Therapeutic Efficacy of Mouse (Mus musculus) Cervical Cancer Therapeutic Vaccine Based on the Attenuated Gene Deletion Listeria monocytogenes
WANG Feng-Ting*, SUN Jing*, LIU Chen, CHENG Yin, JIANG Xin, ZHU Yi-Ran, WEI Fang-Fang, ZHANG Xian, SONG Hou-Hui**, CHENG Chang-Yong**
College of Animal Science ＆ Technology·College of Veterinary Medicine, Zhejiang A＆F University/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Provincial Engineering Research Center for Animal Health Diagnostics＆ Advanced Technology/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China Australia Joint Laboratory for Animal Health Big Data Analytics, Hangzhou 311300, China
Abstract：The attenuated bacterial pathogen Listeria monocytogenes (LM) have been widely studied for cancer immunotherapy as a safe vaccine via presenting tumor-associated antigens. The most common attenuation strategy is to delete the virulence-associated genes. The Listeria attenuated double genes deletion strain ΔactAΔinlB (ΔAB) has become the ideal platform for cancer vaccine research and development. This study aim to develop a ΔAB-based vaccine candidate that can efficiently express and present the cervical cancer-associated E7 antigen (ΔAB-E7) to evaluate the immunotherapeutic efficacy in the mice (Mus musculus) cervical cancer model. The bacterial genetic methods were employed to construct the ΔAB and ΔAB-E7 by introducing E7 with in-frame fusion to the virulence factor listeriolysin O (LLO). The ΔAB-E7 was verified whether the E7 antigen could be expressed in fusion with LLO by Western blot. The infection characteristics of these strains were studied, including in vitro and intracellular growth, bacterial proliferation, and pathogenicity in mice. Then, the immunotherapy efficacy of ΔAB-E7 was evaluated on the mice cervical cancer model by tracking tumor growth. The results showed that ΔAB-E7 could efficiently express and secrete LLO-fused E7, the in vitro and intracellular growth abilities of ΔAB and ΔAB-E7 were comparable to those of wild-type strain, while the virulence was significantly attenuated in mice with the highly-reduced proliferation ability in mice organs. Importantly, vaccination of ΔAB-E7 could inhibit cervical cancer growth in mice, eliciting promising anti-tumor efficacy. Collectively, this study present a favorable Listeria-based vaccine candidate against cervical cancer, which provides new strategies for future research and applications of immunotherapies for human cervical cancer and other cancers.
[1] 陈璐, 汪枫婷, 孙静, 等. 2021. 减毒单增李斯特菌为载体的肿瘤免疫治疗研究进展[J]. 农业生物技术学报, 29(8): 1622-1629. (Chen L, Wang F T, Sun J, et al.2021. Research advances on attenuated listeria monocytogenes as a vaccine platform for cancer immunotherapy[J]. Journal of Agricultural Biotechnology, 29(8): 1622-1629.) [2] Basu P, Mehta A, Jain M, et al.2018. A randomized phase 2 study of ADXS11-001 Listeria monocytogenes-listeriolysin o immunotherapy with or without cisplatin in treatment of advanced cervical cancer[J]. International Journal of Gynecolgical Cancer, 28(4): 764-772. [3] Brahmer J R, Johnson M L, Cobo M, et al.2021. JNJ-64041757 (JNJ-757), a live, attenuated, double-deleted Listeria monocytogenes-based immunotherapy in patients with NSCLC: Results from two phase 1 studies[J]. JTO Clinical And Research Reports, 2(2): 100103. [4] Brockstedt D G, Giedlin M A, Leong M L, et al.2004. Listeria-based cancer vaccines that segregate immunogenicity from toxicity[J]. Proceedings of the National Academy of Sciences of the USA, 101(38): 13832-13837. [5] Cheng C, Jiang L, Ma T, et al.2017. Carboxyl-terminal residues N478 and V479 required for the cytolytic activity of listeriolysin O play a critical role in Listeria monocytogenes pathogenicity[J]. Frontiers of Immunology, 8: 1439. [6] Cheng C, Sun J, Yu H, et al.2020. Listeriolysin o pore-forming activity is required for ERK1/2 phosphorylation during Listeria monocytogenes infection[J]. Frontiers in Immunology, 11: 1146. [7] Deng W, Lira V, Hudson T E, et al.2018. Recombinant Listeria promotes tumor rejection by CD8(+) T cell-dependent remodeling of the tumor microenvironment[J]. Proceedings of the National Academy of Sciences of the USA, 115(32): 8179-8184. [8] Depuydt C E, Thys S, Beert J, et al.2016. Linear viral load increase of a single HPV-type in women with multiple HPV infections predicts progression to cervical cancer[J]. International Journal of Cancer, 139(9): 2021-2032. [9] Duan F, Chen J, Yao H, et al.2021. Enhanced therapeutic efficacy of Listeria-based cancer vaccine with codon-optimized HPV16 E7[J]. Human Vaccines & Immunotherapeutics, 17(6): 1568-1577. [10] Flickinger J C, Rodeck U, Snook A E.2018. Listeria monocytogenes as a vector for cancer immunotherapy: Current understanding and progress[J]. Vaccines (Basel), 6(3): 48. [11] Gaballa A, Guariglia-Oropeza V, Wiedmann M, et al.2019. Cross talk between SigB and PrfA in Listeria monocytogenes facilitates transitions between extra- and intracellular environments[J]. Microbiology and Molecular Biology Reviews, 83(4): e00034-19. [12] Galicia-Carmona T, Arango-Bravo E, Serrano-Olvera J A, et al.2021. ADXS11-001 LM-LLO as specific immunotherapy in cervical cancer[J]. Human Vaccines & Immunotherapeutics, 17(8): 2617-2625. [13] Grupp S A, Kalos M, Barrett D, et al.2013. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia[J]. The New England Journal of Medicine, 368(16): 1509-1518. [14] Gunn G R, Zubair A, Peters C, et al.2001. Two Listeria monocytogenes vaccine vectors that express different molecular 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]. The Journal of Immunology, 167(11): 6471-6479. [15] Hanson W G, Benanti E L, Lemmens E E, et al.2019. A potent and effective suicidal Listeria vaccine platform[J]. Infection and Immunity, 87(8): e00144-19. [16] Hollingsworth R E, Jansen K.2019. Turning the corner on therapeutic cancer vaccines[J]. NPJ Vaccines, 4: 7. [17] Huh W K, Brady W E, Fracasso P M, et al.2020. Phase II study of axalimogene filolisbac (ADXS-HPV) for platinum-refractory cervical carcinoma: An NRG oncology/gynecologic oncology group study[J]. Gynecology Oncology, 158(3): 562-569. [18] Jia Y Y, Tan W J, Duan F F, et al.2017. A genetically modified attenuated Listeria vaccine expressing HPV16 E7 kill tumor cells in direct and antigen-specific manner[J]. Frontiers in Cellular and Infection Microbiology, 7: 279. [19] Kühn S, Enninga J.2020. The actin comet guides the way: How Listeria actin subversion has impacted cell biology, infection biology and structural biology[J]. Cellular Microbiology, 22(4): e13190. [20] Le D T, Dubenksy T W, Brockstedt D G.2012. Clinical development of Listeria monocytogenes-based immunotherapies[J]. Seminars in Oncology, 39(3): 311-322. [21] Li Y R, Zhou Y, Kim Y J, et al.2021. Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy[J]. Cell Reports Medicine, 2(11): 100449. [22] Li Z, Zhao X, Higgins D E, et al.2005. Conditional lethality yields a new vaccine strain of Listeria monocytogenes for the induction of cell-mediated immunity[J]. Infection and Immunity, 73(8): 5065-5073. [23] Maciag P C, Radulovic S, Rothman J.2009. The first clinical use of a live-attenuated Listeria monocytogenes vaccine: A Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix[J]. Vaccine, 27(30): 3975-3983. [24] Musser M L, Berger E P, Tripp C D, et al.2021. Safety evaluation of the canine osteosarcoma vaccine, live Listeria vector[J]. Veterinary and Comparative Oncology, 19(1): 92-98. [25] Nguyen B N, Peterson B N, Portnoy D A.2019. Listeriolysin O: A phagosome-specific cytolysin revisited[J]. Cellular Microbiology, 21(3): e12988. [26] Oladejo M, Paterson Y, Wood L M.2021. Clinical experience and recent advances in the development of Listeria-based tumor immunotherapies[J]. Frontiers in Immunology, 12: 642316. [27] Ott P A, Hodi F S, Kaufman H L, et al.2017. Combination immunotherapy: A road map[J]. Journal for Immunotherapy of Cancer, 5(1): 16. [28] Pizarro-Cerda J, Cossart P.2018. Listeria monocytogenes: Cell biology of invasion and intracellular growth[J]. Microbiology Spectrum, 6(6): 1-16. [29] Rolhion N, Cossart P.2017. How the study of Listeria monocytogenes has led to new concepts in biology[J]. Future Microbiology, 12(7): 621-638. [30] Siegel R L, Miller K D, Jemal A.2020. Cancer statistics, 2020[J]. CA: A Cancer Journal for Clinicians, 70(1): 7-30. [31] Small W, Bacon M A, Bajaj A, et al.2017. Cervical cancer: A global health crisis[J]. Cancer, 123(13): 2404-2412. [32] Stanley M.2016. Preventing cervical cancer and genital warts -How much protection is enough for HPV vaccines?[J]. Journal of Infection, 72: S23-S28 [33] Su L, Zhang Y, Zhang X, et al.2021. Combination immunotherapy with two attenuated Listeria strains carrying shuffled HPV-16 E6E7 protein causes tumor regression in a mouse tumor model[J]. Scientific Reports, 11(1): 13404. [34] Wan X, Cheng C, Lin Z, et al.2015. The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells[J]. Oncotarget, 6(11): 8822-8838. [35] Wood L M, Guirnalda P D, Seavey M M, et al.2008. Cancer immunotherapy using Listeria monocytogenes and listerial virulence factors[J]. Immunologic Research, 42(1-3): 233-245. [36] Wood L M, Paterson Y.2014. Attenuated Listeria monocytogenes: A powerful and versatile vector for the future of tumor immunotherapy[J]. Frontiers in Cellular and Infection Microbiology, 4: 51. [37] Yang Y, Hou J, Lin Z, et al.2014. Attenuated Listeria monocytogenes as a cancer vaccine vector for the delivery of CD24, a biomarker for hepatic cancer stem cells[J]. Cellular & Molecular Immunology, 11(2): 184-196.
