Extraction and biological evaluation of Mycobacterium bovis extracellular vesicles as adjuvant and candidates for bovine tuberculosis vaccine

Abstract

Objectives: BCG vaccine is the only virtual vaccine that has significantly helped control tuberculosis for 80 years. Bacteria naturally release extracellular vesicles (EVs) in different environments during the growth process. The use of extracellular vesicles is an alternative way to transfer ligands that are detected by host cells. Vesicles range in size from 50 nm to 250 nm in diameter and contain phospholipids, proteins, and lipopolysaccharides, and can carry additional factors such as toxins, adhesive, or immune system compounds that are important in pathogens. Therefore, this study aimed to evaluate these compounds as adjuvants or candidates for the bovine tuberculosis vaccine.

Methods: In the present study, Mycobacterium bovis standard CRBIP7.121 was used. Extraction of membrane vesicles after mass culture was performed by a method based on ultracentrifugation and deoxycholate. After preparation and staining, the vesicles were examined by electron microscopy. Sample analysis was also performed by SDS-PAGE. The presence of LPS in the sample was measured by the LAL test. In addition, the harmlessness of bacterial EVs and the absence of any toxic agents in the sample were confirmed by pyrogenic tests in rabbits.

Results: The protein content of membrane vesicles is equal to 1.25 and 1.32 mg/ml. In SDS-page evaluation, bands of 35, 40, 50, and 70 kDa were observed and then membrane vesicles were observed and confirmed by electron microscopy. The amount of vesicle toxin contained by the LAL test was reported in the permissible range.

Conclusions: Discussion of the data obtained from the above research shows that at different stages of the purification process, EVs fully retained their spatial and natural form and lacked impurities. Therefore, due to the importance of external vesicles in developing immune responses, EVs extracted from M. bovis CRBIP7.121 can be considered a useful and effective immunogen against Mycobacterium infections.

Keywords

• Extracellular vesicles
• Mycobacterium bovis CRBIP7.121
• adjuvant and vaccine

References

1. 1. Saad J, Baron S, Lagier JC, Drancourt M, Gautret P. Mycobacterium bovis pulmonary tuberculosis after ritual sheep sacrifice in Tunisia. Emerg Infect Dis 2020;26:1605-7.
2. 2. Pozo P, VanderWaal K, Grau A, de la Cruz ML, Nacar J, Bezos J, et al. Analysis of the cattle movement network and its association with the risk of bovine tuberculosis at the farm level in Castilla y Leon, Spain. Transbound Emerg Dis 2019;66:327-40.
3. 3. Acevedo MA, Dillemuth FP, Flick AJ, Faldyn MJ, Elderd BD. Virulence-driven trade-offs in disease transmission: a meta-analysis. Evolution 2019;73:636-47.
4. 4. Allen AR, Ford T, Skuce RA. Does Mycobacterium tuberculosis var. bovis survival in the environment confound bovine tuberculosis control and eradication? A literature review. Vet Med Int 2021;2021:8812898.
5. 5. Sharifi Yazdi MK, Siadat SD, Khalifeh-Gholi M, Sharifi-Yazdi S, Fayaz-Bakhsh A, Saleh Safari M. Extraction and biological evaluation of external membrane vesicles of Brucella abortus as a candidate for brucellosis vaccine. J Surg Med 2020;4:540-4.
6. 6. Lorente-Leal V, Liandris E, Castellanos E, Bezos J, Domínguez L, de Juan L, et al. Validation of a real-time PCR for the detection of Mycobacterium tuberculosis complex members in bovine tissue samples. Front Vet Sci 2019;6:61.
7. 7. Pozo P, VanderWaal K, Grau A, de la Cruz ML, Nacar J, Bezos J, et al. Analysis of the cattle movement network and its association with the risk of bovine tuberculosis at the farm level in Castilla y Leon, Spain. Transbound Emerg Dis 2019;66:327-40.
8. 8. Trewby H, Wright D, Breadon EL, Lycett SJ, Mallon TR, McCormick C, et al. Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis. Epidemics 2016;14:26-35.

9. 9. Cowie CE, Hutchings MR, Barasona JA, Gortázar C, Vicente J, White PCL. Interactions between four species in a complex wildlife: livestock disease community: implications for Mycobacterium bovis maintenance and transmission. Eur J Wildl Res 2016;62:51-64.
10. 10. Rodriguez-Campos S, Navarro Y, Romero B, de Juan L, Bezos J, Mateos A, et al. Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group. J Clin Microbiol 2013;51:3658-65.
11. 11. Hlokwe TM, de Klerk-Lorist L-M, Michel AL. Wildlife on the move: a hidden tuberculosis threat to conservation areas and game farms through introduction of untested animals. J Wildl Dis 2016;52:837-43.
12. 12. Grange JM, Yates MD, de Kantor IN & World Health Organization. Emerging and other Communicable Diseases, Surveillance and Control. Guidelines for speciation within the Mycobacterium tuberculosis complex, 2nd ed. World Health Organization. 1996.
13. 13. Kronvall G, Stanford JL, Walsh GP. Studies of mycobacterial antigens, with special reference to Mycobacterium. Infect Immun 1976;13:1132-8.
14. 14. Matsuo K, Yasutomi Y. Mycobacterium bovis Bacille Calmette-Gu´erin as a Vaccine Vector for Global Infectious Disease Control. Tuberc Res Treat 2011;2011:574591.
15. 15. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Im Ce Bio 2004;82:488-96.
16. 16. Prados-Rosales R, Baena A, Martinez L, Luque-Garcia J, Kalscheuer R, Veeraraghavan U, et al. Mycobacteria release active membrane vesicles that modulate immune responses In a TLR2 dependent manner in mice. J Clin Invest 2011;121.:1471-83.
17. 17. Kuehn MJ, Kesty NC. Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev 2005;19:2645-55.
18. 18. Nicolson M. Biomedicine in the twentieth century: practices, policies and politics. Soc Hist Med 2009;22:193-4.
19. 19. Rath P, Huang C, Wang T, Wang T, Li H, Prados-Rosales R, et al. Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis. Proc Natl Acad Sci 2013;110:E4790-7.
20. 20. Lee J, Kim SH, Choi DS, Lee JS, Kim DK, Go G, et al. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis. Proteomics 2015;15:3331-7.
21. 21. Delbos V, Lemée L, Bénichou J, Berthelot G, Deghmane AE, Leroy JP, et al. Impact of MenBvac, an outer membrane vesicle (OMV) vaccine, on the meningococcal carriage. Vaccine 2013;31:4416-20.