Aβ单链抗体基因腺相关病毒的包装和纯化

目的 包装和纯化携带Aβ单链抗体基因的腺相关病毒，为阿尔茨海默病的基因治疗创造条件。 方法 用Lipofectamine 2000将pSNAV2.0-Abeta-scFv质粒转染BHK-21细胞并用G418筛选稳定细胞系。培养稳定细胞系用辅助病毒HSV1-rc/ UL2感染包装携带Aβ单链抗体基因的腺相关病毒。用氯仿-PEG/NaCl-氯仿法和离子交换层析法纯化重组腺相关病毒，SDS-PAGE和PCR方法检测纯化的病毒。用地高辛标记探针测定重组腺相关病毒的物理滴度。利用水迷宫测试重组病毒对转基因阿尔茨海默病小鼠模型的治疗效果。结果 带Aβ单链抗体基因的腺相关病毒的纯度为98%，物理滴度为1×1012vg/ml。经过治疗的小鼠模型潜伏期明显缩短。 结论 利用HSV1系统成功包装和纯化了携带Aβ单链抗体基因的腺相关病毒，动物实验表明对阿尔茨海默病具有治疗作用。     

CTLA4-Ig重组腺相关病毒载体的构建及转染表达效果

重组腺相关病毒 (recombinantadeno associatedvirus,rAAV)载体是目前基因治疗领域中倍受瞩目的载体之一。本研究通过构建CTLA4 Ig基因重组腺相关病毒载体 (rAAV CTLA4 Ig) ,研究其对大鼠转染的表达效果及规律。大肠杆菌Sure 2和 2 93细胞均由武汉同济医院心血管内科研究室提供 ;质粒pUF1 CTLA4 Ig、pXX2和pXX6由美国匹兹堡大学分子生物实验室XiaoX教授提供 ;近交系Lewis大鼠 ,2 0 0～ 2 5 0g,雄性 ,购自中国医学科学院实验动物研究所。仓鼠抗小鼠的CTLA4 Ig购自Pharm ingen公司 ,辣根过氧化物酶标记的羊抗人IgG购自Vec…     

反义白介素4重组腺相关病毒载体气道给药对哮喘大鼠的影响

目的 构建携带反义IL-4基因的重组腺相关病毒（rAAV）真核表达载体，并以此干预大鼠哮喘模型，探讨其作为哮喘基因治疗方案的可行性。方法 磷酸钙沉淀法将质粒pasIL4、pXX2及pXX680共转染293T细胞合成携带反义IL-4基因的重组腺相关病毒（rAAV-asILA）。Southem blot法检测合成病毒的滴度。rAAV-asILA在动物实验开始的第1天和第14天2次通过气道给药干预哮喘大鼠（T组），实验中同时设立大鼠正常组（N组）、哮喘组（A组）以及rAAV-GFP干预组（C组）。末次激发后处理大鼠，计数肺泡灌洗液（BALF）中有核细胞以及嗜酸性细胞总数，ELISA检测BALF中IL-4和IgE蛋白量，RT-PCR检测肺组织IL-4 mRNA变化，取肺组织作病理学检查。结果 分析BALF中有核细胞细胞总数和Eos总数，结果表明：T组该2项指标较A组和C组有降低趋势，但这3组间差异无统计学意义（P〉0．05）。BALF中IL-4、IgE的ELISA检测结果以及肺组织IL-4 mRNA RT-PCR半定量检测结果表明：T组的上述指标水平较A组和C组均明显降低（P〈0．01）。T组大鼠肺组织病理学改变较A组和c组轻。结论 携带反义IL-4基因的rAAV载体，对于哮喘的干预具有一定的作用。rAAV-asIL4可能具有一定临床应用前景。     

重组人DcR3腺相关病毒的构建和表达

目的克隆人DcR3基因于腺相关病毒(adeno-associatedvirus,AAV)载体pAAV-IRES-hrGFP中,以获得高感染滴度及纯度的重组人DcR3腺相关病毒,为将其用于感染移植肝,研究其对大鼠移植肝的保护作用打下基础。方法用基因重组的方法构建含全长人DcR3基因的重组腺相关病毒骨架质粒,利用脂质体法将腺相关病毒载体系统共转染入病毒包装细胞AAV-293细胞中,合成重组DcR3腺相关病毒,经PEG/氯仿纯化,用SDS-PAGE鉴定其纯度,扫描电镜观察病毒颗粒形态,倍比稀释法测定重组病毒的感染滴度。并用重组病毒感染COS-7细胞鉴定DcR3的表达。结果正确构建组装重组人DcR3腺相关病毒,纯化后病毒感染滴度为1.1×1010U/mL。在重组DcR3腺相关病毒感染的COS-7细胞上清液中检测到了DcR3的表达。结论成功构建了重组人DcR3腺相关病毒,为下一步研究DcR3对大鼠移植肝的保护作用打下基础。     

大鼠CTLA4-FasL融合基因重组腺相关病毒载体的构建、制备及表达

目的检测大鼠CTLA4-FasL重组腺相关病毒载体感染大鼠关节炎性成纤维样滑膜细胞后目的基因的表达。方法构建大鼠CTLA4-FasL融合基因重组腺相关病毒载体,制备重组病毒,体外感染从佐剂诱导关节炎的大鼠关节中分离的炎性成纤维样滑膜细胞,利用Flag标签纯化目的蛋白,ELISA和Western blot等方法检测目的蛋白的表达。结果获得含有大鼠CTLA4-FasL融合基因的重组腺相关病毒,感染炎性成纤维样滑膜细胞后能够分泌性表达目的蛋白。结论构建并制备的CTLA4-FasL重组腺相关病毒能够有效感染炎性成纤维样滑膜细胞并促使期表达CTLA4-FasL融合蛋白,为今后关节炎治疗的体内实验研究奠定了基础。     

肌肉注射CTLA4-FasL重组腺相关病毒抑制大鼠关节炎的实验研究

目的观察CTLA4-FasL融合基因能否有效抑制大鼠关节炎。方法在动物造模免疫第2天,以肌肉注射方式导入CTLA4-FasL和EGFP(对照)重组腺相关病毒(rAAV),持续观察和测量2组动物的临床指标包括关节指数、踝关节厚度和体质量等,并对踝关节的组织学表现如滑膜增生和炎性细胞浸润等进行了检测。结果应用大鼠佐剂诱导关节炎动物模型,明确了重组AAV病毒可介导目的基因CTLA4-FasL于体内表达;与rAAV.EGFP对照组相比,rAAV.CTLA4-FasL处理组在临床学和组织学表现上均有效抑制了大鼠关节炎。结论 CTLA4-FasL融合分子很可能是具有潜在研究价值的类风湿性关节炎治疗分子。     

抗汉滩病毒双链抗体重组杆状病毒在昆虫细胞中的表达

目的应用Bac—to-Bac杆状病毒表达系统构建具有抗汉滩病毒(HTNV)G2糖蛋白(GP)鼠源性mAb 3G1与抗HTNV核蛋白(NP)鼠源性mAb 1A8嵌合基因的重组杆状病毒,在昆虫细胞中表达抗HTNV双链抗体并对表达产物进行初步鉴定。方法构建含有嵌合基因的杆状病毒表达载体ScFv-pFBD-ScFv‘,转化DH10Bac致敏菌,利用其含有的细菌Tn7转座系统将嵌合基因重组至穿梭质粒Bacmid上,快速筛选出含有抗HTNV双链抗体嵌合基因的重组杆状病毒,在昆虫细胞中进行表达,并利用间接免疫荧光试验检测表达产物。结果构建了含抗HTNV双链抗体嵌合基因之重组杆状病毒,并在昆虫细胞中表达出特异性抗体,该抗体能被兔抗人Q抗体所识别并可与HTNV NP和GP特异性结合。结论成功地在昆虫细胞中表达出抗HTNV双链抗体,且该抗体具有与HTNV NP抗原和GP抗原结合的活性。     

抗阿尔茨海默病重组腺相关病毒的制备与活性研究北大核心CSCD

目的:制备可表达抗阿尔茨海默病单链抗体AT8-scFv的重组腺相关病毒AAV8-AT8-scFv,并探究其体内外活性。方法:利用293T细胞对重组病毒进行包装与纯化;SDS-PAGE、RT-qPCR对重组病毒的理化性质进行表征;细胞感染、Western blot法检测重组病毒的体外感染活性;以BALB/c小鼠为动物模型进行免疫,ELISA法、活体成像检测重组病毒的体内表达情况。结果:成功制备重组腺相关病毒AAV8-AT8-scFv,该病毒具有较好的感染能力,并可在小鼠体内长期稳定表达。结论:成功获得具有良好生物学活性的重组腺相关病毒,为阿尔茨海默病的免疫治疗提供新思路。     

aFGF重组腺相关病毒转染内皮祖细胞

目的研究含分泌型人酸性成纤维细胞生长因子(sp-haFGF)基因的重组腺相关病毒(rAAV)感染内皮祖细胞(EPCs)的可行性。方法用PCR法将FGF-4的信号序列与原始的aFGF基因连接,形成sp-haFGF基因。将此目的基因克隆到AAV载体质粒pAAV-IRES-hrGFP中,并与包装质粒(pAAV-RC)和辅助质粒(pHelper)共转染HEK293细胞,获得编码sp-haFGF的重组腺相关病毒。用浓缩的病毒感染体外培养的EPC,荧光显微镜下观察细胞表达绿色荧光蛋白情况,并用RT-PCR和W estern b lot方法鉴定sp-haFGF在EPC中的表达。结果获得含有sp-haFGF的重组腺相关病毒,将其感染EPC后,约20%~30%的细胞出现绿色荧光。用RT-PCR法从被感染细胞中扩增出一条长约560 bp的基因条带,通过W estern b lot检测到被感染细胞中haFGF蛋白的表达。结论编码sp-haFGF的重组腺相关病毒能够有效地感染EPC,并在EPC中表达haFGF,为进一步研究转基因的内皮祖细胞移植促血管新生奠定基础。     

重组腺相关病毒介导的PD-L1基因在大鼠移植肝中的表达

目的探讨供肝转染PD-L1-AAV2-RFP后基因的表达情况。方法近交系BN大鼠随机分3组:同基因肝移植组(ISO组)、腺相关病毒空载体组(AAV组)、基因转染组(PD-L1组),每组6对。ISO组供肝冷保存期经门静脉注射生理盐水2mL,保存2h后采用改良"二袖套法"行原位肝移植;AAV、PD-L1组所用灌注液分别为AAV2-RFP空病毒颗粒和PD-L1-AAV2-RFP(1×1011v.g./只)。术后7、14d各取3只大鼠采血测定肝功能,取肝组织即刻行冰冻切片在荧光显微镜下观察RFP的表达,免疫组化染色法检测PD-L1蛋白的表达。结果3组术后7d谷丙转氨酶(ALT)、谷草转氨酶(AST)均明显增高,14d后基本恢复正常,各组间相比均无显著性差异(P>0.05);术后7d在荧光显微镜下AAV、PD-L1组移植肝可见少量而微弱的红色荧光,14d可见高亮度的红色荧光,而各组肝外组织及ISO组各时段均未见红色荧光;免疫组化染色结果示未转染组PD-L1蛋白均微弱表达,而PD-L1转染7d后随时间延长靶基因表达逐渐增强(P<0.05)。结论重组腺相关病毒介导,体外冷保存期经门静脉途径供肝转染PD-L1,可以使PD-L...     

Subcutaneous Administration of a Recombinant Vaccinia Virus Vaccine Expressing Multiple Envelopes of HIV-1

A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors. Recombinant vaccinia virus is a highly effective vaccine vector, with demonstrated capacity to protect animals from various viral pathogens, including rabies. Unlike many other candidate vaccine vectors, vast human experience exists with the parenteral smallpox vaccine. However, consideration of recombinant vaccinia virus as a modern vaccine is complicated by the relatively high prevalence of immunocompromised persons compared to such prevalence 4 or more decades ago (when smallpox vaccination was still routine). Administering vaccine by the subcutaneous (SQ) route, rather than the traditional scarification route, could address these concerns. SQ administration could prevent transmission of vaccinia virus to potentially vulnerable persons; it could also avoid the most common adverse events, which are cutaneous in nature. However, previous studies suggest that elicitation of immune response against passenger gene products following SQ administration requires development of a superficial pox lesion, defeating the intention of SQ administration. This is the first report to demonstrate that SQ administration of recombinant vaccinia virus does elicit immune response to the passenger protein in the absence of a cutaneous pox lesion. Results further show that a multi-envelope HIV vaccine can elicit antibody responses toward heterologous HIV-1 not represented by primary sequence in the vaccine. These findings have global implications because they support the consideration of recombinant vaccinia virus as a valuable HIV vaccine vector system.     

Characterization of Host Responses against a Recombinant Fowlpox Virus-Vectored Vaccine Expressing the Hemagglutinin Antigen of an Avian Influenza Virus

There currently are commercial fowlpox virus (FPV)-vectored vaccines for use in chickens, including TROVAC-AIV H5, which expresses the hemagglutinin (HA) antigen of an avian influenza virus and can confer immunity against avian influenza in chickens. Despite the use of recombinant FPV (rFPV) for vaccine delivery, very little is known about the immune responses generated by these viruses in chickens. The present study was designed to investigate host responses to rFPV in vivo and in vitro. In cultured cells infected with TROVAC-AIV H5, there was an early increase in the expression of type I interferons (IFN), Toll-like receptors 3 and 7 (TLR3 and TLR7, respectively), TRIF, and MyD88, which was followed by a decrease in the expression of these genes at later time points. There also was an increase in the expression of interleukin-1β (IL-1β), IL-8, and beta-defensin genes at early time points postinfection. In chickens immunized with TROVAC-AIV H5, there was higher expression of IFN-γ and IL-10 at day 5 postvaccination in spleen of vaccinated birds than in that of control birds. We further investigated the ability of the vaccine to induce immune responses against the HA antigen and discovered that there was a cell-mediated response elicited in vaccinated chickens against this antigen. The findings of this study demonstrate that FPV-vectored vaccines can elicit a repertoire of responses marked by the early expression of TLRs, type I interferons, and proinflammatory cytokines, as well as cytokines associated with adaptive immune responses. This study provides a platform for designing future generations of rFPV-vectored vaccines.Fowlpox virus (FPV) belongs to the genus Avipoxvirus and causes a disease of economic importance in poultry. FPV infection occurs through insect bites, skin abrasions, or the respiratory system, and the disease caused by the virus is presented in two forms: cutaneous and/or diphtheric (5, 48). Attenuated FPVs have been employed as vaccines for chickens or as candidate vectors for the delivery of recombinant proteins in both mammalian and avian species (38, 53). FPV-based vectors have been demonstrated to efficiently elicit both antibody- and T-cell-mediated immune responses against the antigen that they express (23, 34, 43). In mammals, applications of FPV-vectored vaccines include infectious diseases and cancer. Immunization with a FPV vaccine expressing the glycoprotein antigen of rabies virus resulted in conferring protection in mice, dogs, and cats (53). More recently, the safety and efficacy of FPV vaccines against malaria and human immunodeficiency virus (HIV) have been assessed in experimental animal models as well as in human trials (9). In mice, an FPV-vectored vaccine expressing an epitope of the V3 loop of several HIV strains was shown to elicit T helper 1 (Th1) and CD8⁺ T-cell responses to the cognate HIV antigen (57). The delivery of gag proteins of HIV or simian immunodeficiency virus (SIV) by FPV in rabbits resulted in the induction of antibody- and cell-mediated responses to these proteins (44). Moreover, the administration of FPV expressing the E2 protein of bovine viral diarrhea virus (BVDV) to mice led to the induction of gamma interferon (IFN-γ) in spleen mononuclear cells of vaccinated mice following the incubation of their cells with BVDV (34). In the case of malaria, the use of FPV-vectored vaccines in human trials has shown promising results. For instance, Webster and colleagues (59) reported that a prime-boost vaccination regimen, which included FPV expressing the preerythrocytic-stage malaria antigen thrombospondin-related adhesion protein (TRAP) for priming and a vaccinia Ankara virus expressing TRAP for boosting, could confer protection in humans against challenge with Plasmodium falciparum. FPV also has been used in clinical trials of several anticancer vaccines. These FPV vectors express tumor-associated antigens, such as carcinoembryonic antigen, prostate-specific antigen, and NY-ESO-1, which is a malignancy/testis antigen (9, 22). In the case of NY-ESO-1, the delivery of this antigen by FPV resulted in the generation of antibody and T-cell responses against various epitopes of this antigen in the vaccinated patients. Moreover, vaccination appeared to have favorably affected the course of disease in the vaccinated patients (22).In domestic animal species, FPV also has been used as a delivery mechanism for various vaccines, and these vaccines have been shown to induce T-cell responses. Shen and colleagues (47) demonstrated that the coexpression of GP5/GP3 proteins of porcine reproductive and respiratory syndrome virus by recombinant FPV (rFPV) led to enhanced T-cell responses, marked by increased IFN-γ concentrations in sera of immunized pigs. However, there also are some reports that, at least in mammals, FPV may not be able to elicit T-cell responses to same extent as other viruses (12). This may be associated with the suboptimal induction of type I interferons by FPV, leading to the perturbed development and expansion of effector CD8⁺ T cells (12).Several experimental and commercial vaccines for chickens have employed FPV for the delivery of viral antigens. These vaccines are against Marek''s disease virus, infectious bursal disease, Newcastle disease virus (NDV), and avian influenza virus (AIV) (18, 36, 50, 55), of which FPV-vectored vaccines against NDV and AIV are commercially available. Several rFPV vaccines containing the hemagglutinin (HA) gene of AIV have been studied in the past (8, 56). Recombinant FPV expressing HA from a highly pathogenic avian influenza (HPAI) strain, A/turkey/Ireland/1378/83 (H5N8), induced immune responses to and protection against AIV in chickens (52, 60). In addition, a similar construct (TROVAC-AIV H5 vaccine) induced antibodies to HA in cats (26). Swayne et al. (50) showed that the same vaccine prevented the morbidity and mortality of chickens, which were challenged with any of the eight different strains of HPAI virus. However, there also are some caveats associated with the use of FPV-vectored vaccines. For example, the presence of maternal antibodies against FPV could interfere with the vaccination of young birds. Furthermore, similarly to many other viral vectors, secondary immunization with the same vector may not be effective due to the elicitation of a memory immune response following primary immunization. Nevertheless, the efficiency of FPV as a vector for the delivery of chicken vaccines has been underscored by several studies. However, our understanding of avian host immune responses to FPV is very limited. There is only one report of induction cell-mediated responses by FPV in immunized chickens (49). Therefore, the present study was designed to elucidate immune responses to a recombinant FPV in chickens. We reasoned that gaining a better understanding of these responses should lead to the development of better FPV vectors that have a superior capacity of inducing appropriate responses, both quantitatively and qualitatively, against the insert that they deliver. In the present study, we have profiled host responses to FPV at the molecular and cellular levels.     

人乳头瘤病毒HPV6b重组减毒沙门菌粘膜免疫的效果测定

为观察人乳头瘤病毒 6b型 (HPV6b )的重组减毒沙门菌载体疫苗粘膜免疫小鼠的免疫效果及探讨研制治疗尖锐湿疣的治疗性粘膜疫苗的机制 ,我们用构建的HPV6b重组减毒沙门菌粘膜免疫BALB/c小鼠 ,检测了阴道冲洗液中特异的SIgA、血清中IgG、T淋巴细胞增殖以及载体疫苗在机体内的稳定性。结果表明 ,粘膜免疫后 ,实验组与对照组相比较 ,阴道冲洗液中抗HPV6bL1的SIgA差异显著 ,实验组血清中抗HPV6bL1IgG水平低 ,T淋巴细胞增殖明显 ,疫苗在小鼠体内持续存在4 0d以上。研究结果提示我们所构建的重组减毒沙门菌粘膜免疫小鼠后 ,阴道粘膜局部能分泌特异的抗人乳头瘤病毒HPV6bSIgA ,也能激发细胞免疫 ,且此疫苗在小鼠体内能稳定存在。     

Safety and Immunogenicity of a Single Oral Dose of Recombinant Double Mutant Heat-Labile Toxin Derived from Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of traveler''s diarrhea for which there is no licensed vaccine. This phase 1 trial determined the safety and immunogenicity of a recombinantly produced double mutant heat-labile enterotoxin (dmLT) of ETEC. It was administered as a single oral dose of dmLT in escalating doses of 5 μg, 25 μg, 50 μg, and 100 μg, followed by a 72-h inpatient observation, outpatient visits at 8, 14, and 28 days, and telephone calls at 2 and 6 months postvaccination. Safety was assessed by frequency of adverse events, and immune responses determined after immunization included dmLT-specific serum IgA and IgG, fecal IgA, antibody-secreting cells (ASC), and antibodies in lymphocyte supernatant (ALS) responses. All doses were well tolerated by the 36 healthy adults enrolled. Immune responses were limited in the 5- and 25-μg dose recipients. The 50-μg dose recipients trended toward stronger responses than the 100-μg dose recipients by serum IgA (67% versus 33%, P = 0.22), serum IgG (58% versus 33%, P = 0.41), and fecal IgA (58% versus 33%, P = 0.41). By day 14 postvaccination, there were significantly more positive responders (≥4-fold increase from baseline) among the 50- versus 100-μg dose recipients for serum IgA (P = 0.036) but not serum IgG (P = 0.21). In conclusion, a single oral dose of dmLT was well tolerated and immunogenic, with immune responses plateauing at the 50-μg dose. (This clinical trial is registered at www.clinicaltrials.gov, registration number {"type":"clinical-trial","attrs":{"text":"NCT01147445","term_id":"NCT01147445"}}NCT01147445.)     

The Helper Virus Envelope Glycoprotein Affects the Disease Specificity of a Recombinant Murine Leukemia Virus Carrying a v-myc Oncogene

Many retroviruses that carry oncogenes (acute transforming viruses) are generally replication-defective and therefore require co-infection with a replication competent helper retrovirus for infectivity. The helper virus provides the retroviral proteins necessary for particle production and infection. These include the envelope glycoproteins that specifically bind to cell surface receptors and mediate viral adsorption and entry. Thus, a particular helper virus may influence the nature of disease induced by an oncogene-containing retrovirus due to tissue tropism of the helper. In a previous study, a replication-defective recombinant Moloney murine leukemia virus containing the v-myc oncogene was generated (M-MuLV(myc); Brightman B.K., Pattengale P.K., and Fan H., J Virol 60: 68–81, 1986). When M-MuLV(myc) was inoculated into mice using the non-pathogenic amphotropic murine leukemia virus (Am-MuLV 4070) as a helper, T- and B-lymphoblastic lymphomas resulted with the following two surface phenotypes, namely, (1) Thy1.2+, B220- and (2) Thy1.2-, B220+. Thy l.2 surface antigen is characteristic of cells of the lymphoid lineage, whereas B220 surface antigen is characteristic of cells of the B-lymphoid lineage. In these experiments, to assess the influence of the helper virus on the disease specificity of M-MuLV(myc), two weakly pathogenic ecotropic helper MuLVs that interact with different cell surface receptors than Am-MuLV (Mo+PyFl0l and AKV MuLV) were used to pseudotype M-MuLV(myc). In both cases, when inoculated into mice, these pseudotypes induced only T-lymphoblastic lymphoma. These results indicate that for M-MuLV(myc) the types of the tumors induced are influenced by the helper virus utilized, and they suggest that different lymphoid cells may express different levels of retroviral receptors.     

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly+ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ΔureC hly⁺ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.     

The distribution of a specific HLA-B27-associated cell surface component on the tissues of patients with ankylosing spondylitis.

In this paper, we report the presence on Epstein-Barr virus-transformed lymphoblastoid cell lines on platelets and on fibroblasts of an HLA-B27-associated cell surface complex (antigenically related to some antigens of Klebsiella K43 and K21) which is identical to or cross-reactive with the determinant present on the peripheral blood lymphocytes (PBL) of B27-positive patients with ankylosing spondylitis (AS). By contrast, no Klebsiella K43 markers could be demonstrated on the spermatozoa of B27+ AS+ individuals even though these cells expressed the HLA-B27 alloantigen. No B27-associated K43 antigen was detected on the erythrocytes of patients or of normal controls. The B27-associated membrane marker is still detectable on lymphoblastoid cell lines after 20 generations and on fibroblasts after about 10 generations. This finding implies that the continued expression of Klebsiella-modified B27 structure is generally determined and does not require the repeated exposure of the cell surface to Klebsiella antigen. These data suggest that certain non-lymphoid as well as lymphoid cells may be involved in the complex sequence of events leading to the clinical manifestation of AS.     

A Chimeric Influenza Virus Expressing an Epitope of Outer Membrane Protein F of Pseudomonas aeruginosa Affords Protection against Challenge with P. aeruginosa in a Murine Model of Chronic Pulmonary Infection

The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F of Pseudomonas aeruginosa to serve as a protective vaccine against P. aeruginosa was tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains of P. aeruginosa but failed to react with a protein F-deficient strain of P. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity for P. aeruginosa than did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells of P. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 × 10³ CFU of P. aeruginosa in their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused by P. aeruginosa.