Pop-up English-Chinese

Construction and characterization of attenuated recombinant Listeria monocytogenes delivering HPV16 E7

Yanyan Jia1 Yuelan Yin1 Weijun Tan1 Feifei Duan1 Zhiming Pan1 Xiang Chen1 Xin′an Jiao1

(1.Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China 225009)

【Abstract】Listeria monocytogenes (LM) is an excellent tumor vaccine vector. In this study, recombinant LM expressing human papillomavirus type 16 (HPV16) E7 protein, a vaccine candidate was constructed and the biological characteristics were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 hly promoter-signal sequence, and introduced into the LM4 genome. The recombinants, constructed as LM4△hly::E7, were plasmid- and antibiotic-resistant gene-free. LM4△hly::E7 could express and secrete a 66 kDa E7-LLO fusion protein, which had immunological activities. Proliferation of LM4△hly::E7 in RAW264.7 macrophages was observed under confocal laser scanning microscope. Specific anti-E7 antibodies could be detected by ELISA in mice immunized with LM4△hly::E7. The LD50 of LM4△hly::E7 was 3.863 × 109 CFU in C57BL/6 mice with intraperitoneal immunization, which was 104 times higher than that of wild type LM4. No obvious pathological changes were detected in the immunized mice. These results suggest that LM4△hly::E7 expressing E7-LLO fusion protein may be a safe and promising vector-vaccine candidate for cervical cancer, and may also be important biological materials for anti-tumor research.

【Keywords】 attenuated Listeria monocytogenes; HPV16 E7; macrophage cell line; safety; attenuated Listeria monocytogenes; HPV16 E7; macrophage cell line; safety;

【DOI】

【Funds】 National Natural Science Foundation of China (Nos. 31472193, 31101841) National Natural Science Foundation of China(Nos.31472193,31101841) Priority Academic Program Development of Jiangsu Higher Education Institutions Priority Academic Program Development of Jiangsu Higher Education Institutions

Download this article

    References

    [1]Bruni L, Diaz M, Castellsagué X, et al. Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis, 2010, 202 (12): 1789–1799.

    [2]Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN2008. Int J Cancer,2010,127 (12): 2893–2917.

    [3]Zhang HY, Fei MD, Jiang Y, et al. The diversity of human papillomavirus infection among human immunodeficiency virus-infected women in Yunnan, China. Virol J,2014,11(1):202,doi:10.1186/s12985-014-0202-3.

    [4]Honegger A, Schilling D, Bastian S, et al. Dependence of intracellular and exosomal micro RNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells. PLoS Pathog, 2015, 11 (3): e1004712,doi:10.1371/journal.ppat.1004712.

    [5]Zacapala-Gómeza AE, Del Moral-Hernándeza O, Villegas-Sepúlveda N, et al. Changes in global gene expression profiles induced by HPV16 E6 oncoprotein variants in cervical carcinoma C33-A cells. Virology, 2016, 488: 187–195.

    [6]Mitchell G, Ge L, Huang QY, et al. Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages. Infect Immun, 2015, 83 (5): 2175–2184.

    [7]Pillich H, Chakraborty T, Mraheil MA. Cell-autonomous responses in Listeria monocytogenes infection. Future Microbiol, 2015, 10 (4): 583–597.

    [8]Rothman J, Paterson Y. Live-attenuated Listeria-based immunotherapy. Expert Rev Vaccines, 2013, 12 (5): 493–504.

    [9]Wood LM, Paterson Y. Attenuated Listeria monocytogenes: a powerful and versatile vector for the future of tumor immunotherapy. Front Cell Infect Microbiol,2014,4:51,doi:10.3389/fcimb.2014.00051.

    [10]Lin QQ, Zhou MY, Xu ZK, et al. Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes. J Biotechnol, 2015, 196–197: 20–26.

    [11]Abdallah DS, Bitar PA, Oliveira F, et al. A Listeriamonocytogenes-based vaccine that secretes sand fly salivary protein LJM11 confers long-term protection against vector-transmitted Leishmania major. Infect Immun, 2014, 82 (7): 2736–2745.

    [12]Kang MQ, Cai XX, Tan WJ, et al. Construction and characterization of Listeria monocytogenes Δhfq mutant strain. Acta Microbiol Sin, 2015, 55(4): 433–439 (in Chinese).

    [13]Yin YL, Tian DB, Jia YY, et al. Attenuated Listeria monocytogenes, a Mycobacterium tuberculosis ESAT-6 antigen expression and delivery vector for inducing an immune response. Res Microbiol, 2012, 163 (8): 540–549.

    [14]Zamoiskii EA. Evaluation of Reed-Muench method in determination of activity of biological preparations. Zh Mikrobiol Epidemiol Immunobiol, 1956, 27 (1): 77–83.

    [15]Kennedy GT, Judy BF, Bhojnagarwala P, et al. Surgical cytoreduction restores the antitumor efficacy of a Listeria monocytogenes vaccine in malignant pleural mesothelioma. Immunol Lett, 2015, 166 (1):28–35.

    [16]Jia YY, Yin YL, Duan FF, et al. Prophylactic and therapeutic efficacy of attenuated Listeria monocytogenes-based vector vaccine delivering HPV16 E7 antigen in a murine model. Int J of Mol Med, 2012, 30 (6):1335–1342.

    [17]van Pijkeren JP, Morrissey D, Monk IR, et al. A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy. Hum Gene Ther, 2010, 21 (4): 405–416.

    [18]Keenan BP, Saenger Y, Kafrouni MI, et al. A Listeria vaccine and depletion of T-regulatory cells activate immunity against early stage pancreatic intraepithelial neoplasms and prolong survival of mice. Gastroenterology, 2014, 146 (7): 1784–1794.e6

    [19]Hannan R, Zhang HG, Wallecha A, et al. Combined immunotherapy with Listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer. Cancer Immunol Immunother, 2012, 61 (12): 2227–2238.

    [20]Le DT, Brockstedt DG, Nir-Paz R, et al. A live-attenuated Listeria vaccine (ANZ-100)and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase1 studies of safety and immune induction. Clin Cancer Res, 2012, 18 (3): 858–868.

    [21]Walls ZF, Goodell S, Andrews CD, et al. Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules. J Biotechnol, 2013, 164 (4): 500–502.

    [22]Hernández-Flores KG, Vivanco-Cid H. Biological effects of listeriolysin O: implications for vaccination. Biomed Res Int, 2015, 2015: 360741–360749.

    [23]Seveau S. Multifaceted activity of listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes. Subcell Biochem, 2014, 80: 161–195.

    [24]Neeson P, Pan ZK, Paterson Y. Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma. Cancer Immunol Immunother,2008, 57 (4): 493–505.

    [25]Hamon MA, Ribet D, Stavru F, et al. Listeriolysin O: the Swiss army knife of Listeria. Trends Microbiol, 2012, 20 (8): 360–368.

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 683-692

May 2016

Downloads:1

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • References