共表达HIV-1 gag-gp120与IL-6重组鸡痘病毒和核酸疫苗的联合免疫研究

目的探讨共表达HIV-1 gag-gpl20与IL-6重组鸡痘病毒和核酸疫苗联合免疫小鼠的免疫应答。方法以重组鸡痘病毒首免，以表达相同抗原的核酸疫苗质粒追加免疫，检测免疫小鼠脾特异性CTL杀伤活性和血清抗体水平。结果联合免疫组脾特异性CTL杀伤活性比重组鸡痘病毒单独免疫组、核酸疫苗单独免疫组、鸡痘病毒疫苗株对照组和空白质粒对照组高，血清抗体水平显著高于空白质粒对照组和鸡痘病毒疫苗株对照组，但与重组鸡痘病毒单独免疫组、核酸疫苗单独免疫组之间差异无显著性。结论重组鸡痘病毒和核酸疫苗的联合免疫可诱导小鼠产生更强的细胞免疫应答。     

IL-18和HIV-1 gag-gp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答

目的 :探讨IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫小鼠的免疫应答。方法 :构建含IL 18的真核表达质粒pVAXIL18,将他与表达HIV 1gag gp12 0嵌合基因的核酸疫苗质粒pVAXGE共同肌注免疫BALB/c小鼠 ,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体滴度。结果 :联合免疫组小鼠脾特异性CTL杀伤活性和血清抗体水平均显著高于单独免疫组 (P <0 .0 5 ) ,空白质粒对照组 (P <0 .0 1)和PBS对照组 (P <0 .0 1)。结论 :IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫可诱导小鼠产生特异性细胞和体液免疫 ,且IL 18发挥了免疫佐剂的作用。     

HIV DNA疫苗与重组腺病毒伴随病毒联合免疫效果的研究

目的 构建含HIV-1B亚型中国株gagV3基因的DNA疫苗及重组腺病毒伴随病毒(rAAV)疫苗，并研究DNA疫苗和rAAV联合免疫的免疫效果。方法 将HIV-1B亚型中国株gagV3基因克隆入真核表达载体pCI-neo上，构建了含HIV-1 gagV3基因的DNA疫苗pCI-gagV3。采用电击法将pCI-gagV3质粒转染p815细胞，用G418压力筛选，得到转入重组质粒的细胞系p815-gagV3，用免疫酶法检测细胞系中HIV-1基因的表达。用该DNA疫苗进行小鼠免疫实验，检测免疫效果；用该DNA疫苗初次免疫，含同样gagV3基因的重组腺病毒伴随病毒rAAV-gagV3加强免疫，采用免疫酶法检测免疫小鼠血清中HIV-1特异性的抗体水平，用乳酸脱氢酶法检测免疫小鼠的HIV-1特异性CTL水平。结果 pCI-gagV3可以在p815细胞中表达HIV-1的基因，免疫BALB／c小鼠后可以在小鼠体内诱发HIV-1特异性的细胞和体液免疫反应。HIV-1特异性抗体滴度为1：20；效靶比为50：1时，CTL平均杀伤率为41．7％。pCI-gagV3与rAAV-gagV3联合免疫并不能明显提高抗体水平，但可以提高CTL反应，效靶比为50：1时，CTL平均杀伤率为61.3％，高于单独用DNA疫苗或重组AAV疫苗免疫后产生的CTL活性。结论 DNA疫苗与重组腺病毒伴随病毒联合免疫可以提高免疫小鼠产生的HIV-1特异性CTL反应。     

HIV-2 gag rFPV与rDNA疫苗的联合免疫研究

目的 探讨HIV-2核心蛋白基因gag重组DNA疫苗与重组鸡痘病毒进行联合免疫引起小鼠的免疫应答，为研究HIV-2基因重组疫苗的免疫策略提供实验基础。方法 大量制备并纯化HIV-2 gag重组DNA疫苗和重组鸡痘病毒，以肌肉注射的方式免疫BALB／c小鼠，ELISA法检测小鼠血清HIV-2抗体，流式细胞仪测定CD4^＋、CD8^＋T淋巴细胞亚类数量，乳酸脱氢酶（LDH）释放法检测脾CTL对HIV-2靶细胞的杀伤活性。结果 重组DNA疫苗和重组鸡痘病毒单独免疫及二者联合免疫均刺激小鼠产生HIV-2特异性抗体，脾T细胞亚类数量增加，并产生针对HIV-2靶细胞的特异性CTL杀伤活性，但联合免疫组在各项指标上均高于单独免疫组。结论 以HIV-2gag重组DNA疫苗进行基础免疫、以HIV-2gag重组鸡痘病毒进行加强免疫能诱导小鼠产生更强的特异性细胞和体液免疫应答。     

表达HIV-1 gag-gp120嵌合基因的DNA疫苗在小鼠体内的免疫应答

目的 :检测表达HIV 1gag gp12 0嵌合基因的DNA疫苗在小鼠体内的免疫应答效果。方法 :将真核表达质粒pVAXGE肌肉注射BALB C小鼠 ,观察免疫小鼠脾T淋巴细胞亚群的数量、特异性CTL杀伤率及小鼠免疫后不同时间点血清中IgG抗体滴度。结果 :重组质粒pVAXGE免疫组小鼠脾淋巴细胞进行了增殖 ,脾特异性CTL杀伤率显著高于对照组 (P <0 0 1) ;小鼠免疫后于第 8周血清抗体达到最高。结论 :表达HIV 1gag gp12 0嵌合基因的DNA疫苗质粒可诱导BALB C小鼠发生免疫应答     

人IL-12表达质粒联合含信号肽Mtb8.4基因疫苗对小鼠结核杆菌感染的免疫保护效果

目的研究结核分枝杆菌含信号肽Mtb8.4(MS)基因疫苗与人白细胞介素12(hIL-12)联合免疫小鼠后,诱导的细胞免疫应答及对小鼠结核杆菌感染的免疫保护效果。方法将40只C57BL/6N小鼠随机分为联合免疫组(MS基因疫苗 hIL-12组)、MS基因疫苗组、卡介苗(BCG)组、空载体组和PBS组。将基因疫苗、空载体和PBS,经肌内注射免疫各组小鼠,每隔3周免疫1次,共免疫3次;BCG组经尾部皮下注射1×106CFU BCG免疫1次。采用ELISA法检测小鼠脾细胞培养上清中细胞因子的水平;用乳酸脱氢酶(LDH)释放法检测免疫小鼠细胞毒性T细胞的杀伤活性。用结核杆菌强毒株H37Rv静脉攻击小鼠后,计数肺和脾组织中结核杆菌的菌落数。对小鼠的部分肺和脾组织作病理切片,经HE染色观察组织病变的程度,经ZN染色检查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液中IFN-γ和IL-2的水平,与BCG组相当,显著高于MS基因疫苗组,IL-4分泌减少,特异性CTL的杀伤活性增强,对小鼠结核杆菌感染有较好的免疫保护效果。表现为小鼠肺和脾组织中结核杆菌的菌落数显著减少,组织病变明显减轻,其效果与卡介苗(BCG)组相当,但优于MS基因疫苗组。结论以hIL-12表达质粒与MS基因疫苗联合免疫后,能显著增强MS基因疫苗对小鼠结核杆菌感染的免疫保护效果。     

共表达HIV-1 gp120与IFN-α重组鸡痘病毒诱导特异性CTL的初步研究

目的:探讨共表达HIV -1gp12 0与IFN- α重组鸡痘病毒诱导小鼠产生特异性的CTL杀伤活性。方法:将重组鸡痘病毒经肌肉注射免疫BALB c小鼠后,制备小鼠脾淋巴细胞悬液。以免疫小鼠的脾淋巴细胞为效应细胞,以表达HIV- 1结构蛋白的P815细胞为靶细胞,用乳酸脱氢酶释放法测定免疫小鼠脾特异性CTL杀伤活性。结果:重组病毒可有效地诱导特异性CTL的产生,且重组病毒免疫组小鼠脾特异性CTL对靶细胞的杀伤活性显著高于FPV对照组(P <0 .0 1)和PBS对照组(P <0. 0 1)。结论:共表达HIV- 1gp12 0和IFN -α的重组鸡痘病毒可激发强烈的细胞免疫,可作为我国HIV- 1疫苗候选株。     

IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响

目的：观察IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠（H-2d）特异性体液免疫和细胞免疫应答的影响.方法：用肌肉注射法将HBV核心区DNA疫苗、IL-12质粒和IL-18 质粒接种BALB/c小鼠;ELISA法检测小鼠血清抗-HBc（IgG）及IgG亚类（IgG1、IgG2a）;LDH释放法检测小鼠脾细胞HBcAg特异性CTL活性.结果：免疫6周后,HBcAg DNA疫苗联合IL-12质粒、IL-18质粒和IL-12＋IL-18质粒组小鼠的血清抗HBc终点滴度均明显高于单纯注射HBcAg DNA疫苗组小鼠（P＜0.05）,抗HBc IgG亚类以IgG2a占优.DNA疫苗免疫的各组小鼠,HBcAg特异性细胞毒性T淋巴细胞杀伤率均高于对照组（P组）,其中C＋IL-18组和C＋IL-12＋IL-18组中CTL值明显高于C组,尤以C＋IL-12＋IL-18组中的CTL杀伤率最高.结论：IL-12和IL-18基因与HBcAg DNA疫苗联合免疫,不仅能增强HBcAg特异性体液免疫应答,而且能增强HBcAg特异性CTL的杀伤活性.     

IL-2和GM-CSF表达质粒对pcDNA3/MDC-VP1 DNA疫苗作用的比较

目的:比较IL-2与GM-CSF两种细胞因子对pcDNA3/MDC-VP1 DNA疫苗免疫的免疫增强效果.方法:4～6周龄雄性BALB/c小鼠随机分成pcDNA3组、pcDNA3/MDC-VP1组、pcDNA3/MDC-VP1与pcDNA3/hIL-2混合注射组、pcDNA3/MDC-VP1与pcDNA3/mGM-CSF混合注射组,每组10只.每3周接种1次,共3次.每次接种后的第20天眼眶采血,用微量中和试验(固定病毒-稀释血清法)检测血清中和抗体效价.第3次免疫后3周,每组取3只小鼠脾脏制备淋巴细胞悬液,检测淋巴细胞增殖活性与特异性细胞毒性T淋巴细胞(CTL)杀伤活性.结果:pcDNA3/MDC-VP1+pcDNA3/mGM-CSF组的血清中和抗体滴度明显提高,小鼠脾脏淋巴细胞增殖活性和特异性CTL杀伤活性均有增强.结论:GM-CSF作为本疫苗分子佐剂能诱导小鼠产生较强的体液和细胞免疫,免疫效果优于IL-2.     

结核菌Mtb8.4基因疫苗对结核杆菌感染小鼠的免疫保护效果研究

为研究Mtb8.4基因疫苗的免疫原性及对小鼠结核杆菌感染的免疫保护效果,将雌性C57BL/6N小鼠32只,随机分为4组,即Mtb8.4基因疫苗组、BCG组、pcDNA3.1(+)组和PBS组。小鼠脾细胞培养上清检测细胞因子水平;并按效靶比例分别为100∶1、50∶1、10∶1进行CTL杀伤活性检测。用结核杆菌H37Rv强毒株静脉攻击小鼠,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果表明,Mtb8.4基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果,能够诱导较强的抗原特异性Th1型细胞免疫应答,细胞因子IFN-γ和IL-2分泌增加,IL-4分泌减少,特异性CTL活性增加;使小鼠肺和脾组织中的结核杆菌菌落数较空载体组显著减少,组织病变明显减轻。     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

Comparison of Immune Responses Induced in Mice by Vaccination with DNA Vaccine Constructs Expressing Mycobacterial Antigen 85A and Interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.     

Comparison of immune responses induced in mice by vaccination with DNA vaccine constructs expressing mycobacterial antigen 85A and interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.     

Novel constructs of tuberculosis gene vaccine and its immune effect on mice

A novel tuberculosis(TB)gene vaccine containing mouse granulocyte macrophage-colony stimulating factor(mGM-CSF)and a TB antigen(Ag85A)was developed in this study.The genes encoding Ag85A and mGM-CSFwere amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF.The genes were thencloned into two different polylinker sites of plasmid pIRES,forming a novel TB gene vaccine construct pI85AGM.Following transfection of pI85AGM plasmid into 7721 cell line by Lipofectamine~(TM),the expression of Ag85A andGM-CSF proteins was identified by Western blotting or RT-PCR.Then Balb/c mice were inoculated with therecombinant pI85AGM,pI85A,pIGM or plasmid alone,respectively.The activities of CTL,NK cells and theAg85A-stimulated proliferation of spleen cells were measured by MTT method.The serum antibody against Ag85Awas detected by ELISA.The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cellline and the activity of CTLs and the proliferation of spleen cells were significantly increased in thepI85AGM-immunized mice,indicating that the pI85AGM-immunized mice could generate specific immuneresponses to Ag85A.This study might provide possibility for developing novel anti-TB gene vaccine.Cellular &Molecular Immunology.2005;2(1):57-62.Cellular & Molecular Immunology.2005;2(1):57-62.     

Tuberculosis DNA vaccine encoding Ag85A is immunogenic and protective when administered by intramuscular needle injection but not by epidermal gene gun bombardment

Immunogenicity and protective efficacy of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis were compared in BALB/c and C57BL (B6 and B10) mice immunized by intramuscular (i.m.) needle injection or epidermal gene gun (gg) bombardment. In BALB/c mice, gg immunization could induce elevated antibody and cytotoxic T lymphocyte responses with plasmid doses 50-fold lower than those required for i.m. immunization. Interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion, however, was much lower in gg-immunized than in i.m.-immunized BALB/c mice. On the other hand, C57BL mice reacted only very weakly to gg immunization, whereas elevated Ag85A-specific antibody, IL-2, and IFN-gamma responses (significantly higher than in BALB/c mice) were detected following vaccination by the i.m. route. Antibody isotypes were indicative of Th2 activation following gg injection of BALB/c and of Th1 activation following i.m. injection of C57BL mice. Finally, C57BL but not BALB/c mice were protected by i.m. Ag85A DNA immunization against intravenous M. tuberculosis challenge, as measured by reduced numbers of CFU in spleen and lungs, compared to animals vaccinated with control DNA. Gene gun immunization was not effective in either BALB/c or C57BL mice. These results indicate that i.m. DNA vaccination is the method of choice for the induction of protective Th1 type immune responses with the Ag85A tuberculosis DNA vaccine.     

Enhanced cellular immune response elicited by a DNA vaccine fused with Ub against Mycobacterium tuberculosis

This study evaluated the immune response elicited by a Ub-fused Ag85A DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding Ag85A protein, Ub-fused Ag85A DNA vaccine (UbGR-Ag85A) and negative DNA vaccines, respectively. Ag85A DNA vaccine immunization induced a Th(l)-polarized immune response. The production of Th(l)-type cytokine (IFN-γ) and proliferative T cell responses was enhanced significantly in mice immunized with UbGR-Ag85A fusion DNA vaccine, compared with non-fusion DNA vaccine. Moreover, this fusion DNA vaccine also resulted in an increased relative ratio of IgG(2a) to IgG(l) and the cytotoxicity of T cells. IFN-γ intracellular staining of splenocytes indicated that UbGR-Ag85A fusion DNA vaccine activated CD4(+) and CD8(+) T cells, particularly CD8(+) T cells. Thus, this study demonstrated that the UbGR-Ag85A fusion DNA vaccine inoculation could improve antigen-specific cellular immune responses, which is helpful for protection against TB infection.     

Evaluation of a needle-free delivery platform for prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus 2 glycoprotein D

A previous report described a prime-boost immunization strategy using plasmid and modified vaccinia virus Ankara (MVA) vectors expressing herpes simplex virus 2 glycoprotein D (gD). Enhanced humoral and cellular immune responses were elicited by the prime-boost combination compared to plasmid DNA immunization alone. Surprisingly, a more diverse antibody isotype response, and a greater antibody and cellular immune response, was obtained if the gD MVA vector was used as the priming immunization rather than the gD plasmid vector. The present report evaluates the use of a needle-free delivery platform (Biojector) for delivery of plasmid and MVA gD-expressing vectors in a prime-boost immunization strategy. Needle-free delivery of both plasmid and MVA gD expression vectors was efficient, reproducible, and elicited a strong immune response in immunized mice. Biojector delivery of plasmid DNA was able to evoke a broader isotype response and cellular immune response than that obtained by gene gun delivered plasmid DNA. Further, DNA priming by Biojector delivery as part of a prime-boost procedure with MVA-gD2 resulted in a diverse antibody isotype distribution and enhanced cellular immune responses, similar to the responses obtained when MVA-gD2 was used as the priming immunization. Thus, needle-free delivery of plasmid DNA may provide additional flexibility and options for effective prime-boost vaccination.     

结核分枝杆菌Ag85B-MPT64融合基因疫苗的免疫效果观察

目的:研究并比较结核分枝杆菌(H37Rv)Ag85B、MPT64DNA.以及两者的融合基因(AM)的免疫原性。方法:雌性C57BL/6小鼠25只,随机分为5组,即A组(PBS)、B组(peDNA3.1)、C组(pcDNA/Ag85B)、D组(pcDNA/MPT64)和E组(pcDNA/AM)。分别于胫前肌注射7.5 g/L利多卡因和质粒混合物(1:4,100μL,含质粒70μg/次),间隔2 wk免疫 1次,共3次。末次免疫后4 wk取血分离血清测定总IgG,同时分离脾淋巴细胞,用PPD刺激后分别做脾淋巴细胞增殖实验(MTT比色法)和测定脾淋巴细胞培养上清中IFN-γ的水平。结果:Ag85B、MPT64和AM质粒DNA,均能诱导小鼠产生较高水平的PPD特异性IgG。免疫小鼠脾淋巴细胞体外经PPD刺激后,能产生特异性淋巴细胞增殖和分泌IFN-γ。peDNA/AM组IFN-γ的分泌水平明显高于pcDNA/Ag85B和pcDNA/MPT64免疫组(P<0.05)。结论:结核分枝杆菌Ag85B-MPT64融合基因疫苗,能在小鼠体内诱导特异性细胞和体液免疫。     

Pneumococcal IgA1 Protease Activity Interferes with Opsonophagocytosis of Streptococcus Pneumoniae Mediated by Serotype-Specific Human Monoclonal IgA1 Antibodies

Immunity against tuberculosis (TB), caused by Mycobacterium tuberculosis , depends largely on activation and maintenance of strong cell-mediated immune responses involving both CD4⁺ and CD8⁺ T cells and the ability to respond with Th1-type cytokines, particularly IFN-γ. Recent studies suggested that BCG, the only licensed vaccine against M. tuberculosis , may fail to induce T-cell responses in the lung mucosa and may therefore not protect against pulmonary TB. A decrease in TB mortality may be achieved by enhancing immunity in the lung. The present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein I (OprI) from Pseudomonas aeruginosa . OprI has shown to be a Toll-like receptor 2/4 agonist that, when given subcutaneously, induces Type-1 immune responses against heterologous antigens. Here, a fusion of OprI to Ag85A of Mtb (OprI-Ag85A) was used as a subunit vaccine in homologous prime-boost immunizations. In addition, OprI-Ag85A was combined with an Ag85A-encoding DNA vaccine (Ag85A DNA) or with BCG in heterologous prime-boost vaccinations. Intranasal and parenteral delivery with OprI-Ag85A elicited comparable T-cell responses in the spleen; in addition, i.n. delivery elicited specific T-cell responses in the lung lymph nodes (LLNs). Intramuscular delivery of Ag85A DNA induced significant systemic Th1 immune responses. Intranasal boosting with OprI-Ag85A enhanced this response and in addition induced an antigen-specific IFN-γ response in LLN. OprI may therefore be an efficient adjuvant for mucosal boosting. We continue to evaluate the protection induced by OprI-based prime-boost vaccinations against pulmonary TB. Results on the immunogenicity and protection against intravenous Mtb H37Rv infection will be presented.