Jingjing REN, Mingwei YANG, Pengyan WANG, Jianjun JIANG, Genqiang YAN

A Mutant of Listeria monocytogenes Shows Decreased Virulence and Confers Protection Against Listeriosis in Mice

This research was performed to obtain a safe and highly immunogenic Listeria strain and evaluate the biological characteristics of the deletion mutant. Based on homologous recombination technology, we constructed a deletion mutant Lm90-ΔinlB of L. monocytogenes. Meanwhile, we characterized its safety and protective efficacy against listeriosis infection in mice. The results showed that the virulence of Lm90-ΔinlB could significantly decrease compared with the parental strain (Lm90). The deletion strain retained hemolytic activity and induced CD8+ T cell response comparable to that of Lm90. Mice immunized with Lm90-ΔinlB were capable of stimulating specific CD8+ T cells to the listerial epitopes LLO91-99 and P60217-225 at levels equivalent to Lm90. Importantly, immunization of mice with Lm90-ΔinlB displayed good protection against listeriosis. In conclusion, strain Lm90-ΔinlB is a vaccine candidate with the potential to be more immunogenic yet considerably less toxic than the parental strain.

Farelerde Düşük Virulans Gösteren ve Listeriosise Karşı Koruma Sağlayan Listeria monocytogenes Mutantı

Bu araştırma güvenli ve yüksek derecede immunojenik bir Listeria suşu elde etmek ve delesyon mutantının biyolojik özelliklerini değerlendirmek için yapıldı. Homolog rekombinasyon teknolojisi kullanılarak, L. monocytogenes’in bir delesyon mutantı olan Lm90-ΔinlB oluşturuldu. Aynı zamanda, farelerde listeriosis enfeksiyonuna karşı güvenliği ve koruyucu etkinliği belirlendi. Sonuçlar, Lm90-ΔinlB’in virülansının, parental suşa (Lm90) kıyasla önemli ölçüde azalabileceğini gösterdi. Delesyon mutantı suş hemolitik aktiviteyi korudu ve CD8+ T hücre tepkisini Lm90 ile karşılaştırılabilir düzeyde uyardı. Lm90- ΔinlB ile immünize aşılanmış farelerde, Lm90’a eşdeğer seviyelerde LLO91-99 ve P60217-225 listeria epitoplarına spesifik CD8+ T hücrelerini uyarabilme kapasitesi mevcuttu. Farelerde Lm90-ΔinlB ile aşılanması sonrası listeriyoza karşı iyi bir koruma görülmesi önemli bir bulgudur. Sonuç olarak, Lm90-ΔinlB suşu, parental suşundan daha fazla immünojenik ancak önemli ölçüde daha az toksik olması nedeniyle potansiyel bir aşı adayıdır.

PDF

___

1. Jacks A, Pihlajasaari A, Vahe M, Myntti A, Kaukoranta SS, Elomaa N, Salmenlinna S, Rantala L, Lahti K, Huusko S, Kuusi M, Siitonen A, Rimhanen-Finne R: Outbreak of hospital-acquired gastroenteritis and invasive infection caused by Listeria monocytogenes, Finland, 2012. Epidemiol Infect, 144 (13): 2732-2742, 2016. DOI: 10.1017/s0950268815002563

2. Radoshevich L, Cossart P: Listeria monocytogenes: Towards a complete picture of its physiology and pathogenesis. Nat Rev Microbiol, 16 (1): 32-46, 2018. DOI: 10.1038/nrmicro.2017.126

3. Pagliano P, Ascione T, Boccia G, De Caro F, Esposito S: Listeria monocytogenes meningitis in the elderly: Epidemiological, clinical and therapeutic findings. Infez Med, 24 (2): 105-111, 2016.

4. Pucci L, Massacesi M, Liuzzi G: Clinical management of women with listeriosis risk during pregnancy: A review of national guidelines. Expert Rev Anti-Infect Ther, 16 (1): 13-21, 2018. DOI: 10.1080/14787210.2018.1417837

5. Charlier C, Perrodeau É, Leclercq A, Cazenave B, Pilmis B, Henry B, Lopes A, Maury MM, Moura A, Goffinet F, Dieye HB, Thouvenot P, Ungeheuer MN, Tourdjman M, Goulet V, de Valk H, Lortholary O, Ravaud P, Lecuit M; MONALISA study group: Clinical features and prognostic factors of listeriosis: The MONALISA national prospective cohort study. Lancet Infect Dis, 17, 510-519, 2017. DOI: 10.1016/S1473- 3099(16)30521-7

6. Harty JT, Lenz LL, Bevan MJ: Primary and secondary immune responses to Listeria monocytogenes. Curr Opin Immunol, 8 (4): 526-530, 1996. DOI: 10.1016/S0952-7915(96)80041-0

7. Pamer EG: Immune responses to Listeria monocytogenes. Nat Rev Immunol, 4, 812–823, 2004. DOI: 10.1038/nri1461

8. Jia Q, Bowen R, Dillon BJ, Maslesagalic S, Chang BT, Kaidi AC, Horwitz MA: Single vector platform vaccine protects against lethal respiratory challenge with tier 1 select agents of anthrax, plague, and tularemia. Sci Rep, 8:7009, 2018. DOI: 10.1038/s41598-018-24581-y

9. Yin Y, Tian D, Jiao H, Zhang C, Pan Z, Zhang X, Wang X, Jiao X: Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model. Clin Vaccine Immunol, 18 (3): 500-505, 2011. DOI: 10.1128/CVI.00445-10

10. Cruz R, Pereira-Castro I, Almeida MT, Moreira A, Cabanes D, Sousa S: Epithelial keratins modulate cMet expression and signaling and promote InlB-Mediated Listeria monocytogenes infection of Hela cells. Front Cell Infect Microbiol, 8:146, 2018. DOI: 10.3389/fcimb.2018.00146

11. Van Ngo H, Bhalla M, Chen DY, Ireton K: A role for host cell exocytosis in InlB-mediated internalization of Listeria monocytogenes. Cell Microbiol, 19:e12768, 2017. DOI: 10.1111/cmi.12768

12. Bolhassani A, Naderi N, Soleymani S: Prospects and progress of listeria-based cancer vaccines. Expert Opin Biol Ther, 17 (11): 1389-1400, 2017. DOI: 10.1080/14712598.2017.1366446

13. Horton RM, Cai Z, Ho SN, Pease LR: Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction. Biotechniques, 8 (5): 528-535, 2013. DOI: 10.2144/000114017

14. Soni DK, Ghosh A, Chikara, SK, Singh KM, Joshi CG, Dubey, SK: Comparative whole genome analysis of Listeria monocytogenes 4b strains reveals least genome diversification irrespective of their niche specificity. Gene Rep, 8, 61-68, 2017. DOI: 10.1016/j.genrep.2017.05.007

15. Lingnau A, Domann E, Hudel M, Bock M, Nichterlein T, Wehland J, Chakraborty T: Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and-independent mechanisms. Infect Immun, 63 (10): 3896-3903, 1995.

16. Portnoy DA, Jacks PS, Hinrichs DJ: Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med, 167 (4): 1459- 1471, 1988. DOI: 10.1084/jem.167.4.1459

17. Bull RA, Leung P, Gaudieri S, Deshpande P, Cameron B, Walker M, Chopra A, Lloyd AR, Luciani F and on Behalf of the HITS-p Investigators: T transmitted/founder viruses rapidly escape from CD8+ T cell responses in acute hepatitis c virus infection. J Virol, 89 (10): 5478- 5490, 2015. DOI: 10.1128/JVI.03717-14

18. Fuchs YF, Jainta GW, Kühn D, Wilhelm C, Weigelt M, Karasinsky A, Upadhyay B, Ziegler AG, Bonifacio E: Vagaries of the ELISpot assay: Specific detection of antigen responsive cells requires purified CD8+ T cells and MHC class I expressing antigen presenting cell lines. Clin Immunol, 157 (2): 216-225, 2015. DOI: 10.1016/j.clim.2015.02.012

19. Sun P, Simmons M: Dendritic cell-based ELISpot assay for assessing T-Cell IFN-γ responses in human peripheral blood mononuclear cells to dengue envelope proteins. Methods Mol Biol, 1808, 187-196, 2018. DOI: 10.1007/978-1-4939-8567-8_17

20. Youn JC, Kim JY, Jung MK, Yu HT, Park SH, Kim IC, Lee SK, Choi SW, Han S, Ryu K, Park S, Shin EC: Analysis of cytomegalovirus-specific T-cell responses in patients with hypertension: Comparison of assay methods and antigens. Clin Hypertens, 24:5, 2018. DOI: 10.1186/s40885-018-0090-8

21. Ding C, Ma J, Dong Q, Liu Q: Live bacterial vaccine vector and delivery strategies of heterologous antigen: A review. Immunol Lett, 197, 70-77, 2018. DOI: 10.1016/j.imlet.2018.03.006

22. Flickinger JC, Rodeck U, Snook AE: Listeria monocytogenes as a vector for cancer immunotherapy: Current understanding and progress. Vaccines, 6:48, 2018. DOI: 10.3390/vaccines6030048

23. Gaillard JL, Jaubert F, Berche P: The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J Exp Med, 183, 359-369, 1996. DOI: 10.1084/jem.183.2.359

24. Pizarro-Cerdá J, Cossart P: Listeria monocytogenes membrane trafficking and lifestyle: The Exception or the rule? Ann Rev Cell Dev Biol, 25, 649-670, 2009. DOI: 10.1146/annurev.cellbio.042308.113331

25. Pentecost M, Kumaran J, Ghosh P, Amieva MR: Listeria monocytogenes internalin B activates junctional endocytosis to accelerate intestinal invasion. Plos Pathog, 6(5):e1000900, 2010. DOI: 10.1371/journal.ppat.1000900

26. Dramsi S, Biswas I, Maguin E, Braun L, Mastroeni P, Cossart P: Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigene family. Mol Microbiol, 16, 251- 261, 1995. DOI: 10.1111/j.1365-2958.1995.tb02297.x

27. Stachowiak R, Jagielski T, Roeske K, Osińska O, Gunerka P, Wiśniewski J: Lmo0171, a novel internalin-like protein, determines cell morphology of listeria monocytogenes and its ability to invade human cell lines. Curr Microbiol, 70 (2): 267-274, 2015. DOI: 10.1007/s00284-014-0715-4

28. Chiba S, Nagai T, Hayashi T, Baba Y, Nagai S, Koyasu S: Listerial invasion protein internalin B promotes entry into ileal Peyer’s patches in vivo. Microbiol Immunol, 55 (2): 123-129, 2011. DOI: 10.1111/j.1348- 0421.2010.00292.x

29. Uchiyama R, Yonehara S, Taniguchi S, Ishido S, Ishii KJ, Tsutsui H: Inflammasome and Fas-Mediated IL-1β contributes to Th17/Th1 cell induction in pathogenic bacterial infection in vivo. J Immunol, 199 (3): 1122-1130, 2017. DOI: 10.4049/jimmunol.1601373

30. Geginat G, Nichterlein T, Kretschmar M, Schenk S, Hof H, LalicMülthaler M, Goebel W, Bubert A: Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua. J Immunol, 162 (8): 4781-4789, 1999.

31. Chandrabos C, Soudja SM, Weinrick B, Gros M, Frangaj A, Rahmoun M, Jacobs WR, Lauvau G: The p60 and NamA autolysins from Listeria monocytogenes contribute to host colonization and induction of protective memory. Cell Microbiol, 17 (2): 147-163, 2015. DOI: 10.1111/ cmi.12362

32. Yamamoto K, Kawamura I, Tominaga T, Nomura T, Kohda C, Ito J, Mitsuyama M: Listeriolysin O, a cytolysin derived from Listeria monocytogenes, inhibits generation of ovalbumin-specific Th2 immune response by skewing maturation of antigen-specific T cells into Th1 cells. Clin Exp Immunol, 142 (2): 268-274, 2005. DOI: 10.1111/j.1365- 2249.2005.02922.x

33. Kono M, Nakamura Y, Suda T, Uchijima M, Tsujimura K, Nagata T, Giermasz AS, Kalinski P, Nakamura H, Chida K: Enhancement of protective immunity against intracellular bacteria using type-1 polarized dendritic cell (DC) vaccine. Vaccine, 30 (16): 2633-2639, 2012. DOI: 10.1016/j.vaccine.2012.02.026

34. Villanueva MS, Sijts AJ, Pamer EG: Listeriolysin is processed efficiently into an MHC class I-associated epitope in Listeria monocytogenes-infected cells. J Immunol, 155 (11): 5227-5233, 1995.

35. Köhler S, Leimeister-Wächter M, Chakraborty T, Lottspeich F, Goebel W: The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes. Infect Immun, 58(6): 1943-1950, 1990.

36. Bahey-El-Din M, Casey PG, Griffin BT, Gahan CGM: Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8+ T cells against Listeria monocytogenes in the murine infection model. Vaccine, 26, 5304-5314, 2008. DOI: 10.1016/j.vaccine.2008.07.047

37. Bahey-El-Din M, Casey PG, Griffin BT, Gahan CG: Expression of two Listeria monocytogenes antigens (P60 and LLO) in Lactococcus lactis and examination for use as live vaccine vectors. J Med Microbiol, 59, 904-912, 2010. DOI: 10.1099/jmm.0.018770-0

38. Mclaughlin HP, Bahey-El-Din M, Casey PG, Hill C, Gahan CGM: A mutant in the Listeria monocytogenes Fur-regulated virulence locus (frvA) induces cellular immunity and confers protection against listeriosis in mice. J Med Microbiol, 62, 185-190, 2013. DOI: 10.1099/jmm.0.049114-0