|
|
|
| Immune responses induced by mucosal immunization with Mycobacterium tuberculosis EsxV subunit vaccine |
| BAI Lu1,2, NING Huan-huan2, KANG Jian2, LIANG Xuan3, XIE Yan-ling1,2, PENG Yu-jun2, ZHANG Jing-yao2, LU Yan-zhi2, BAI Yin-lan2 |
1. School of Life Sciences,Yan’an University,Yan’an 716000,China;
2. Department of Microbiology and Pathogen Biology, Basic Medical School, Air Force Medical University,Xi’an 710032,China;
3. College of Life Sciences,Northwest University,Xi’an 710069, China; |
|
|
|
|
Abstract This study investigated the immune responses induced by mucosal immunization with Mycobacterium tuberculosis antigen Rv3619c (EsxV) in mice. The esxV gene was amplified by PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant E. coli were induced to express the EsxV protein. Protein expression was identified by SDS-PAGE electrophoresis and western blotting. The recombinant EsxV protein was purified by affinity chromatography. BALB/c mice were immunized with EsxV and/or cyclic diadenosine monophosphate (c-di-AMP) adjuvant via the nasal mucosa. Specific antibody levels and subclasses were detected with ELISA. Splenocyte proliferation was detected with MTS assays. Cytokine expression levels in the spleen and lung were detected by qRT-PCR. Cytokines secreted by splenocytes were detected by ELISA. The prokaryotic expression vector pET28a (+)-esxV was successfully constructed. The recombinant EsxV protein was induced and expressed, then purified by affinity chromatography. EsxV induced significant humoral immune responses, but modest cellular immune responses, through mucosal immunization. Mucosal inoculation of EsxV with c-di-AMP induced an increase in specific IgG levels, EsxV protein-specific splenocyte proliferation, and spleen and lung IFN-γ transcription. c-di-AMP significantly enhanced EsxV-specific secretion of the IFN-γ, IL-2, IL-10 and IL-17 cytokines, but did not induce the expression of the inflammatory factors TNF-α and IL-6. The subunit vaccine constructed by EsxV and c-di-AMP induced specific humoral and cellular immune responses, which should be further used in the development of novel TB subunit vaccines.
|
|
Received: 21 January 2022
|
|
|
| Fund:Supported by the National Science Project of Major Infectious Diseases during the 13th Five-Year Plan (No.2018ZX10302302002004),the National Natural Science Foundation of China (No.81971560, No.81671638), the and Key Research and Development Program of Shaanxi Province (No.2019ZDLSF02-04) |
|
Corresponding Authors:
Bai Yin-lan, Email: yinlanbai@fmmuedu.cn; Ning Huan-huan, Email: ninghuanhuan2014@163.com
|
|
|
|
[1] WHO. Global tuberculosis report 2020[R]. Geneva: WHO, 2021.
[2] Abdallah AM, Behr MA.Evolution and strain variation in BCG[J]. Adv Exp Med Biol, 2017, 1019(8):155-169. DOI:10.1007/978-3-319-64371-7_8
[3] Nieuwenhuizen NE, Kaufmann S.Next-generation vaccines based on Bacille Calmette-Guérin[J]. Front Immunol, 2018, 9(3):121. DOI:10.3389/fimmu.2018.00121
[4] Zhang W, Zhang Y, Zheng H, et al.Genome sequencing and analysis of BCG vaccine strains[J]. PLoS One, 2013, 8(8):e71243. DOI:10.1371/journal.pone.0071243
[5] Ruhwald M, de Thurah L, Kuchaka D, et al. Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6[J]. Sci Rep, 2017, 7(1):45969. DOI:10.1038/srep45969
[6] Ates LS, Brosch R.Discovery of the type VII ESX-1 secretion needle[J]. Mol Microbiol, 2017, 103(1):7-12. DOI:10.1111/mmi.13579
[7] Bitter W, Houben EN, Bottai D, et al.Systematic genetic nomenclature for type VII secretion systems[J]. PLoS Pathog, 2009, 5(10):e1000507. DOI:10.1371/journal.ppat.1000507
[8] Safar HA, Mustafa AS, Amoudy HA, et al.The effect of adjuvants and delivery systems on Th1, Th2, Th17 and Treg cytokine responses in mice immunized with Mycobacterium tuberculosis-specific proteins[J]. PLoS One, 2020, 15(2):e0228381. DOI:10.1371/journal.pone.0228381
[9] Hanif SN, Al-Attiyah R, Mustafa AS.Molecular cloning, expression, purification and immunological characterization of three low-molecular weight proteins encoded by genes in genomic regions of difference of Mycobacterium tuberculosis[J]. Scand J Immunol, 2010, 71(5):353-361. DOI:10.1111/j.1365-3083.2010.02388.x
[10] Ansari MA, Zubair S, Mahmood A, et al.RD antigen based nanovaccine imparts long term protection by inducing memory response against experimental murine tuberculosis[J]. PLoS One, 2011, 6(8):e22889. DOI:10.1371/journal.pone.0022889
[11] Hanif SN, Al-Attiyah R, Mustafa AS.Species-specific antigenic Mycobacterium tuberculosis proteins tested by delayed-type hypersensitivity response[J]. Int J Tuberc Lung Dis, 2010, 14(4):489-494.
[12] Chai Q, Lu Z, Liu CH.Host defense mechanisms against Mycobacterium tuberculosis[J]. Cell Mol Life Sci, 2020, 77(10):1859-1878. DOI:10.1007/s00018-019-03353-5
[13] Vasiyani H, Shinde A, Roy M, et al.The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells[J]. Apoptosis, 2021, 26(5/6):293-306. DOI:10.1007/s10495-021-01669-x
[14] Abdul-Sater AA, Tattoli I, Jin L, et al.Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome[J]. EMBO Rep, 2013, 14(10):900-906. DOI:10.1038/embor.2013.132
[15] 宁唤唤, 徐志凯, 柏银兰. c-di-AMP在细菌感染与免疫中的作用[J]. 微生物学通报, 2019, 46(9):2362-2369. DOI:10.13344/j.microbiol.china.180785
[16] Ebensen T, Libanova R, Schulze K, et al.Bis-(3',5')-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant[J]. Vaccine, 2011, 29(9):5210-5220. DOI:10.1016/j.vaccine.2011.05.026
[17] Ning H, Zhang W, Kang J, et al.Subunit vaccine ESAT-6:c-di-AMP delivered by intranasal route elicits immune responses and protects against Mycobacterium tuberculosis infection[J]. Front Cell Infect Microbiol, 2021, 11(1):647220. DOI:10.3389/fcimb.2021.647220
[18] Baldwin SL, Bertholet S, Kahn M, et al.Intradermal immunization improves protective efficacy of a novel TB vaccine candidate[J]. Vaccine, 2009, 27(23):3063-3071. DOI:10.1016/j.vaccine.2009.03.018
[19] Hart BE, Lee S.Overexpression of a Mycobacterium ulcerans Ag85B-EsxH fusion protein in recombinant BCG improves experimental buruli ulcer vaccine efficacy[J]. PLoS Negl Trop Dis, 2016, 10(12):e0005229. DOI:10.1371/journal.pntd.0005229
[20] Safar HA, El-Hashim AZ, Amoudy H, et al.Mycobacterium tuberculosis-specific antigen Rv3619c effectively alleviates allergic asthma in mice[J]. Front Pharmacol, 2020, 11(3):532199. DOI:10.3389/fphar.2020.532199
[21] Lu Y, Kang J, Ning H, et al.Immunological characteristics of Mycobacterium tuberculosis subunit vaccines immunized through different routes[J]. Microb Pathog, 2018, 125(10):84-92. DOI:10.1016/j.micpath.2018.09.009
[22] Pulendran B, Arunachalam PS, O’Hagan DT, et al. Emerging concepts in the science of vaccine adjuvants[J]. Nat Rev Drug Discov, 2021, 20(6):454-475. DOI:10.1038/s41573-021-00163-y
[23] Ashhurst AS, McDonald DM, Hanna CC, et al. Mucosal vaccination with a self-adjuvanted lipopeptide is immunogenic and protective against Mycobacterium tuberculosis[J]. J Med Chem, 2019, 62(17):8080-8089. DOI:10.1021/acs.jmedchem.9b00832
[24] Sanchez MV, Ebensen T, Schulze K, et al.Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge[J]. PLoS One, 2014, 9(8):e104824. DOI:10.1371/journal.pone.0104824
[25] Ning H, Wang L, Zhou J, et al.Recombinant BCG with bacterial signaling molecule cyclic di-AMP as endogenous adjuvant induces elevated immune responses after Mycobacterium tuberculosis infection[J]. Front Immunol, 2019, 10(8):1519. DOI:10.3389/fimmu.2019.01519
[26] Devaux L, Kaminski PA, Trieu-Cuot P, et al.Cyclic di-AMP in host-pathogen interactions[J]. Curr Opin Microbiol, 2018, 41(11):21-28. DOI:10.1016/j.mib.2017.11.007 |
|
|
|
2022, Vol. 38 
