单核细胞增生性李斯特菌作为肿瘤疫苗运送载体的研究进展

段斐斐，殷月兰，康美琴，谈卫军，陶成武，潘志明，黄金林，焦新安

1 LM感染后诱导的免疫应答

1.1天然免疫应答 LM感染宿主细胞以后可以导致多种前炎性因子以及炎症趋化因子的产生，其中最重要的早期细胞因子有IL-12p70、γ-干扰素(IFN-γ)及白细胞介素18(IL-18)[4]。这些细胞因子及级联反应产生的趋化因子会吸引嗜中性粒细胞浸润至感染部位，以此来活化细胞介导的杀伤作用[5]。白细胞介素12(IL-12)可刺激NK细胞和T细胞产生IFN-γ，而产生的IFN-γ反过来又可以活化巨噬细胞，同时刺激携带细菌抗原的树突状细胞(DC)聚集至感染部位，从而进一步的诱发调节抗原特异性的细胞毒性T淋巴细胞效应(CTL)[6]。LM感染宿主后诱导产生的IFN-γ可触发携带细菌抗原的DC细胞正调节共刺激分子，并产生功能性的IL-12，而IL-12作为重要的信号分子可以诱导未致敏的辅助性T细胞分化成辅助性T细胞，以此来推进细胞介导的免疫应答[7]。

2 减毒LM作为外源抗原表达载体的优点

研究者将弱毒化的LM改造成为可以容纳外源性抗原的载体，从而成为可诱导较强细胞免疫应答的预防性或治疗性活载体疫苗。减毒李斯特菌作为疫苗载体具有以下优势：直接感染抗原递呈细胞，具有两类抗原加工和提呈途径；利用染色体系统可以稳定表达多个基因产物，操作容易；作为革兰阳性菌，不含有内毒素，使用方便[10]。鉴于LM作为外源抗原表达载体的这些优点，近几年来，LM已成为分子生物学和免疫学的研究热点。LM已被广泛作为携带病毒和肿瘤抗原的载体来激发细胞介导的免疫应答[11]。以减毒LM为运送载体，在小鼠、兔、猫、鸡和猴子等实验动物模型，已对许多外源抗原进行了研究，并取得较好的结果。

3 LM作为肿瘤疫苗载体的应用

1992年，首次报道应用LM运送外源抗原能够诱导抗原特异性CTL应答[12]。Pan 等最早报道应用LM运送模式肿瘤抗原，可以保护小鼠抵抗致死性肿瘤细胞的攻击，并且能引起肉眼可见的肿瘤消退，证明LM作为载体运送肿瘤抗原方面具有很强的优势[13]。目前，运用LM作为多种病毒或肿瘤相关抗原的疫苗载体，已如火如荼进行，这些相关抗原主要包括人乳头瘤病毒16型E7蛋白(HPV16 E7)、酪氨酸酶相关蛋白(Trp2)、高分子量黑色素瘤相关抗原(HMW-MAA)、前列腺特异性抗原(PSA)以及酪氨酸激酶表皮生长因子(HER-2/neu)等[14-16]。

3.2HER-2/neu抗原 Her-2/neu是酪氨酸激酶表皮生长因子家族中的一员，在20%～40%的乳腺癌中过度表达，并且还可引发卵巢癌、肺癌、胰腺癌及胃肠道癌症[24]。Paterson等构建了5种表达不同片段的鼠Her-2/neu和LLO融合蛋白的LM载体疫苗，研究表明这5种重组疫苗株能够诱导小鼠体内抗Her-2/neu的CTL应答，并且对NT-2肿瘤显示出有效的预防性和治疗性效果[16]。研究还报道这些疫苗同时对表达Her-2/neu的转基因小鼠肿瘤模型有同样的作用，该转基因小鼠对Her-2/neu有显著耐受。免疫LM重组疫苗后可延迟Her-2/neu转基因小鼠中乳头瘤的出现。为了使这些研究成果更好的应用于临床，Seavey等进一步完善疫苗，研发出表达Her-2/neu的HLA表位和LLO融合蛋白的嵌合疫苗[25-26]。

3.5PSA抗原 PSA抗原又被称为前列腺特异抗原，是由前列腺腺泡和导管的上皮细胞分泌的一种单链糖蛋白，由于PSA在恶性的前列腺细胞中过量表达，而在正常的前列腺上皮细胞和其他器官中低表达，因此已成为前列腺癌的免疫靶抗原[31]。Shahabi等构建表达融合蛋白的重组李斯特菌，并在pCa小鼠模型上鉴定LM-LLO-PSA疫苗株，结果显示该疫苗株能诱导肿瘤浸润性T调节型细胞数量的下降，导致80%小鼠的肿瘤完全消退。ELISPOT和胞内染色试验表明LM-LLO-PSA能够诱导小鼠产生分泌IFN-γ细胞的数量增加，另外，CTL实验表明PSA特异性的T细胞能够识别和裂解PSA靶细胞[32]。对于应用于临床的疫苗而言，安全性是一个重要问题。Wallecha等对减毒重组疫苗的安全性进行了有意义的探索，他们应用减毒李斯特菌(LM△dal/dat/actA)运送PSA抗原，该菌必须在添加丙氨酸的条件下才能生存。研究表明虽然减毒重组菌在体内的存活率降低，但是仍然能有效诱导清除肿瘤所必须的细胞免疫应答。实验结果进一步表明，用该减毒疫苗免疫小鼠后，可诱导产生大量的PSA特异性浸润T细胞，同时研究显示该疫苗还可引起表达PSA抗原的肿瘤消退，表明该减毒重组疫苗是非常有效和安全的前列腺癌免疫治疗候选疫苗，并且LM△dal/dat/actA是非常有潜力的疫苗载体，可以运送其它单抗原或融合抗原用于癌症免疫治疗[33]。

4 展 望

LM独特的胞内生活史及其诱导强烈的天然免疫应答和特异性免疫应答，使得它具有活载体疫苗的众多优势，运用LM作为多种肿瘤疫苗载体的研究也在进行，并且取得了一定的预防性和治疗性作用。但是也存在需进一步克服的问题，例如疫苗的抗性、外源抗原的表达水平等，此外在应用LM做载体时如何将其毒力降至最低，这也是研究工作者主要考虑的问题。我们相信随着研究的不断深入，LM作为疫苗载体将具有更广阔的应用前景。

参考文献：

[1]Lecuit M. Human listeriosis and animal models[J]. Microbes Infect, 2007, 9: 1216-1225.

[2]Stavru F, Archambaud C, Cossart P. Cell biology and immunology ofListeriamonocytogenesinfections:novel insights[J]. Immunol Rev, 2011, 240 (1): 160-184. DOI: 10.1111/j.1600-065X.2010.00993.x

[3]Wallecha A, Carroll KD, Maciag PC, et al. Multiple effector mechanisms induced by recombinantListeriamonocytogenesanticancer immunotherapeutics[J]. Adv Appl Microbiol, 2009, 66: 1-27. DOI: 10.1016/S0065-2164(08)00801-0

[4]Lara-Tejero M, Pamer EG. T cell responses toListeriamonocytogenes[J]. Curr Opin Microbiol, 2004, 7(1): 45-50.

[5]Pamer EG. Immune responses toListeriamonocytogenes[J]. Nat Rev Immunol, 2004, 4(10): 812-823.

[6]Pamer EG. The innate and adaptive response toListeriamonocytogenes: a short overview[J]. Microbes Infect, 2007, 9(10): 1208-1215.

[7]Bruhn KW,Craft N，Miller JF, et al.Listeriaas a vaccine vector[J]. Microbes Infect, 2007, 9(10): 1226-1235.

[9]Flutter B, Gao B. MHC class I antigen presentation e recently trimmed and well presented[J]. Cell Mol Immunol, 2004, 1(1): 22-30.

[10]Schoen C, Loeffler DI, Frentzen A, et al.Listeriamonocytogenesas novel carrier system for the development of live vaccines[J]. Int J Med Microbiol, 2008, 298(1-2): 45-58.

[11]Wallecha A, Carroll KD, Maciag PC, et al. Multiple effector mechanisms induced by recombinantListeriamonocytogenesanticancer immunotherapeutics[J]. Adv Appl Microbiol, 2009, 66: 1-27. DOI: 10.1016/S0065-2164(08)00801-0

[12]Schafer R, Portnoy DA, Brassell SA, et al. Induction of a cellular immune response to a foreign antigen by a recombinantListeriamonocytogenesvaccine[J]. J Immunol, 1992, 149(1): 53-59.

[13]Pan ZK, Ikonomidis G, Lazenby A, et al. A recombinantListeriamonocytogenesvaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours[J]. Nat Med, 1995, 1(5): 471-477.

[14]Gunn GR, Zubair A, Peters C, et al. TwoListeriamonocytogenesvaccine vectors that express differentmolecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16[J]. J Immunol, 2001, 167(11): 6471-6479.

[15]Shahabi V, Reyes-Reyes M, Wallecha A, et al. Development of aListeriamonocytogenesbased vaccine against prostate cancer[J]. Cancer Immunol Immunother, 2008, 57(9): 1301-1313. DOI: 10.1007/s00262-008-0463-z

[16]Singh R, Dominiecki ME, Jaffee EM, et al. Fusion to Listeriolysin O and delivery byListeriamonocytogenesenhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse[J]. J Immunol, 2005, 175(6): 3663-3673.

[17]Scheurer ME, Tortolero-Luna G, Adler-Storthz K. Human papilloma virus infection: biology, epidemiology, and prevention[J]. Int J Gynecol Cancer, 2005, 15: 727-746.

[18]Ling M, Kanayama M, Roden R, et al. Preventive and therapeutic vaccines for HPV-associated cervical cancers[J]. J Biomed Sci, 2000, 7: 341-356.

[19]Sewell DA, Pan ZK, Paterson Y.Listeria-based HPV-16 E7 vaccines limit autochthonous tumor growth in a transgenic mouse model for HPV-16 transformed tumors[J]. Vaccine, 2008, 26 (41): 5315-5320. DOI: 10.1016/j.vaccine.2008.07.036

[20]Jia Y, Yin Y, Duan F, et al. Prophylactic and therapeutic efficacy of an attenuatedListeriamonocytogenes-based vaccine delivering HPV16 E7 in a mouse model[J]. Int J Mol Med, 2012, 30(6): 1335-1342. DOI: 10.3892/ijmm.2012.1136

[21]Hussain SF, Paterson Y. What is needed for effective antitumor immunotherapy? Lessons learned usingListeriamonocytogenesas a live vector for HPV-associated tumors[J]. Cancer Immunol Immunother, 2005, 54(6): 577-586.

[22]Maciag PC, Radulovic S, Rothman J. The first clinical use of a live-attenuatedListeriamonocytogenesvaccine: A Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix[J]. Vaccine, 2009, 27(30): 3975-3983. DOI: 10.1016/j.vaccine.2009.04.041

[23]Singh R, Paterson Y.Listeriamonocytogenesas a vector for tumor-associated antigens for cancer immunotherapy[J]. Expert Rev Vaccines, 2006, 5: 541-552.

[24]Disis ML, Cheever MA. HER-2/neu protein: a target for antigen-specific immunotherapy of human cancer[J]. Adv Cancer Res, 1997, 71: 343-371.

[25]Singh R, Paterson Y. Immunoediting sculpts tumor epitopes during immunotherapy[J]. Cancer Res, 2007,67(5):1887-1892.

[27]Bruhn KW, Craft N, Nguyen BD, et al. Characterization of anti-self CD8 T-cell responses stimulated by recombinantListeriamonocytogenesexpressing the melanoma antigen TRP-2[J]. Vaccine, 2005,23 (33):4263-4272.

[28]Prins RM, Bruhn KW, Craft N, et al. Central nervous system tumor immunity generated by a recombinantListeriamonocytogenesvaccine targeting tyrosinase related protein-2 and real-time imaging of intracranial tumor burden[J]. Neurosurgery, 2006,58 (1):169-178.

[29]Campoli MR, Chang CC, Kageshita T, et al. Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance[J]. Crit Rev Immunol, 2004, 24(4):267-296.

[30]Maciag PC, Seavey MM, Pan ZK, et al. Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature[J]. Cancer Res, 2008, 68(19):8066-8075. DOI: 10.1158/0008-5472.CAN-08-0287.

[31]Cunha AC, Weigle B, Kiessling A, et al. Tissue-specificity of prostate specific antigens: comparative analysis of transcript levels in prostate and non-prostatic tissues[J]. Cancer Lett, 2006, 236:229-238.

[32]Shahabi V, Reyes-Reyes M, Wallecha A, et al. Development of aListeriamonocytogenesbased vaccine against prostate cancer[J]. Cancer Immunol Immunother, 2008,57 (9):1301-1313. DOI: 10.1007/s00262-008-0463-z.

[33]Wallecha A, Maciag PC, Rivera S, et al. Construction and characterization of an attenuatedListeriamonocytogenesstrain for clinical use in cancer immunotherapy[J]. Clin Vaccine Immunol, 2009,16 (1):96-103. DOI: 10.1128/CVI.00274-08.