Original Article

Cellular Immunity in Mice Vaccinated with Recombinant Phospholipase D Toxoid of Hemiscorpius lepturus Scorpion


Background: Hemiscorpius lepturus is one of the most dangerous scorpions in Iran and the world. Numerous studies have been conducted on phospholipases, especially phospholipase D, in this scorpion’s venom, and the results have shown this protein to be the main cause of death. Therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. The present study was conducted to assess the non-toxicity of this toxoid and the safety of the vaccine candidate in BALB/c mice.

Methods: The production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using ELISpot assay 28 days following immunization with rPLD toxoid.

Results: The unpaired t-test results showed a significant increase in the concentration of IFN-γ cytokine in the vaccinat­ed mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant differ­ence was observed in the levels of IL-4 (P= 0.16) despite an increase in this cytokine. The in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the LD100 of the venom. Moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin.

Conclusion: As a vaccine, the toxoid has the potential to increase the Th1 cytokine response and, subsequently, in­crease acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the ven­om of Hemiscorpius lepturus.

1. Jalali A, Rahimi F (2014) Epidemiological review of scorpion envenomation in Iran. J Iran Pharm Res. 13(3): 743–756.
2. Latifi M, Tabatabai M (1979) Immunological studies on Iranian scorpion venom and antiserum. J Toxicon. 17(6): 617–621.
3. Shahbazzadeh D, Srairi‐Abid N, Feng W, Ram N, Borchani L, Ronjat M, Akbari A, Pessah IN, De Waard M, El Ayeb M (2007) Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine‐sensitive Ca2+ channels. J Biochem. 404(1): 89–96.
4. Seyedian R, Jalali A, Babaee MH, Pipelzadeh MH, Rezaee SA (2012) A biodistri-bution study of Hemiscorpius lepturus scorpion venom and available polyclonal antivenom in rats. J Venom Anim Toxins Incl Trop Dis. 18(1): 375–383.
5. Pipelzadeh MH, Dezfulian AR, Jalali MT, Mansouri AK (2006) In vitro and in vivo studies on some toxic effects of the venom from Hemiscorpious lepturus scorpion. J Toxicon. 48(1): 93–103.
6. Jalali A, Pipelzadeh MH, Sayedian R, Rowan EG (2010) A review of epidemiological clinical and in vitro physiological studies of envenomation by the scorpion Hemiscorpius lepturus (Hemiscorpiidae) in Iran. J Iran Pharm Res. 55(2): 173–179.
7. Pipelzadeh MH, Jalali A, Taraz M, Pour-abbas R, Zaremirakabadi A (2007) An epidemiological and a clinical study on scorpionism by the Iranian scorpion Hemiscorpius lepturus. J Toxicon. 50(7): 984–992.
8. Jalali A, Pipelzadeh MH, Taraz M, Khodadadi A, Makvandi M, Rowan EG (2011) Serum TNF- a levels reflect the clinical severity of envenomation following a Hemiscorpius lepturus sting. Eur Cytokine Netw. 22(1): 5–10.
9. Borchani L, Sassi A, Shahbazzadeh D, Strub JM, Tounsi H, Boubaker MS, Akbari A, Van Dorsselaer A, El Ayeb M (2011) Heminecrolysin the first hemo-lytic der¬monecrotic Toxin purified Scorpion venom. J Toxicon. 58(1): 130–139.
10. Safari Foroushani N, Modarressi MH, Behdani M, Shahbazzadeh D, Torabi E, Pooshang Bagheri K (2018) Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/ c mice. J Toxicon. 152(15): 30–36.
11. Torabi E, Shahbazzadeh D, Behdani M, Hosseininejad M, Moazzami R, Sabatier JM, Khalaj V, Bagheri KP (2017) Char-acteristics and lethality of a novel recombinant dermonecrotic venom phospholipase D from Hemiscorpius lep¬turus. J Toxins. 9(3): 102–119.
12. Tambourgi DV, Magnoli, FC, Van den Berg CW, Morgan BP, De A raujo PS, Alves EW, Da Silva WD (1998) Sphin-gomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis. Biochem Biophys Res Commun. 251(1): 366–373.
13. Mortimer N, Nathan T (2015) Parasitoid wasp venom SERCA regulates drosophila calcium levels and inhibits cellular immunity. Proceeding of the National Acadamy of Sciences. 110(23): 9427–9432.
14. Metz BW, Jiskoot WE, Hennink DJA, Crommelin, Kersten GFA (2003) Physicochemical and immunochemical techniques predict the quality of diphtheria toxoid vaccines. Vaccine. 22(1): 156–167.
15. Farizo KM (2007) Diphtheria and Tetanus toxoids absorbed FDA. Iran Academy Press. 8(2): 33–34.
16. Erlich P (1897) Die werthbemessung des diphtheria heil-serum und deren theoretische grundlagen. Klin Jahrb Jena. 6: 299–326.
17. Moloney PG (1926) The preparation and testing of Diphtheria toxoid (Anatoxin-Ramon). Am J Public Health. 16(12): 1208–1210.
18. Lonard AS (2009) Botulism and vaccines for its prevention. J Vaccine. 27(5): 33–39.
19. Weinberg M, Goy P (1924) Recherches sur la toxin botulinque. Compt Rend Soc Biol. 90: 269–271.
20. El-Maghraby AS, Abd El-hakim MA (2020) Sensitivity of different methods used in determination of formalin resi-dues in inactivated veterinary vaccines. Glob Vet. 14(4): 546–552.
21. Teakston RDG, Warrell DA, Griffiths E (2003) Report of a WHO 574 workshop on the standardization and control of antivenoms. J Toxicon. 41(5): 541–557.
22. Ramon G (1924) Sur la toxin et sur l, anatoxine diphteriques. Ann. Inst. Pasteur. 38: 1–10.
23. Glenny AT, Hopkins, BE (1923) Diphtheria toxoid as an immunizing agent. Br Exp Pathol J. 4(5): 283–288.
24. Someya S, Murata R, Sawai Y, Kondo H, Ishii A (1972) Active immunization of man with toxoid of trimeresurus flavoviridis (Habu) venom. Jpn Med Sci Biol J. 25(1): 47–51.
25. Kondo S, Sadahiro S, Yamauchi K, Kon-do H, Murata R (1971) Preparation and standardization of toxoid from the venom of Trimeresurus flavoviridis. Jpn Med Sci Biol J. 24(5): 281–294.
26. Piersma Sytse J, Leenaars Marlies PPAM, Guzylack-Piriou Laurence, Summerfield Artur, Hendriksen Coenraad FM, Mc-Cullough Ken C (2006) An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria. Vaccine. 24(2): 3076–3083.
27. Bizzini B, Raynaud M (1974) Detoxification of protein toxins by formol: Supposed mechanisms and new developments. Biochimie. 56(2): 297–303.
28. Borchani L, Sassi A, Yehlefa R, Safra E, Ayeb M (2011) Heminecrolysin a potential immunogen for monospecific anti-venom production against Hemiscorpius lepturus scorpion. J Toxicon. 58(8): 681–688.
29. Alvarenga LM, Martins MS, Moura JF, Kalapothakis E, Oliveira JC, Mangili OC, Granier C, Chávez-Olórtegui C (2003) Production of monoclonal anti-bodies capable of neutralizing dermonecrotic activity of Loxoceles intermedia spider venom and their use in a specific immunometric assay. J Toxicon. 42(7): 725–731.
30. Dragulev B, Bao Y, Ramos-Cerrillo B, Vazquez H, Olvera A, Stock R, Algaron A, Fox JW (2007) Upregulation of IL-6, IL-8, CXCL1, and CXCL2 dominates gene expression in human fibroblast cells exposed to Loxosceles reclusa sphingo-myelinase D: insights into spider venom dermonecrosis. J Invest Dermatol. 127(5): 1264–1266.
31. Khosravi A, Motevalibashi M, Babaahmadi E, Davodian A, Jafarzadeh L, Manian Azodi M (2012) The effect of car smoke on the level of TNF-α, INF-γ and IL-2 in rat serum. Res Med. 36(5): 99–131.
32. Kashiwada M, Deborah M, McKeag L, Murray K, Schröder AJ, Canfield SM, Geri Traver G, Rothman PB, Coffman RL (2009) IL-4-induced transcription factor NFIL3/E4BP4 controls IgE class switching. Proc Natl Acad Sci USA. 107(2): 821–826.
33. Saba MA, Akbari H, Banihashemian SM, Jazayeri H, Talaei SA, Banihashemian SH, Ghaforian S (2013) Relationship between serum levels of IL-4 and IgE with disease severity in allergic asthma. Feyz J Kashan Univ Med Sci. 17(4): 366–372.
34. Haas H, Falcone F, Holland M, Schramm G, Haisch K, Gibbs B (2000) Early interleukin–4: its role in the switch to-wards a th2 response and IgE mediated allergy. Int Arch Allergy Immunol. 119(2): 86–94.
35. Fietta P, Delsante G (2009) The effector T helper cell triade. Riv Biol. 102(1): 61–74.
36. Holgate S, Polosa R (2008) Treatment strategies for allergy and asthma. Nat Rev Immunol. 8(3): 218–229.
37. Paul WE, Zhu J (2010) How are TH2-type immune responses initiated and ampli-fied? Nat Rev Immunol. 10(4): 225–235.
38. Teixeira L, Fonseca B, Barboza B, Viola J (2005) The role of interferon-γ on immune and allergic responses. Mem Inst Oswaldo Cruz. 1(1): 137–144.
39. Torre D, Speranza F, Giola M, Matteelli A, Tambini R, Biondi G (2002) Role of Th1 and Th2 cytokines in immune response to uncomplicated Plasmodium Falciparum Malaria. Clin Diagn Lab Immunol. 9(2): 348–351.
40. Rosloniec EF, Latham K, Guedez YB (2002) Paradoxical roles of INF-γ in models of Th1-mediated auto immunity. Arthritis Res. 4(6): 333–336.
41. Boulet S, Ndongala M, Peretz Y, Boisvert MP, Boulassel MR, Tremblay C, Routy JP, Sekaly RP, Bernard NF (2007) A Dual Color ELISPOT Method for the Simultaneous Detection of IL-2 and IFN-γ HIV-Specific Immune Responses J Immunol Methods. 320(1): 18–29.
42. Borchani L, Sassi A, Gharsa HB, Safra I, shahbazzadeh D, Lasfar ZB, El-Ayeb M (2013) The pathological effects of Heminecrolysin, a dermonecrotic toxin from Hemiscorpius lepturus scorpion venom are mediated through its lyso-phospholipase D activity. J Toxicon. 68: 30–39.
43. Adib-Conquy M, Cavaillon JM (2009) Compensatory anti-inflammatory re-sponse syndrome. Thromb. Haemost. 101(1): 36–47.
44. Homayoon M, Tahamtan Y, Kargar M (2020) The comparison detection of cy-tokines (IL-6 and IL-12) from spleen cells and serums in BALB/c mice after immunization with killed P. multocida vaccines co formulated with bacterial DNAs as adjuant. Arch Clin Microbiol. 11(1): 1105–1109.
45. Roberts MT, B-Stober C, Mckezie A, Blackwell JM (2005) Interleukin-4 (IL-4) and IL-10 collude in Vaccine failure for novel exacerbatory antigens in murine leshmania major infection. Infect Immun. 73(11): 7620–7628.
IssueVol 16 No 1 (2022) QRcode
SectionOriginal Article
DOI https://doi.org/10.18502/jad.v16i1.11187
Hemiscorpius lepturus Toxoid Interferon-gamma (IFN-γ) Interleukin 4 (IL-4) Cellular im¬munity

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Safari-Foroushani N, Modarressi MH, Bagheri KP, Behdani M, Shahbazzadeh D. Cellular Immunity in Mice Vaccinated with Recombinant Phospholipase D Toxoid of Hemiscorpius lepturus Scorpion. J Arthropod Borne Dis. 2022;16(1):1-12.