等浓度混合作为刺激原,初步临床验证试验表明,其灵敏度为95.3％,特异性为96.2％.结论 基于蛋白ESAT-6和CFP-10的序列设计、筛选、制备的混合型模拟多肽抗原,有望建立一种临床结核分枝杆菌鉴别诊断的新方法.     

Combination of three virus-derived nanoparticles as a vaccine against enteric pathogens; enterovirus,norovirus and rotavirus

Enteric viruses cause diverse infections with substantial morbidity and mortality in children, rotavirus (RV) and norovirus (NoV) being the leading agents of severe pediatric gastroenteritis. Coxsackie B viruses (CVB) are common enteroviruses (EV), associated with increased incidence of severe neonatal CVB disease with potentially fatal consequences. To prevent majority of childhood gastroenteritis, we have developed a non-live NoV–RV combination vaccine consisting of NoV virus-like particles (VLPs) and RV oligomeric rVP6 protein that induced protective immune responses to NoV and RV in mice. Moreover, rVP6 acted as an adjuvant for NoV VLPs. Here, we investigated a possibility to include a third enteric virus-derived antigen in the candidate NoV–RV vaccine, by adding recombinant nanoparticles derived from EV CVB1. To examine immunogenicity of EV-NoV-RV vaccine, BALB/c mice were immunized intramuscularly twice with 10 µg CVB1 VLPs, GII.4 VLPs and rVP6 nanotubes, either separately or combined. To evaluate the adjuvant effect of rVP6 on EV responses, mice received 0.3 µg CVB1 VLPs with or without 10 µg rVP6. Comparable serum IgG antibodies were detected whether the antigens were administered separately or in combination. Each formulation generated IgG1 and IgG2a antibodies, indicating a mixed Th2/Th1-type response. CVB1 VLPs skewed the isotype distribution slightly towards IgG1 subtype, while EV-NoV-RV combination vaccine induced unbiased Th1/Th2 responses to CVB1. Each antigen also induced T cell mediated immunity measured by IFN-γ secretion to specific stimulants ex vivo. Antisera raised by single antigens and combined formulation also exhibited strong neutralizing ability against CVB1 and NoV GII.4. Further, rVP6 showed an adjuvant effect on CVB1 responses, sparing the VLP dose and homogenizing the responses. Finally, the results support inclusion of additional antigens in the candidate NoV-RV combination vaccine to combat severe childhood infections and confirm adjuvant effect of rVP6 nanostructures.     

Delivery of a multivalent scrambled antigen vaccine induces broad spectrum immunity and protection against tuberculosis

The development of effective anti-Tuberculosis (TB) vaccines is an important step towards improved control of TB in high burden countries. Subunit vaccines are advantageous in terms of safety, particularly in the context of high rates of HIV co-infection, but they must contain sufficient Mycobacterium tuberculosis antigens to stimulate immunity in genetically diverse human populations. We have used a novel approach to develop a synthetic scrambled antigen vaccine (TB-SAVINE), comprised of overlapping, recombined peptides from four M. tuberculosis proteins, Ag85B, ESAT-6, PstS3 and Mpt83, each of which is immunogenic and protective against experimental TB. This polyvalent TB-SAVINE construct stimulated CD4 and CD8T cell responses against the individual proteins and M. tuberculosis in C57BL/6 and Balb/c mice, when delivered as DNA, Fowl Pox Virus or Vaccinia Virus vaccines. In addition, the DNA-TBS vaccine induced protective immunity against pulmonary M. tuberculosis infection in C57BL/6 mice. Co-immunization of Balb/c mice with virally expressed TBS and HIV1-SAVINE vaccine stimulated strong T cell responses to both the M. tuberculosis and HIV proteins, indicating no effects of antigenic competition. Further development of this TB-SAVINE vaccine expressing components from multiple M. tuberculosis proteins may prove an effective vaccine candidate against TB, which could potentially form part of a safe, combined preventative strategy together with HIV immunisations.