人乳头状瘤病毒16型不同基因疫苗联合免疫的实验研究

目的 探索联合免疫策略在预防和治疗人乳头状瘤病毒16型（HPV16）相关肿瘤中的作用。方法 在C57BL／6动物模型中，观察了表达HPV16基因的融合蛋白L2E7疫苗和重组痘苗病毒mE67疫苗的不同联合免疫方式在预防和治疗HPV16相关肿瘤中的作用。用酶联免疫斑点（ELISPOT）和细胞毒T淋巴细胞（CTL）反应评价它们在诱发机体产生细胞免疫应答中的作用。结果 我们发现以HPV16 L2E7融合蛋白＋佐剂（CpG）初免，用重组痘苗病毒rVVmE67加强免疫的联合免疫方式在C57BL／6小鼠实验中可以有效预防和治疗HPV16相关肿瘤的攻击。ELISPOT可以检测到高水平的E749-57肽特异性，分泌IFN-γ的效应T细胞。CTL检测同样反应出这种联合免疫方式所诱发的CTL细胞可以有效识别并杀伤含HPV16E6／E7的靶细胞。结论 以HPV16L2E7融合蛋白＋CpG初免，用重组痘苗病毒rVVmE67加强的联合免疫策略可以有效防治HPV16相关肿瘤，为进一步研究提供了科学基础。     

表达野生型和突变型HPV16-E7重组痘苗病毒诱发的抗肿瘤免疫反应

目的 选出适合于治疗性疫苗研制的HPV16E7突变基因。方法 对表达野生型和突变型E7蛋白的重组痘苗病毒所诱发的细胞免疫反应和抗肿瘤活性进行比较研究。结果 表达突变型ME7-1（24G26G）的重组痘苗病毒VmE7-1与表达野生型E7的VwE7相同,可诱发特异性抗体和CTL的产生,明显推迟成瘤时间并且保护部分小鼠抵抗肿瘤细胞的攻击;而表达突变型ME7-2（24G26G91G）的重组痘苗病毒VmE7-2免疫小鼠后难以有效的激发细胞免疫反应,在抗肿瘤移植实验中也不具明显的免疫保护作用。结论 E7突变基因ME7-1可作为候选基因用于HPV16治疗性疫苗的研制。     

表达HPV16 L1、L2和E7蛋白的非复制重组痘苗病毒诱发的抗肿瘤免疫反应

目的:评价表达HPV16 L1、L2E7的非复制重组痘苗病毒疫苗的抗肿瘤免疫反应.方法:采用肿瘤预防、肿瘤治疗和肿瘤切除等方法,观察疫苗NTVJL1/L2E7的抗肿瘤效果,用酶联免疫斑点(ELISPOT)和CTL检测该疫苗在小鼠体内诱发的细胞免疫应答.结果:在肿瘤预防和肿瘤治疗试验中,疫苗可以分别使60%和50%的小鼠免受HPV16阳性的治疗细胞攻击,肿瘤切除试验中,疫苗可以使70%的小鼠免于肿瘤复发,与对照组之间的差异具有显著性.ELISPOT和CTL均检测出强的特异性细胞免疫水平.结论:NTVJL1/L2E7能够在小鼠体内诱发出理想的抗肿瘤免疫反应,可以作为防治宫颈癌的候选疫苗.     

共表达HPV16型E6和E7突变基因重组痘苗病毒的构建及其抗肿瘤免疫效果的观察

目的：构建用于子宫颈癌治疗的HPV16型E6和E7重组痘苗病毒实验性疫苗株，并对其抗肿瘤免疫效果进行初步评价。方法：以痘苗病毒为载体、利用同源重组技术构建共表达HPV16 E6和E7基因的重组痘苗病毒。该病毒免疫C57BL／6小鼠后，检测其免疫原性和抗移植瘤生长情况。结果：PCR结果显示，重组病毒VmE6E7的TK基因内插入了分别由痘苗病毒早晚期启动子H6和7．5K表达的ME6和ME7－1基因。动物实验结果表明，rVmE6E7在C57BL／6小鼠体内可诱发E6和E7特异性抗体产生，被免疫小鼠能够抵抗HPV16 E6E7转化的同系肿瘤细胞的攻击。结论：获得1株用于宫颈癌治疗的HPV16型实验疫苗株，为进一步研制人用HPV16型疫苗株奠定了基础。     

表达HPV18E7E6的重组痘苗病毒构建及免疫效果的初步研究

目的 构建表达HPV18E7E6融合蛋白的重组痘苗病毒,并对E7E6蛋白的免疫原性进行研究.方法 将去除了转化活性的HPV18E6、E7基因融合,插入痘苗病毒重组质粒,通过同源重组构建表达HPV18E7E6的重组痘苗病毒,观察其免疫效果.结果 构建了表达E7E6融合蛋白的重组痘苗病毒,PCR鉴定及测序表明融合基因序列与设计相符,正确插入到痘苗病毒TK区域;Western-Blot检测表明该重组病毒能表达HPV18E7E6融合蛋白.免疫后的小鼠可产生E6、E7特异性抗体,但ELISPOT没检测到E7肽库刺激小鼠脾细胞产生分泌IFN-丫的阳性反应.结论 构建了一株表达HPV18E7E6融合蛋白的重组痘苗病毒,可以有效诱发小鼠产生针对E6、E7的体液免疫,但不能诱发产生相应的细胞免疫,为进一步研究不同动物模型中HPV18E6E7的细胞免疫特点提供了实验基础.     

人乳头瘤病毒18型E6、E7蛋白T细胞表位筛选

目的 筛选人乳头瘤病毒18型E6、E7蛋白在小鼠中的T细胞表位.方法 以重组痘苗病毒rVVJ18 E7、E6分别免疫C57BL/6和BALB/c小鼠,利用覆盖E6和E7蛋白全长序列的肽库或截短的多肽,用酶联免疫斑点方法 （ELISPOT）和细胞内因子染色检测其所诱发的细胞免疫反应.结果 重组痘苗病毒rVVJ18 E7、E6免疫的两种品系小鼠均可检测到E6肽库刺激产生的特异性细胞免疫反应,经筛选确定E667-75（KCIDFYSRI）为C57BL/6小鼠、E660-68（IPHAAGHKC）为BALB/c小鼠识别的CD8＋的T细胞表位.两种小鼠中均未检测到E7蛋白诱发的细胞免疫反应.结论 筛选到分别被BALB/c和C57BL/6小鼠识别的两条针对E6蛋白的不同T细胞表位,为今后评价HPV18疫苗中E6蛋白的细胞免疫效果提供了实验依据.     

小鼠对HPV16L1-E7重组腺病毒和重组质粒的免疫应答

对比研究人乳头瘤病毒16型L1-E7重组腺病毒(rAd5HPV16L1-E7)和重组质粒经不同途径免疫小鼠所产生的免疫效应.将rAd5HPV16L1-E7病毒在293细胞中扩增,并制备rAd5HPV16L1-E7重组质粒,分别以肌肉注射、滴鼻途径免疫小鼠,采用ELISA法测其血清IgG抗体水平;制备脾细胞悬液,加入到96孔无菌培养板中,分别加入rAd5HPV16L1-E7特异性蛋白和10%FCS RPMI1640,加入3H-TdR 1μCi/孔,做T-细胞增殖实验;取经过培养56 h的培养液进行IFN-γ的测定.不同疫苗和不同途径免疫的BALB/c小鼠血清IgG抗体水平均高于对照组小鼠(P＜0.05),IFN-γ和T-细胞增殖亦高于对照组(P＜0.05),且各实验组中病毒特异性蛋白刺激组均高于非刺激组(P＜0.05).提示rAd5HPV16L1-E7重组质粒和rAd5HPV16L1-E7重组活病毒一样既能刺激T-细胞产生细胞免疫应答,又能刺激B-细胞产生体液免疫应答,说明其具有很好的免疫原性,是一种很有发展前景的防治疫苗.     

HPV16E7E6重组腺病毒的构建及免疫效果评价

目的 构建用于宫颈癌治疗的HPV16E7E6重组腺病毒并评价其免疫效果.方法 根据哺乳细胞使用密码子的偏嗜性设计HPV16E7E6融合蛋白表达优势密码子基因序列并合成.将合成的E7E6基因插入腺病毒重组穿棱质粒pCD316后,与Ad5腺病毒骨架质粒共转染293细胞,重组病毒经单斑纯化并进行目的基因插入及表达的鉴定.扩增纯化重组病毒后免疫小鼠,评价其免疫活性.结果 PCR试验证明E7E6密码子优化基因成功插入Ad5病毒;Western blot检测结果表明重组腺病毒中E7E6优化基因能高效表达,该重组腺病毒免疫C57小鼠后,可诱发强特异性T细胞免疫应答,在小鼠体内能完全抑制5×104 TC-1移植瘤细胞的生长.结论 重组HPV16E7E6腺病毒可作为治疗HPV慢性感染或宫颈癌的候选疫苗.     

共表达HPV16L1、L2、E6、E7蛋白的重组非复制型痘苗病毒构建及鉴定

目的构建共表达人乳头瘤病毒16型(HPV16)L1、L2、E6、E7蛋白的非复制型重组痘苗病毒人用疫苗株。方法以痘苗病毒为载体、利用同源重组技术筛选共表达HPV16L1、L2、E6、E7蛋白的重组痘苗病毒并对其进行鉴定。结果该病毒在CEF细胞上连续传至第15代,经斑点杂交结果表明重组病毒基因组中有L1、L2、E6、E7基因插入;经WesternBlot检测,重组病毒能稳定表达HPV16L1、L2、E6、E7蛋白。结论非复制型重组痘苗病毒NTVJE6E7CKL1L2可作为预防和治疗HPV16相关肿瘤及其癌前病变候选疫苗。     

HPV16E7E6抗原表位嵌合体DNA抗肿瘤疫苗的研究

目的:探讨HPV16 E7、E6抗原表位与HSP70 N端重组DNA疫苗抗肿瘤活性.方法:以重叠PCR将HPV16 E7基因N端60个氨基酸的编码序列与E6基因的10个(48～57)氨基酸的编码序列及HSP70 N端序列进行融合.以pcDNA3.0为载体,构建真核表达载体pcD-E76HSP.重组质粒免疫C57BL/6小鼠后,通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度;观察该疫苗对C57BL/6小鼠TC-1肿瘤细胞移植瘤的治疗效果.结果:重组DNA疫苗免疫C57BL/6小鼠后,小鼠脾淋巴细胞体外增殖明显,并可诱导产生针对TC-1肿瘤细胞的特异性CTL反应;体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有抑制作用.结论:该疫苗能激发特异性细胞免疫反应,显著抑制HPV16转化的TC-1肿瘤细胞生长.     

Workshop L: Tolerance, Abstract L1 bis L14

L. 1 Retrovirally transduced T lymphocytes expressing viral interleukin-10 modulate allogeneic immune responsesL. 2 Functional involvement of vascular adhesion receptor ligand pairs in leukocyte traffic to the maternal/fetal interfaceL. 3 Zinc as an immunosuppressant maintaining the capacity of the host to react on antigenL. 4 Infectious tolerance: A joint venture of T cells and antigen presenting cellsL. 5 Antigen presenting cells of nickel-tolerant mice can transfer tolerance and are hypostimulatory in a mixed lymphocyte reactionL. 6 A role for liver endothelial cells in the induction of tolerance to oral antigens and apoptotic cellsL. 7 The anti-CD3 mediated T cell response in cord blood, maternal blood, and peripheral bloodL. 8 CD8 T cell tolerance induction in the absence of CD4 T cellsL. 9 Cytokine production of decidual large granular lymphocytes is influenced by HLA-G and HLA-E transfected K-562 cellsL. 10 Naturally occurring CTLA-4 positive, antigen-specific T regulatory-1 cells in a chronic helminth infection (onchocerciasis) are associated with immunosuppressionL. 11 Crucial role of IL-10 in the maintenance but not induction of peripheral CD4⁺ T cell toleranceL. 12 Modulation of the T cell alloresponse with an MHC class I immunodominant peptide and its analoguesL. 13 MHC haplotypes HLA-G and HLA-E modulate cytokine secretion of monocyte-derived dendritic cellsL. 14 Oral administration of nickel induces a high frequency of anergic T cells with persistent suppressor activity     

Natural antibiotic susceptibility of Listeria species: L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri strains

Objective   To investigate the natural susceptibility to 71 antimicrobial agents of 103 Listeria strains belonging to all known Listeria species ( L. monocytogenes ( N  = 21), L. innocua ( N  = 21), L. seeligeri ( N  = 21), L. ivanovii ( N  = 19), L. welshimeri ( N  = 11), and L. grayi ( N  = 10)).
Methods   MICs were determined using a microdilution procedure in H-Medium.
Results   All listeriae were naturally sensitive or intermediate to tetracyclines, aminoglycosides, penicillins (except oxacillin), loracarbef, cefazoline, cefaclor, cefotiam, cefoperazone, carbapenems, macrolides, lincosamides, glycopeptides, dalfopristin/quinupristin, chloramphenicol and rifampicin (probably except L. grayi ). Listeria spp. were naturally resistant or intermediate to most 'modern' cephalosporins (cefetamet, cefixime, ceftibuten, ceftazidime, cefdinir, cefpodoxime, cefotaxime, ceftriaxone, cefuroxime), aztreonam, pipemidic acid, dalfopristin quinupristin and sulfamethoxazole. Significant differences in natural susceptibility among the species were seen with the quinolones, trimethoprim, co-trimoxazole, rifampicin, fosfomycin and fusidic acid. It seems likely that L. grayi is naturally resistant to all antifolates; the species was least susceptible to rifampicin and most susceptible to quinolones, whereas L. ivanovii was naturally resistant to most quinolones. L. ivanovii was naturally sensitive to fosfomycin, whereas L. innocua and L. monocytogenes were naturally resistant. L. ivanovii was also the most susceptible species to fusidic acid.
Conclusions   The present study describes a database on the natural susceptibility of Listeria spp. to a wide range of antibiotics, which can be used to validate susceptibility testing results of these microorganisms.     

Development of human papillomavirus chimaeric L1/L2 candidate vaccines

Recombinant human papillomavirus (HPV) virus-like particle (VLP) vaccines based on the L1 capsid protein have been shown to be efficient prophylactic vaccines, albeit type-specific. As a first step to investigate the feasibility of extending protection against non-vaccine types, HPV-16 L1 chimaeras were generated. The region downstream of L1 amino acid (aa) 413 was replaced with selected cross-neutralising epitopes (aa 108-120; 56-81 and 17-36) derived from the HPV-16 L2 protein, generating proteins designated SAF, L2.56 and L2.17, respectively. The chimaera L1BPV containing BPV-1 L2 peptide aa 1-88 was similarly constructed. The chimaeras were evaluated for expression in insect cells; their ability to form particles was studied by electron microscopy, and their immunogenicity was evaluated in mice. SAF, L2.56 and L2.17 proteins were expressed to high concentrations in insect cells and elicited HPV-16 pseudovirus-neutralising anti-L1 antibodies. L2.56 and L2.17 also elicited anti-L2 antibodies. L1BPV was a poor vaccine candidate due to low levels of expression with concomitant lack of immunogenicity. All chimaeras assembled into tertiary structures. The results indicate that chimaeric L1 vaccines incorporating cross-neutralising L2 peptides could be promising second-generation prophylactic HPV vaccine candidates.     

Studying the Copy Number of Ribosomal Protein L7/L12

Studying the copy number of ribosomal protein L7/L12 was performed using monoclonal antibody 3G9 to the linear epitope on the C-terminal domain of this protein from Escherichia coli. Immunohistochemical study showed that Agrobacterium tumefaciens ribosomes include 6 copies of protein L7/L12. Our results suggest that the copy number of this protein has an evolutionary role.     

Antimutagenic activity of some saponins isolated from Calendula officinalis L., C. arvensis L. and Hedera helix L

Thirteen saponins were isolated and identified from Calendula officinalis, C. arvensis and Hedera helix. Mutagenic and antimutagenic activities of these products were investigated using a modified liquid incubation technique of the Salmonella/microsomal assay. The Salmonella tester strain TA98 +/- S9 mix was used. Screening of the antimutagenic activity was performed with a known promutagen: benzo-[a]pyrene (BaP) and a mutagenic urine concentrate from a smoker (SU). Antimutagenic activities were also compared with the activity of chlorophyllin. All the saponins were found to be non-toxic and non-mutagenic for doses of 400 micrograms. Chlorophyllin inhibited the mutagenic activities of BaP (1 microgram) and SU (5 microliters) in a dose-dependent manner. The four saponins from C. arvensis and the three saponins from H. helix showed antimutagenic activity against BaP (1 microgram) and SU (5 microliters) with a dose-response relationship. The possible mechanism of the antimutagenic activity of saponins is discussed.     

HPV16L1／L2蛋白单克隆抗体的制备

用真核表达的ＨＰＶ１６Ｌ１／Ｌ２蛋白作为抗原，经免疫、融合、选择性培养、克隆化等过程，我们建立了两株抗ＨＰＶ１６Ｌ１／Ｌ２蛋白的杂交瘤细胞株（另有数株仍在建株中）。免疫斑点法检测证明，它们产生的抗体，只与ＨＰＶ１６Ｌ１／Ｌ２蛋白起反应，而不与ＨＰＶ１６Ｅ６、Ｅ７蛋白、人血清蛋白和牛血清蛋白起反应。另外，免疫组化试验证明，这种抗体可应用于临床ＨＰＶ１６感染的检验，具有敏感、特异、准确、快捷、经济的优点。     

Comparison of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii by transmission electron microscopy

Clinical isolates of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii were grown on charcoal-yeast extract agar from a living-medium inoculum and prepared for transmission electron microscopy by three different methods. Cells of all four Legionella species possessed cytoplasmic vacuoles, a gram-negative type of cell envelope with a dense peptidoglycan-like layer, a ruthenium red-positive polysaccharide capsule, and a single subpolar flagellum. The dense polysaccharide capsule seen on cells of L. micdadei was separated from the outer membrane by an extra layer of electron-lucent material that was not present on cells of the other species examined.