Parasitology Research

, Volume 118, Issue 6, pp 1849–1863 | Cite as

Troponin 1 of human filarial parasite Brugia malayi: cDNA cloning, expression, purification, and its immunoprophylactic potential

  • Vikas Kushwaha
  • Prachi Tewari
  • Payal Mandal
  • Anurag Tripathi
  • P. Kalpana MurthyEmail author
Immunology and Host-Parasite Interactions - Original Paper


In the search for immunoprophylactics for the control of human lymphatic filariasis, we recently identified troponin 1 (Tn1) in Brugia malayi adult worms. The present study reports the cloning and expression of the B. malayi Tn1 (Tn1bm), its immunoprophylactic efficacy against B. malayi infection, and the immunological responses of the host. The Tn1bm gene was cloned (Acc no. JF912447) and expressed, and the purified recombinant Tn1bm (rTn1bm) presented a single ~ 27 kDa band. Parasite load in rTn1bm-immunized BALB/c mice challenged with B. malayi infective larvae (L3) was assessed. In rTn1bm-immunized animals, IgE, IgG, and IgG subclasses in the serum, cell proliferative response, Th1 and Th2 cytokine secretion (from splenocytes), and NO release (from peritoneal macrophages) were determined. Antibody-dependent cell-mediated cytotoxicity (ADCC) to L3 was assayed using rTn1bm-immune serum. The innate immune response markers MHC class-I, MHC class-II, TLR2, TLR4, and TLR6 expression in peritoneal macrophages and CD3+, CD4+, CD8+, and CD19+ in the splenocyte population were determined in Tn1bm-exposed cells from naïve mice. rTn1bm-immunized L3-challenged animals showed a 60% reduction in parasite burden. Immunization upregulated cellular proliferation, cytokine (IFN-γ, TNF-α, IL-1β, IL-4, IL-6, and IL-10) secretion, NO release, and antigen-specific IgG, IgG1, and IgG2b antibody levels. rTn1bm-immune serum killed > 65% of L3 in the ADCC assay. Increased MHC class-II, TLR2, and TLR6 expression and the relative CD4+ and CD19+ cell populations of naïve animal cells indicated the ability of rTn1bm to mobilize innate immune responses. This is the first report of the immunoprophylactic potential of rTn1bm against B. malayi.


Troponin 1 Brugia malayi Immunoprophylaxis Th1 and Th2 responses ADCC MHC class-I MHC class-II TLRs CD3+ CD4+ CD8+ CD19+ 



Thanks are due to the Council of Scientific and Industrial Research, New Delhi, India, for the Emeritus Scientist Project award (21(0963)/13/EMR-II; 29-10-2014) to PKM and for the Research Associateship award to PT and VK. The authors thank the Honorable Vice Chancellor, of Lucknow University, and Head of the Department of Zoology, Lucknow University, Lucknow, for providing laboratory facilities and the Director of CSIR-CDRI, Lucknow, for allowing the use of the experimental animal wing for animal experiments. The authors also thank the Director of the CSIR-Indian Institute of Toxicology Research, Lucknow, India, for allowing for part of the work to be carried out in his institute and Dr. Mukesh Srivastava, Biometry section, Division of Clinical and Experimental Medicine, CSIR-CDRI, Lucknow, for help with the statistical analyses.

Compliance with ethical standards

Ethical approval

The animal study protocol was approved by the National Laboratory Animal Centre (NLAC), CSIR-Central Drug Research Institute (CDRI), Lucknow, India, following the rules and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environments and Forests, Govt. of India, New Delhi, for use and handling of animals. The animals used in the study were housed in climatically controlled pathogen-free animal quarters (Temp.: 23 ± 2 °C; RH: 60%; and photoperiod: 12 h light–dark cycles) at NALC, CSIR-CDRI, Lucknow, India, and they were fed standard rodent maintenance diet (prepared in-house) and water ad libitum. The animal study protocol was approved (approval number: IAEC/2011/145 renewed dated 03/07/2014).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Artis D, Humphreys NE, Bancroft AJ, Rothwell NJ, Potten CS, Grencis RK (1999) Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection. J Exp Med 190:953–962PubMedPubMedCentralGoogle Scholar
  2. Babayan SA, Attout T, Harris A, Taylor MD, Le Goff L, Vuong PN, Rénia L, Allen JE, Bain O (2006) Vaccination against filarial nematodes with irradiated larvae provides long-term protection against the third larval stage but not against subsequent life cycle stages. Int J Parasitol 36:903–914PubMedGoogle Scholar
  3. Babu S, Nutman TB (2014) Immunology of lymphatic filariasis. Parasite Immunol 36:338–346PubMedPubMedCentralGoogle Scholar
  4. Babu S, Nutman TB (2012) Immunopathogenesis of lymphatic filarial disease. Semin Immunopathol 34:847–861PubMedPubMedCentralGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedPubMedCentralGoogle Scholar
  6. Chandrashekar R, Rao UR, Subrahmanyam D (1990) Antibody-mediated cytotoxic effects in vitro and in vivo of rat cells on infective larvae of Brugia malayi. Int J Parasitol 20:725–730PubMedGoogle Scholar
  7. Chauhan N, Khatri V, Banerjee P, Kalyanasundaram R (2018) Evaluating the vaccine potential of a tetravalent fusion protein (rBmHAXT) vaccine antigen against lymphatic filariasis in a mouse model. Front Immunol 9:1520. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen Q, Ghilardi N, Wang H, Baker T, Xie MH, Gurney A, Grewal IS, de Sauvage FJ (2000) Development of Th1-type immune responses requires the type I cytokine receptor TCCR. Nature 407:916–920PubMedGoogle Scholar
  9. Dakshinamoorthy G, Samykutty AK, Munirathinam G, Reddy MV, Kalyanasundaram R (2013) Multivalent fusion protein vaccine for lymphatic filariasis. Vaccine 31:1616–1622PubMedPubMedCentralGoogle Scholar
  10. Dakshinamoorthy G, von Gegerfelt A, Andersen H, Lewis M, Kalyanasundaram R (2014) Evaluation of a multivalent vaccine against lymphatic filariasis in rhesus macaque model. PLoS One 9:e112982PubMedPubMedCentralGoogle Scholar
  11. Dimock KA, Eberhard ML, Lammie PJ (1996) Th1-like antifilarial immune responses predominate in antigen-negative persons. Infect Immun 64:2962–2967PubMedPubMedCentralGoogle Scholar
  12. Dixit S, Gaur RL, Khan MA, Saxena JK, Murthy PS, Murthy PK (2004) Inflammatory antigens of Brugia malayi and their effect on rodent host Mastomys coucha. Parasite Immunol 26:397–407PubMedGoogle Scholar
  13. Dixit S, Gaur RL, Sahoo MK, Joseph SK, Murthy PS, Murthy PK (2006) Protection against L3 induced Brugia malayi infection in Mastomys coucha pre-immunized with BmAFII fraction of the filarial adult worm. Vaccine 24:5824–5831PubMedGoogle Scholar
  14. Doetze A, Satoguina J, Burchard G, Rau T, Loliger C, Fleischer B, Hoerauf A (2000) Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-beta but not by a T(h)1 to T(h)2 shift. Int Immunol 12:623–630PubMedGoogle Scholar
  15. Elson LH, Calvopina M, Paredes W, Araujo E, Bradley JE, Guderian RH, Nutman TB (1995) Immunity to onchocerciasis: putative immune persons produce a Th1-like response to Onchocerca volvulus. J Infect Dis 171:652–658PubMedGoogle Scholar
  16. Furchgott RF (1999) Endothelium-derived relaxing factor: discovery, early studies, and identification as nitric oxide. Biosci Rep 19:235–251PubMedGoogle Scholar
  17. Gomase VS, Chitlange NR, Changbhale SS, Kale KV (2013) Prediction of Brugia malayi antigenic peptides: candidates for synthetic vaccine design against lymphatic filariasis. Protein Pept Lett 20:864–887PubMedGoogle Scholar
  18. Gupta J, Pathak M, Misra S, Misra-Bhattacharya S (2015) Immunogenicity and protective efficacy of Brugia malayi heavy chain myosin as homologous DNA, protein and heterologous DNA/protein prime boost vaccine in rodent model. PLoS One 10:e0142548PubMedPubMedCentralGoogle Scholar
  19. Harrison RA, Wu Y, Egerton G, Bianco AE (1999) DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice. Vaccine 18:647–655PubMedGoogle Scholar
  20. Joseph SK, Sambanthamoorthy S, Dakshinamoorthy G, Munirathinam G, Ramaswamy K (2012) Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2. Vaccine 30:6477–6482PubMedPubMedCentralGoogle Scholar
  21. Joseph SK, Verma SK, Sahoo MK, Dixit S, Verma AK, Kushwaha V, Saxena K, Sharma A, Saxena JK, Murthy PK (2011) Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha. Acta Trop 120:191–205PubMedGoogle Scholar
  22. Kanai N, Min WP, Ichim TE, Wang H, Zhong R (2007) Th1/Th2 xenogenic antibody responses are associated with recipient dendritic cells. Microsurgery 27:234–239PubMedGoogle Scholar
  23. Khatri V, Chauhan N, Vishnoi K, von Gegerfelt A, Gittens C, Kalyanasundaram R (2018) Prospects of developing a prophylactic vaccine against human lymphatic filariasis - evaluation of protection in non-human primates. Int J Parasitol 48:773–783. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kushwaha V, Kumar V, Verma SK, Sharma R, Siddiqi MI, Murthy PK (2014) Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning, expression, purification, structural modeling and its potential as vaccine candidate for human filarial infection. Vaccine 32:1693–1699PubMedGoogle Scholar
  25. Li BW, Chandrashekar R, Alvarez RM, Liftis F, Weil GJ (1991) Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds. Mol Biochem Parasitol 49:315–323PubMedGoogle Scholar
  26. Li BW, Hoppe PE, Weil GJ (1995) Cloning of an early immunodominant filarial antigen: a member of the Brugia malayi myosin heavy chain gene family. Int J Parasitol 25:611–619PubMedGoogle Scholar
  27. Li BW, Zhang S, Curtis KC, Weil GJ (1999) Immune responses to Brugia malayi paramyosin in rodents after DNA vaccination. Vaccine 18:76–81PubMedGoogle Scholar
  28. Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, Allen JE (2004) Helminth parasites--masters of regulation. Immunol Rev 201:89–116PubMedPubMedCentralGoogle Scholar
  29. Molyneux DH, Savioli L, Engels D (2017) Neglected tropical diseases: progress towards addressing the chronic pandemic. Lancet 389:312–325PubMedGoogle Scholar
  30. Muhsin (2013) Role of interleukin-6 during infection with the filarial nematode Litomosoides sigmodontis. Universitäts-und Landesbibliothek, BonnGoogle Scholar
  31. Murthy PK, Dennis VA, Lasater BL, Philipp MT (2000) Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins. Infect Immun 68:6663–6669PubMedPubMedCentralGoogle Scholar
  32. Netea MG, Van De Veerdonk F, Verschueren I, Van Der Meer JW, Kullberg BJ (2007) Role of TLR1 and TLR6 in the host defense against disseminated candidiasis. FEMS Immunol Med Microbiol 52:118–123PubMedGoogle Scholar
  33. Rao RU, Samarasekera SD, Nagodavithana KC, Dassanayaka TDM, Punchihewa MW, Ranasinghe USB, Weil GJ (2017) Reassessment of areas with persistent lymphatic Filariasis nine years after cessation of mass drug administration in Sri Lanka. PLoS Negl Trop Dis 11:e0006066PubMedPubMedCentralGoogle Scholar
  34. Roy R, Tripathi A, Das M, Dwivedi PD (2011) Cytotoxicity and uptake of zinc oxide nanoparticles leading to enhanced inflammatory cytokines levels in murine macrophages: comparison with bulk zinc oxide. J Biomed Nanotechnol 7:110–111PubMedGoogle Scholar
  35. Roy R, Parashar V, Chauhan L, Shanker R, Das M, Tripathi A, Dwivedi PD (2014) Mechanism of uptake of ZnO nanoparticles and inflammatory responses in macrophages require PI3K mediated MAPKs signaling. Toxicol in Vitro 28:457–467PubMedGoogle Scholar
  36. Sahoo MK, Sisodia BS, Dixit S, Joseph SK, Gaur RL, Verma SK, Verma AK, Shasany AK, Dowle AA, Murthy PK (2009) Immunization with inflammatory proteome of Brugia malayi adult worm induces a Th1/Th2-immune response and confers protection against the filarial infection. Vaccine 27:4263–4271PubMedGoogle Scholar
  37. Sabesan S, Vanamail P, Raju K, Jambulingam P (2010) Lymphatic filariasis in India: epidemiology and control measures. J Postgrad Med 56:232–238PubMedGoogle Scholar
  38. Samykutty A, Dakshinamoorthy G, Kalyanasundaram R (2010) Multivalent vaccine for lymphatic Filariasis. Procedia Vaccinol 3:12–18PubMedPubMedCentralGoogle Scholar
  39. Snapper C, Finkelman F (1999) In: Paul WE (ed) Fundamental Immunology. Lippincott, PhiladelphiaGoogle Scholar
  40. Taylor MJ, Cross HF, Mohammed AA, Trees AJ, Bianco AE (1996) Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell-free culture and from IFN gamma-activated macrophages. Parasitology 112(Pt 3):315–322PubMedGoogle Scholar
  41. Taylor MJ, Hoerauf A, Bockarie M (2010) Lymphatic filariasis and onchocerciasis. Lancet 376:1175–1185PubMedGoogle Scholar
  42. Tewari P, Mandal P, Roy R, Asthana S, Dwivedi PD, Das M, Tripathi A (2017) A novel function of TLR4 in mediating the immunomodulatory effect of Benzanthrone, an environmental pollutant. Toxicol Lett 276:69–84PubMedGoogle Scholar
  43. Thirugnanam S, Pandiaraja P, Ramaswamy K, Murugan V, Gnanasekar M, Nandakumar K, Reddy MV, Kaliraj P (2007) Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model. Exp Parasitol 116:483–491PubMedPubMedCentralGoogle Scholar
  44. Thomas GR, McCrossan M, Selkirk ME (1997) Cytostatic and cytotoxic effects of activated macrophages and nitric oxide donors on Brugia malayi. Infect Immun 65:2732–2739PubMedPubMedCentralGoogle Scholar
  45. Vanam U, Pandey V, Prabhu PR, Dakshinamurthy G, Reddy MV, Kaliraj P (2009) Evaluation of immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) in single and multiple antigen vaccination with BmALT-2 and BmTPX for human lymphatic filariasis. Am J Trop Med Hyg 80:319–324PubMedGoogle Scholar
  46. Veerapathran A, Dakshinamoorthy G, Gnanasekar M, Reddy MV, Kalyanasundaram R (2009) Evaluation of Wuchereria bancrofti GST as a vaccine candidate for lymphatic filariasis. PLoS Negl Trop Dis 3:e457PubMedPubMedCentralGoogle Scholar
  47. Verma SK, Arora A, Murthy PK (2017) Recombinant Calponin of human filariid Brugia malayi: secondary structure and immunoprophylactic potential. Vaccine 35:5201–5208PubMedGoogle Scholar
  48. Verma SK, Joseph SK, Verma R, Kushwaha V, Parmar N, Yadav PK, Thota JR, Kar S, Murthy PK (2015) Protection against filarial infection by 45-49 kDa molecules of Brugia malayi via IFN-gamma-mediated iNOS induction. Vaccine 33:527–534PubMedGoogle Scholar
  49. Volkmann L, Bain O, Saeftel M, Specht S, Fischer K, Brombacher F, Matthaei KI, Hoerauf A (2003) Murine filariasis: interleukin 4 and interleukin 5 lead to containment of different worm developmental stages. Med Microbiol Immunol 192:23–31PubMedGoogle Scholar
  50. WHO (2014) Global Programme to eliminate lymphatic Filariasis: progress report. WHO Wkly Epidemiol Rec 90:489–504Google Scholar
  51. WHO (2017) Alternative mass drug administration regimens to eliminate lymphatic filariasis. World Health Organization, Geneva Licence: CC BY-NC-SA 3.0 IGOGoogle Scholar
  52. WHO (2018) Three more countries eliminate lymphatic filariasis. World Health Organization.
  53. Yadav A, Kumar A, Tripathi A, Das M (2013) Sunset yellow FCF, a permitted food dye, alters functional responses of splenocytes at non-cytotoxic dose. Toxicol Lett 217:197–204PubMedGoogle Scholar
  54. Yadav A, Kumar A, Dwivedi PD, Tripathi A, Das M (2012) In vitro studies on immunotoxic potential of Orange II in splenocytes. Toxicol Lett 208:239–245PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Vikas Kushwaha
    • 1
    • 2
  • Prachi Tewari
    • 1
  • Payal Mandal
    • 3
  • Anurag Tripathi
    • 3
  • P. Kalpana Murthy
    • 1
    Email author
  1. 1.Department of ZoologyUniversity of LucknowLucknowIndia
  2. 2.Postdoctoral Fellow, Zoology DepartmentPanjab UniversityChandigarhIndia
  3. 3.Food Toxicology Lab, Food, Drug and Chemical Toxicology GroupCSIR-Indian Institute of Toxicology ResearchLucknowIndia

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