目的 探讨虚拟仿真实验项目与CBL及TBL相结合的混合式教学方法在“医学形态学实验”教学中的应用效果.方法 以2016级和2017级选课学生为研究对象,随机分为对照组和实验组,对照组采用传统教学方法,实验组采用混合式教学方法,分别进行“医学形态学实验”教学;课程结束后,对学生进行问卷调查及综合考核,比较两种方法的教学效果.结果 实验组学生的综合考核成绩显著高于对照组(P＜0.05);问卷调查显示,实验组学生对混合式教学方法满意度较高(P＜0.05).结论 在“医学形态学实验”教学中,混合式教学的教学效果明显优于传统教学方法.

The application-based flipped classroom (APP-FC) is an innovative teaching-learning model that has not been applied and assessed in basic medical curricula teaching in China. The aim of this investigation is to assess students’ perceptions to the APP-based flipped classroom (APP-FC) teaching model in an immunology course. The data of this study were collected from second-year medical students (n = 92) at Lanzhou University. One class (n = 50), as a control group, was offered lecture-based learning (LBL), while the other class (n = 42), as the APP-FC group, was given lecture-based instruction and the APP-FC teaching model during September–November 2017. Afterward, the perceptions of students on APP-FC teaching model were evaluated using questionnaires. Students responded that APP-FC improves their motivation (83%) and interest in learning immunology (81%), as well as their self-directed learning skills (81%). Compared to the traditional lecture-based instruction, the APP-FC noticeably improved students’ motivation in learning (P = 0.011), self-directed learn skills (P = 0.001), memory abilities (P = 0.009), and problem-solving abilities (P = 0.010). Most medical students’ scores (60%) in the final examination were more than 80 points after implementing an APP-FC model as compared to the control group (40%). The majority of students (70%) preferred the APP-FC teaching approach over traditional lecture-based pedagogy. The implementation of the APP-FC teaching model could improve students’ learning motivation, self-directed learn skills, and problem-solving abilities, which is a preferable teaching model for medical immunology courses in China.

"课程思政"是加强高等学校大学生思想政治教育的重要途径,为更好地发挥"课程思政"的协同育人效应,基于医学免疫学的课程特点,把"思政元素"有机融入医学免疫学课程教学中,实施"知识传授"与"价值引领"同行并重,培养德才兼备的高素质医学人才.

Parasite infections of humans and animals remain a major global health problem, with limited choice of drugs being available to the treatment of parasitosis in the clinic. Sophora moorcroftiana (S. moorcroftiana) is a shrub that grows in Tibet Plateau of China. Decoction of the seeds has been used as a traditional Tibetan medicine to treat parasitosis for years. But the anti-parasitic effects of water-soluble fractions in the seeds need further investigation. In the present study, the water-soluble alkaloid fractions (E2) were obtained from S. moorcroftiana seeds by refluxing extraction with 60% ethanol and low polarity fraction (E2-a) and high polarity fraction (E2-b) were subsequently isolated from E2 using column chromatography. As a parasite model, Caenorhabditis elegans (C. elegans) were treated with different fractions and their survivals were recorded. The results showed that that E2-a induced a lower survival rate in C. elegans than E2-b and E2. The protoscoleces of Echinococcus granulosus (E. granulosus) were cultured in the presence of E2-a. Compared with E2-b and E2, protoscoleces exhibited decreased survival rate following E2-a treatment. Furtherly, the effects of E2-a on the behavior, brood size, and lifespan of the worms were investigated. Body bend frequencies of the worms treated with the high concentration of E2-a were reduced by two-thirds compared with the control group (P < 0.01). Compared with non-E2-a-treated group, exposure of nematodes to E2-a led to a decrease in head thrashes and pharyngeal pumps frequency (P < 0.01). E2-a treatment resulted in a significantly lower brood size (P < 0.01). Additional E2-a treatment induced a significantly shortened lifespan, compared with the control (P < 0.05). These findings indicated that water-soluble fraction E2-a from S. moorcroftiana seeds was a potential helminthic agent.

In this study, Mtb8.4 and HspX, which are expressed at proliferating and dormant stages of Mycobacterium tuberculosis (M. tuberculosis), respectively, were chosen to construct two fusion proteins, Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4), and we investigated whether the antigen dose and protein sequential order could impact the immunogenicity and protective efficacy of these fusion protein vaccines against M. tuberculosis. C57BL/6 mice were vaccinated with new constructions containing a fusion protein with adjuvant of N, N′-dimethyl-N, N′-dioctadecylammonium bromide (DDA) or a mixed adjuvant composed of DDA, polyribocytidylic acid and gelatin (DPG), and the antigen specific immune responses and protective efficacy against M. tuberculosis H37Rv were evaluated. The results showed that both antigens, Mtb8.4-HspX and HspX-Mtb8.4, could elicit strong human T cell responses. With the existing of DDA adjuvant, HspX-Mtb8.4 induced significantly higher secretion level of IFN-γ and TNF-α in spleen cells than Mtb8.4-HspX (p<0.05). In its protective efficacy study, the isolated bacterial Colony Form Unit (CFU) in H8.4-DPG group was significantly reduced compared to 8.4H–DPG group (p<0.05). Furthermore, with the stimulation of Mtb8.4 in vitro, the secretion of IFN-γ and TNF-α from mice immunized with 20μg of H8.4 exhibited relative higher level than the group immunized by 7μg of H8.4 (p<0.05), whereas, IL-2 secreting showed contrary result. The data suggest that the antigen sequential order and dose selection should be considered when a tuberculosis protein vaccine is to be constructed and its immune strategy is to be planned.

Abstract Context: Gastric cancer remains highly prevalent, but treatment options are limited. Natural products have proved to be a rich source of anticancer drugs. Chrysosplenium nudicaule Ledeb. (Saxifragaceae) is a perennial herb that grows in the highlands of China. It has been used as a traditional Chinese medicine to treat digestive diseases for hundreds of years. Recent studies revealed that this herb had anticancer activity, and the flavonoids were speculated to be the effective components. 6,7,3′-Trimethoxy-3,5,4′-trihydroxy flavone (TTF) and 5,4′-dihydroxy-3,6,3′trimethoxy-flavone-7-O-β-d-glucoside (DTFG) are flavonoid compounds isolated from Chrysosplenium nudicaule. Objective: This study examined the effect of TTF and DTFG on SGC-7901 human stomach cancer cell in vitro to determine the anticancer and induction of apoptosis properties of TTF. Materials and methods: The proliferation of cells treated with 32, 16, 8, 4, and 2 μg/mL of TTF or DTFG for 24, 48, and 72 h was assessed by the MTT assay. After being treated with TTF, the apoptosis of SGC-7901 cells was assessed by acridine orange staining, ultrastructure, electrophoresis of DNA fragmentation, and flow cytometry. Results: Results indicated that TTF inhibited the growth of cancer cells with an IC50 value of 8.33 μg/mL after 72 h incubation. However, DTFG showed no inhibitory effect on the growth of the cancer cell. Further studies on TTF also confirmed that it was able to induce apoptosis of SGC-7901 cells at a concentration as low as 4 μg/mL. Discussion and conclusion: The apoptotic effect of TTF makes it a promising candidate for future chemotherapeutic application in treating stomach cancer.

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38log10CFU) than traditional vaccine Bacillus Calmette-Guerin (BCG) (6.01 ± 0.33log10CFU) and PBS control (6.53 ± 0.26log10CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.

目的 利用pYr-adshuttle-4质粒构建重组腺病毒载体pAd-4-hMASP-2.方法 应用PCR技术扩增甘露糖凝集素相关丝氨酸蛋白酶-2(human mannan-binding lectin associated serine protease-2,hMASP-2)基因,经BamH Ⅰ和EcoR Ⅰ双酶切后与pYr-adshuttle-4质粒连接,构建穿梭质粒pYr-ads-4-hMASP-2,在LR酶作用下与腺病毒骨架载体pAd/PL-DEST进行体外同源重组,获得重组腺病毒载体pAd-4-hMASP-2,转染HEK293细胞,获得重组腺病毒rAd-hMASP-2,应用酶切、PCR扩增及基因测序进行鉴定,并测定病毒滴度.将rAd-hMASP-2感染小鼠,实时荧光定量PCR法检测hMASP-2基因mRNA在小鼠肺组织中的表达.结果 重组穿梭质粒pYr-ads-4-hMASP-2经双酶切及测序证实构建正确,通过同源重组获得了重组腺病毒载体pAd-4-hMASP-2,扩增出滴度为1.5×109 PFU/ml的重组腺病毒颗粒rAd-hMASP-2,其能在小鼠肺组织中表达.结论 成功获得重组腺病毒载体pAd-4-hMASP-2和重组腺病毒颗粒rAd-hMASP-2,为体内外研究hMASP-2的活性提供了有效的转基因载体.

ABSTRACT To develop an effective subunit vaccine which could target tubercle bacilli with different metabolic states and provide effective protective immunity, we fused antigens ESAT6, Ag85B, peptide 190–198 of MPT64, and Mtb8.4 mainly expressed by proliferating bacteria and latency-associated antigen Rv2626c together to construct a multistage protein ESAT6-Ag85B-MPT64(190–198)-Mtb8.4-Rv2626c (LT70 for short) with the molecular weight of 70 kDa. The human T-cell responses to LT70 and other antigens were analyzed. The immune responses of LT70 in the adjuvant of DDA and Poly I:C and its protective efficacy against Mycobacterium tuberculosis (M. tuberculosis) infection in C57BL/6 mice were evaluated. The results showed that LT70 was stably produced in Escherichia coli and could be purified by successive salting-out and chromatography. LT70 could be strongly recognized by human T cells from TB patients and persons who are supposed latently infected with M. tuberculosis. The subunit vaccine LT70 generated strong antigen-specific humoral and cell-mediated immunity, and induced higher protective efficacy (5.41±0.37 Log10 CFU in lung) than traditional vaccine Bacillus Calmette-Guerin (6.01±0.33 Log10 CFU) and PBS control (6.53±0.26 Log10 CFU) at 30 weeks post vaccination (10 weeks post-challenge) against M. tuberculosis infection (p < 0.05). These findings suggested that LT70 would be a promising subunit vaccine candidate against M. tuberculosis infection.

Abstract Background: Case-based learning (CBL), an educational method of problem-based learning, provides students with a venue to relate content learned in the classroom to performance in professional practice. This study compared CBL in the teaching of a clinical laboratory immunology (CLI) course to lecture-based learning (LBL), and evaluated the effect on students regarding the CBL. Methods: Data were collected from senior students (n=85; 46% males, 54% females) at Lanzhou University in China. The students were divided into two groups, one group was offered CBL, while the other LBL as a teaching instrument. After intervention, perceptions of both the groups about their respective teaching method were evaluated using questionnaires, the resulting scores were compared to those obtained in the LBL group. Results: The CBL group showed significantly better scores in course examination (p<0.05) as compared to the LBL group. Seventy-seven (90.6%) students in the CBL group opined that CBL improved their learning and clinical problem-solving skills. CBL also provided them with better understanding (90.6%) and preparation for examinations (90.6%). CBL group improved markedly in comparison to the LBL group with regard to learning motivation (p=0.040), clinical reasoning ability (p=0.023) and clinical problem-solving ability (p=0.022). Conclusions: Our findings demonstrate that CBL is a more effective teaching strategy as compared to LBL in a CLI course. Consequently, the implementation of CBL in teaching a CLI course helps students to improve their learning motivation, problem solving abilities and mastery of knowledge.

Experimental education is the basic way to cultivate students' innovative spirit.In order to meet the urgent requirement of the social development for the innovative medical personnel,the experiment teaching of medical immunology was reformed and explored in Lanzhou University.We revised and edited medical immunology experiment teaching materials,optimized the experimental project,increased the practical and comprehensive content,strengthened the experimental operation,so as to improve medical undergraduates' ability to analyze and solve problems,their clinical thinking and reasoning ability,experimental operation ability and their interest in medical immunology.We obtained good teaching effect.

Regulatory T cells (Tregs), which could be down-regulated by IL-28B, were reported to suppress T-cell-mediated immunity. The aim of this study was to investigate the role of IL-28B on the immune responses and protective efficacy of a tuberculosis (TB) subunit vaccine. First, a recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) was constructed; then C57BL/6 mice were immunized with subunit vaccine ESAT6-Ag85B-Mpt64(190-198)-Mtb8.4-HspX (EAMMH) and rAd-mIL-28B together thrice or primed with Mycobacterium bovis bacillus Calmette-Gue'rin (BCG) and boosted by EAMMH and rAd-mIL-28B twice. At last the immune responses were evaluated, and the mice primed with BCG and boosted by subunit vaccines were challenged with virulent Mycobacterium tuberculosis H37Rv to evaluate the protective efficacy. The results showed that rAd-mIL-28B treatment significantly down-regulated the frequency of Tregs at 4 weeks after the last immunization but did not increase the Th1-type immune responses. Moreover, in the regimen of BCG priming and EAMMH boosting, rAd-mIL-28B treatment did not increase the antigen-specific cellular and humoral immune responses, and consequently did not reduce the bacteria load following H37Rv challenge. Instead, it induced more serious pathology reaction. In conclusion, IL-28B down-regulates Tregs following EAMMH vaccination but does not improve the protective immune responses.

OBJECTIVE:To investigate the correlation among serum levels of manning-binding lectin (MBL), MBL-associated serine proteases-2 (MASP-2), complement C3 and high-sensitive C reactive protein (HsCRP) in patients with rheumatoid arthritis (RA).METHODS:Fasting venous blood were collected from 50 RA patients (25 in active stage and 25 in remission) and 40 healthy subjects for detecting serum levels of MBL, MASP-2, complement C3 and HsCRP using enzyme-linked immunosorbent assay (ELISA) and immune turbidity assay.RESULTS:The serum levels of MBL and MASP-2 were significantly lower and HsCRP level was significantly higher in patients with RA (in both acute stage and remission) than in the healthy control group (P<0.05), but complement C3 level was similar between the RA patients and control group. Bivariate Pearson correlation analysis showed that in RA patients, MBL was positively correlated with MASP-2 level (r=0.550, P=0.001) and negatively with HsCRP (r=-0.323, P=0.022) but not correlated with C3 (r=-0.022, P=0.882); MASP-2 was negatively correlated with HsCRP (r=0.453, P=0.453) and was not correlated with C3 (r=0.049, P=0.738). ROC curve analysis revealed the largest area under curve (AUC) of HsCRP (0.844, P=0.001) and smaller AUCs of MBL (0.025, P=0.001) and MASP-2 (0.266, P=0.001). HsCRP had a much higher sensitivity (84%) than MBL (10%) and MASP-2 (40%) in the diagnosis of RA.CONCLUSION:In RA patients, MBL and MASP-2 are negatively correlated with HsCRP level. Serum MBL and MASP-2 levels decrease with the progression of joint injury in RA patients, suggesting their involvement in the pathological process of RA; but due to their low sensitivity, they are not appropriate indicators for evaluating the disease activity of RA.

Background: The role of MASP-2 is vital in the process of complement activation by the lectin pathway. It is generally considered that the functional activation of MASP-2 contribute to the infection disease development process.Aims: To analyze the association between MASP-2 functional gene (rs72550870) polymorphism and the infection disease risk by a meta-analysis.Method: Relevant case-control studies were identified by searching Cochrane Library, PubMed, Emabase, DOAJ, CAB Abstracts, CSA, CINAHL, EBSCO, Scopus, Global Health, Index Copernicus, CA, China National Knowledge Infrastructure (CNKI) databases up to 10th January 2016. The data were extracted and the methodological quality of studies were evaluated. The STATA 12.0 software was used to perform statistical analysis.Results: 9 studies were included. There was no significant association between masp-2 gene (p.D120G, rs72550870) polymorphism and the risk of infection disease under the allele model (G vs. A: OR = 0.89, 95%CI = 0.66-1.21)(P = 0.445>0.05) and the recessive model (AG + GG vs.AA: OR = 0.88, 95%CI = 0.65-1.20) (P = 0.428>0.05).Conclusion: This is the first comprehensive meta-analysis indicates that the MASP-2 functional gene (rs72550870) polymorphism is not associated with the infection diseases, and the key functional gene polymorphism of rs72550870 did not increase susceptibility to the infection diseases. Similarly, there were no obvious difference in subgroup analysis based on geographical areas and pathogenic microorganisms. (C) 2016 Elsevier Ltd. All rights reserved.

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.

本研究旨在探讨黄芪多糖对免疫抑制模型小鼠CD4+ CD25+调节性T细胞(Treg细胞)和CD4+ Th17细胞亚群功能的影响.通过小鼠腹腔注射环磷酰胺建立免疫抑制动物模型,应用黄芪多糖对免疫抑制小鼠进行治疗,采用流式细胞仪分析脾脏中CD4+T细胞、CD4+ CD25+Treg细胞的比例,ELISA检测血清IL-17的水平.结果表明:与模型对照组比较,黄芪多糖高、中剂量明显降低小鼠脾脏CD4+T细胞数的比例(P＜0.05),同时黄芪多糖上调小鼠脾脏CD4+ CD25+Treg细胞数的比例(P＜0.05),降低免疫抑制小鼠血清IL-17的水平,尤其1.0 g/(kg·d)剂量组的IL-17水平下降较为明显(P＜0.01).提示黄芪多糖具有提高免疫抑制小鼠CD4+CD25+ Treg细胞数的比例、降低CD4 +T细胞的数量及抑制IL-17分泌活性的作用.

To search for more effective tuberculosis (TB) subunit vaccines, antigens expressed in different growth stages of Mycobacterium tuberculosis (M. tuberculosis), such as RpfE (Rv2450c) produced in the stage of resuscitation, Mtb10.4 (Rv0288), Mtb8.4 (Rv1174c), ESAT6 (Rv3875), Ag85B (Rv1886c) mainly secreted by replicating bacilli, and HspX (Rv2031c) highly expressed in dormant bacilli, were selected to construct six fusion proteins: ESAT6-Ag85B-MPT64190-198-Mtb8.4 (EAMM), Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B, and ESAT6-RpfE. The six fusion proteins were separately emulsified in an adjuvant composed of N,N'-dimethyl-N, N'-dioctadecylammonium bromide (DDA), polyribocytidylic acid (poly I:C) and gelatin to construct subunit vaccines, and their protective effects against M. tuberculosis infection were evaluated in C57BL/6 mice. Furthermore, the boosting effects of EAMM and MH in the adjuvant of DDA plus trehalose 6,6'-dimycolate (TDM) on BCG-induced immunity were also evaluated. It was found that the six proteins were stably produced in E. coli and successfully purified by chromatography. Among them, EAMM presented the most effective protection against M. tuberculosis. Interestingly, the mice that received EAMM+MH had significantly lower bacterial counts in the lungs and spleens than the single protein vaccinated groups, and had the same effect as those that received BCG. In addition, EAMM and MH could improve BCG-primed protective efficacy against M. tuberculosis infection in mice. In conclusion, the combination of EAMM and MH containing antigens from both replicating and dormant stages of the bacilli could induce robust immunity against M. tuberculosis infection in mice and may serve as promising subunit vaccine candidate.

To better control tuberculosis (TB) epidemics in developing countries a real need exists to study the liquefaction and cavity formation that occur in pulmonary TB lesions. This report is the first to evaluate the effects of immunomodulators on these two processes in a rabbit skin model. The effects of recombinant human interferon-γ (rIFN-γ), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) were evaluated in TB lesions produced by intradermal injection of 5 × 10(6) viable BCG bacilli. Recombinant IL-2 and rIFN-γ accelerated the liquefaction and healing of the lesions, and reduced the bacterial load. In contrast, dexamethasone inhibited the liquefaction of the lesions, and increased the bacterial load. The effect of CTX was similar to dexamethasone but not as pronounced. Serum levels of IL-2 were higher during the liquefaction and healing phases in the rIL-2 and rIFN-γ groups. Therefore, immunomodulators affect both the development of TB lesions and the survival of the mycobacteria within them. This study suggests that the rabbit skin model can be a valuable method to select therapeutic agents that could inhibit liquefaction and cavity formation in pulmonary tuberculosis.

Subunit vaccines have the potential advantage to boost Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-primed immunity in adults. However, most candidates are antigens highly expressed in replicating bacilli but not in dormant or persisting bacilli, which exist during Mycobacterium tuberculosis infection. We constructed M. tuberculosis fusion protein Ag85B-Mpt64(190-198)-HspX (AMH) and Ag85B-Mpt64(190-198)-Mtb8.4 (AMM), which consist of Ag85B, the 190-198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4 (Rv1174c), respectively. AMH and/or AMM were mixed with adjuvants composed of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA-BCG PSN) to construct subunit vaccines. Mice were immunized thrice with Ag85B, AMH and AMM vaccines and the immunogenicity of the fusion protein vaccines was determined. Then, mice were primed with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH vaccines, respectively, followed by challenging with M. tuberculosis virulent strain H37Rv, and the immune responses and protective effects were measured. It was found that mice immunized with AMH vaccine generated high levels of antigen-specific cell-mediated responses. Compared with the group injected only with BCG, the mice boosted with AMM, AMH and AMM + AMH produced higher levels of Ag85B-specific IgG1 and IgG2a and IFN-gamma-secreting T cells upon Ag85B and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation. It is interesting that only mice boosted with AMM + AMH had significantly lower bacterial count in the lungs than those receiving BCG, whereas mice boosted with AMH or AMM did not. The results suggest that AMH consisting of HspX, the antigen highly expressed in dormant bacilli, could be combined with antigens from replicating bacilli to enhance BCG primed immunity so as to provide better protection against both growing and non-growing bacteria that occur during the infection process.

To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection including the persister bacteria, antigens of Mtb10.4 (Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) without affinity tag with potential advantage for clinical use. The human T-cell responses to MH were evaluated for its immunogenicity. Furthermore, MH was emulsified in an adjuvant composed of N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalose-6,6-dimycolate (TDM) to construct subunit vaccine, whose immunogenicity and potency to boost BCG primed immunity against M. tuberculosis infection were evaluated in mice. The results showed that the fusion protein MH without affinity tag was stably produced in Escherichia coli and was successfully purified by chromatography. MH was strongly recognized by human T cells from TB patients and persons latently infected with M. tuberculosis. In conclusion, MH in adjuvant DDA-TDM generated strong antigen-specific humoral and cell-mediated immunity, and had the capability to enhance BCG-primed immunity and the protective efficacy against M. tuberculosis in mice. These findings suggest that MH in DDA-TDM have the potential to be a good multistage tuberculosis vaccine candidate.