J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 1 http://jad.tums.ac.ir Published Online: March 31, 2022 Original Article Cellular Immunity in Mice Vaccinated with Recombinant Phospholipase D Toxoid of Hemiscorpius lepturus Scorpion Narges Safari-Foroushani1, Mohammad Hossein Modarressi2, Kamran Pooshang Bagheri3, Mahdi Behdani3, *Delavar Shahbazzadeh3 1Islamic Azad University, Science and Research Branch, Tehran, Iran 2Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3Venom and Bio therapeutics Molecules Lab, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran *Corresponding author: Dr Delavar Shahbazzadeh, Email: shahbazzadeh@yahoo.com (Received 15 June 2021; accepted 22 Sep 2021) Abstract 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 vaccinat- ed mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant differ - ence 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, in- crease acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the ven- om of Hemiscorpius lepturus. Keywords: Hemiscorpius lepturus; Toxoid; Interferon-gamma (IFN-γ); Interleukin 4 (IL-4); Cellular immunity Introduction Scorpion sting is one of the main public health problems in the world (1). The six dan- gerous scorpion species in Iran include Hemiscorpius lepturus (Scorpionidae), An- droctonus crassicauda, Hottentotta saulcyi, Hottentotta schach, Odontobuthus doriae, and Mesobuthus eupeus (Buthidae) and among them, H. lepturus is considered the most dan- gerous (2). Hemiscorpius lepturus, or Gadim scorpion (its local name), is blamed for 50% of all scorpion stings nationwide, while it is responsible for 89% of the reported deaths (3). In addition to adults, this scorpion’s sting is highly deadly in sensitive groups, particu- larly children, and causes 60 times more deaths than other scorpion species in Iran (3, 4). Sting- ing by this scorpion causes clinical signs and symptoms not previously described for other scorpion species (5). Unlike other scorpions, whose venom causes neurotoxic signs and symp- toms, this scorpion’s venom produces cytotox- ic signs and symptoms with systemic effects such as dermonecrosis, inflammation, arterial leak, and renal failure (6, 7). These signs and symptoms are attributed to the venom’s enzy- matic compounds (5, 8). Recently, a 32-kDa Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:shahbazzadeh@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 2 http://jad.tums.ac.ir Published Online: March 31, 2022 protein, called phospholipase D, has been iden- tified in H. lepturus scorpion’s venom (9). It is a dermonecrotic toxin that causes signs and symptoms fully similar to those of the com- plete venom of H. lepturus and is the most important molecule in the scorpion venom (10, 11). These signs and symptoms are fully similar to the main clinical signs and symptoms of the brown Loxosceles intermedia sting, which is responsible for many signs and symptoms and deaths in humans (9, 12). The treatment currently used for Gadim venom in Iran is the equine polyvalent antiserum against the six venomous scorpions in the country but there is still no standard protocol for the manage- ment of people afflicted with the venom of this scorpion (2). Moreover, there are reports of anaphylactic shock and death in the people stung by this scorpion, especially young chil- dren (7). The use of antiserum is particularly effective against pathogens that can activate the immune system in the unstimulated state (13). Based on a previous study, we assumed that toxoid (the first non-toxic form of phos- pholipase D toxin produced using formalin) would be able to induce active immunity in BALB/c mice and, consequently, could be used as a vaccine (10). This hypothesis has previ- ously been confirmed by using toxoids as vac- cines for the prevention of diphtheria (14, 15, 16), tetanus (15, 17), and botulism (18, 19). Accordingly, the present study was conducted to test the non-toxicity of the toxoid and as- sess the efficiency of the immunogenicity of the produced toxoid for its use as a vaccine. This study assesses the vaccine by inves- tigating inflammatory and anti-inflammatory cytokines in the immunized mice. The authors believe that if it is demonstrated that pro in- flammatory cytokines are significantly increased in the present study, the results were confirmed by repeated tests, it can then be concluded that the present vaccine increases cellular immun- ity in the mice and can also cause acquired im- munity of the cellular type. Materials and Methods Expression and purification of recombinant phospholipase D The phospholipase D (PLD) gene of the Iranian venomous scorpion H. lepturus was expressed and the product purified based on a previous design (10, 11). Briefly, pET-22b plas- mid was used in gene expression. This gene has the gene bank accession number: KY287766. E. coli BL21 bacterium was transformed using the PLD gene. The transformed bacterium was cultured in LB medium containing ampicillin at 37 °C until optical absorption at 600nm wavelength reached 0.6. The induction was carried out using 0.1mM isopropyl beta-d-1- thiogalactosidase (IPTG) (Thermo Fisher Sci- entific Co. Waltham, MA, USA). The cells were kept at 30 °C for 3.5 hours, then centrifuged at 10000×g for 10 minutes at 4 °C and suspend- ed in the lysis buffer. To purify the recombi- nant protein, bacterial cells were first dis- solved in 1mL of the lysis buffer per 0.1g of the precipitate and placed on ice for 30 minutes. Then, the host cell membrane was disintegrat- ed by sonication (Hielscher co. Teltow, Germa- ny) over 15 minutes at 30% amplitude with a cycle frequency of 30. Next, the lysed cells were centrifuged, and the supernatant contain- ing the soluble protein was purified by affinity chromatography with Ni-NTA agarose column (Qiagen co. Hilden, Germany). The recombi- nant protein was dialyzed using PBS and ana- lyzed by SDS-PAGE (20). Toxoid production from recombinant phos- pholipase D To produce the toxoid, detoxification, re- combinant PLD was first diluted in sterile PBS and then added to a 10kD dialysis sac (10). The protein was exposed to 0.25 methanol forma- lin for 10 days at room temperature. The sam- ples were gently dialyzed with PBS buffer at 4 °C, over 24 hours (the buffer was changed twice during this procedure) and filtered through a 0.22μm membrane to remove excess residue. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 3 http://jad.tums.ac.ir Published Online: March 31, 2022 The toxoid concentration was determined using the BCA kit following the manufacturer’s in- structions (i NtRoN Biotechnology Co. Seoul, Korea). The residual formalin test was per- formed to ensure the absence of formalin in the toxoid samples (20). Animals The present study had a code of ethics from Pasteur Institute of Iran for using animals in research (No: PII.REC.1394.38). Two 3±1 months-old New Zealand white rabbits weigh- ing 2kg each, and one hundred- twenty 6±1 months-old female BALB/c mice weighing 20– 25g each were procured from the Pasteur In- stitute of Iran. Before the experiments, the an- imals were kept in standard laboratory condi- tions at a temperature of 22–25 °C with 12 hourly alternating light/dark cycles and rela- tive humidity of 40–60% with free access to food and water for one week, after which time, the mice were randomly divided into 2 groups (60 mice per group). Toxoid toxicity test The toxoid toxicity was assessed using der- monecrotic activity so that 3μg of the toxoid was brought to a volume of 100μl of sterile PBS and injected subcutaneously into a rabbit with the hair on its back shaven. The presence of inflammation and necrosis was assessed af- ter 4, 8, 16, 24, 48, 72, and 96 hours, and the injection site diameter was measured with a cal- iper. PBS and rPLD1 toxin were used as nega- tive and positive controls. Toxoid immunogenicity in mice One hundred-twenty female BALB/c mice were assigned into two sixty mice groups. The first group was chosen for vaccinating with toxoid- Freund’s adjuvant and the second group for toxoid- Alum adjuvant as described in de- tail below. Immunization by toxoid-Alum adjuvant In this series of experiments each of sixty other mice received 80μg of toxoid. This amount of toxoid was mixed with 100μl of sterile PBS and then mixed with 100μl of Alum adjuvant (Thermo Fisher Scientific Co. Waltham, MA, USA.) followed by stirring until a homogenous solution was obtained. 200μl of this solution was subcutaneously injected to each mouse on days 1, 14, 28, 42, 56 and 70. Here also, 6 mice that only received sterile PBS-alum under the same conditions were considered as negative controls. Immunization by toxoid-Freund’s adjuvant Based on previous studies (Safari et al. 2018), mice were immunized in 10 groups with two-weeks intervals on days 15, 29, 43, 57 and 71. The first injection was administered using complete Freund’s adjuvant and the following injections using incomplete Freund’s adjuvant. 80μg of toxoid was homogenized with 100μl of Freund’s adjuvant at 4 °C for 4 hours, so that the antigen could completely mix with the adjuvant, diluted in PBS to 200μl, and inject- ed subcutaneously. The control group was in- jected with 100μl of PBS and 100μl of Freund’s adjuvant. The animals were examined in terms of health and weight in all stages of the ex- periment. Here also, 6 mice that only received sterile PBS-Freund under the same conditions were considered as negative controls. Challenging tests in immunized mice The mice immunized by toxoid were chal- lenged one week after the last injection. In the case of the mice which have been immunized by toxoid- Freund’s adjuvant, the first group of them was divided into five groups of six mice. Each mouse in group 1 received 10 (37µg), group 2; 20 (74µg), group 3; 50 (185µg), group 4; 100 (370µg) and group 5; 200 (740µg) LD 100 of the rPLD. Also, the other 30 mice from these mice were assigned to five groups of six mice and received different doses of crude ven- om, as group 1 received 1 (100µg), group 2= 2 (200µg), group 3= 5 (500µg), group 4= 7 (700 µg) and the group 5= 10 (1000µg) LD 100 of the crude venom. The other 60 mice that were immunized by toxoid-alum adjuvant received http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 4 http://jad.tums.ac.ir Published Online: March 31, 2022 the same doses of rPLD and crude venom re- spectively as mentioned above. All the injec- tions were done intraperitoneal, and mice were monitored for 2 weeks. Those that survived were regarded as the survivors and non-im- munized mice were regarded as the negative controls. Isolation of splenocytes from mouse spleen The vaccinated mice were put down by spi- nalization four weeks after the last injection. Then, they were submerged in 70% alcohol for a few seconds. The spleen was removed from each mouse by opening the peritoneum in fully sterile conditions under a microbiological hood and was completely cleaned of excess matter and adipose tissue. The spleen was quickly transferred to a small sterile petri dish with 5 ml of RPMI culture medium containing 0.5% FBS and penicillin and streptomycin antibiot- ics. The spleen cells were suspended using sev- eral injections of RPMI culture medium. The resulting suspension was centrifuged at 600×g and 4 °C for 10 minutes and Tris-NH4Cl was then added to it. The cell mixture was centri- fuged after inverting it several times. The cell deposit was suspended in the RPMI culture me- dium containing antibiotics. Cells were count- ed using a hemocytometer slide, and the per- centage of the surviving cells was determined. The resulting cell supernatant was kept at -20 °C until analysis. Counting was carried out using the following formula: Number of cells counted in 4 squares in the corners of the Ne- oBar slide × 2 (dilution coefficient) × 104 (vol- ume coefficient) Measurement of IL-4 and IFN-γ cytokines To place the mouse anti-IL-4 and anti- IFN-γ monoclonal antibodies, first, 100μl of the antibody in question at a concentration of 5μg/ml in sterile PBS buffer was added to each well in a 96-well plate and kept at 4 °C overnight for 16 hours. The wells were rinsed three times with PBS and kept with 200μl of complete RPMI culture medium at laboratory temperature for 2 hours. The spleen cells iso- lated from each mouse were diluted, and 2× 105 cells in a volume of 100μl were added to wells in triplicates. A volume of 50μl of the toxoid at a concentration of 10μg/ml was add- ed to each well to stimulate cells. Only sterile PBS was added to the control wells. Plates were kept at 37 °C for 48 hours. The culture medium was drawn from each well, and wells were rinsed once with cold sterile distilled wa- ter and three times with PBS buffer containing 0.05% Tween 20; then, 100μl of the mouse biotin-avidin detection MBA in PBS buffer with 1% BSA at a concentration of 2μl was added to the wells. Plates were kept at labor- atory temperature for 2 hours, and then un- bound antibodies were removed and each well was rinsed with PBS buffer containing 0.05% tween 20. Diluted streptavidin-HRP was added to each well at a 1:1000 ratio in PBS contain- ing 1% BSA and then kept at 37 ºC for 1 hour. The wells were rinsed three to five times with PBS buffer, and 100μl of the final substrate solution BCIB-BCIP/NBT Blue One Compo- nent AP Membrane Substrate (Surmodics IVD, Inc., USA) was added. One hour after incuba- tion at 37 °C, the wells were rinsed with ster- ile distilled water, and plates were air-dried. The spots that were developed were counted au- tomatically through Enzyme-Linked Immuno- Spot (ELISpot) assay. IL-4 and IFN-γ cytokines were measured using ELISpot IFN-γ (ab 64029) and IL-4 (ab 64033) kits purchased from ABCAM Company, USA. Each test was repeat- ed three times, and the results were assessed as mean ± SD. Statistical analysis The statistical analysis was performed us- ing IBM SPSS 19.0 (New York, USA). The results were expressed as the mean ± SD and were compared using Student’s t-test. Differ- ences were considered statistically significant at values of P≤ 0.05. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 5 http://jad.tums.ac.ir Published Online: March 31, 2022 Results Detection of dermonecrotic activity of phos- pholipase D toxoid Given that the main indicator of the effect of H. lepturus scorpion venom is phospho- lipase D with dermonecrotic properties, this test was used to assess the toxic properties of the produced toxoid, and the results were as- sessed after conducting the dermonecrotic test. The results showed that in 96 hours, the tox- oid injection had no dermonecrotic effect in rabbits and no wounding or inflammation signs were observed on rabbit skin (Fig. 1A). There- fore, detoxification of phospholipase D to pro- duce toxoid was successful, while 72 hours af- ter phospholipase D injection (positive control), the necrosis area reached 0.72±0.3cm3 (Fig. 1B), and PBS was considered as the negative control (Fig. 1C). Immune response of cytokines IL-4 and IFN- γ to toxoid vaccine Following immunization of the mice with toxoid -Freund/Alum adjuvants, the superna- tant obtained from their spleen was used to evaluate the anti-inflammatory IL-4 cytokine and the inflammatory IFN-γ cytokine. No fa- talities occurred in the course of the study. The mean immunity indices of IFN-γ and IL- 4 cytokines were measured in the study and control groups. The mean concentrations of IFN-γ cytokine in the vaccinated group with toxoid -Freund/Alum adjuvants increased com- pared to the control. As a result, IFN-γ cyto- kine in the vaccinated group with toxoid - Al- um adjuvant, extremely close results were ob- served with toxoid- Freund adjuvant. The mean concentration of IL-4 cytokine in the vaccinat- ed group with toxoid -Freund/Alum adjuvants slightly increased compared to the control. There was a significant difference between all vac- cinated groups in terms of the concentration of the inflammatory IFN-γ cytokine (P≤ 0.05) but no significant difference was observed in the concentration of anti-inflammatory IL-4 cy- tokine between these groups (P≥ 0.05) (Table 1). The concentration of inflammatory and an- ti-inflammatory cytokines was determined in vaccinated and control groups using the ELISpot technique, and the results were compared. The results showed an increase of 400–500 units or 400–500% in the inflammatory IFN-γ level in the study group compared to the control, and this increase was significant (P= 0.001). The level of the anti-inflammatory IL-4 cytokine increased by 1.1–1.33 units or 1.1–1.3% in the study groups compared to the control group. Despite this slight increase, the mean level of IL-4 showed no significant difference between the study and control groups (P= 0.16). The com- parison of the levels of inflammatory IFN-γ and anti-inflammatory IL-4 cytokine with each other in the study groups showed an increase of 400– 432 units or 400–432% in the IFN-γ level com- pared to the IL-4 level, and the difference be- tween them was significant (P= 0.001) (Fig. 2). Thus, an increase in IFN-γ activity leads to a decrease in IL-4 activity, and they have a reg- ulatory effect on each other. Comparisons were based on the unpaired t-test. P≤ 0.05 indicates a significant difference in all cases. Only IFN- γ concentration from Freund/Alum -immunized mice showed statistically significant results (P= 0.001) (Fig. 2). Thus, an increase in IFN-γ ac- tivity leads to a decrease in IL-4 activity, and they have a regulatory effect on each other. Vaccination Trials in the Mice The results indicated that the mice vaccinat- ed with Toxoid-Freund/Alum adjuvants chal- lenged either by rPLD or crude venom of H. lepturus, survived throughout the study. The results obviously indicated that all mice vac- cinated with the toxoid were completely pro- tected against a wide range of rPLD or crude venom concentrations, using either of these two adjuvants (Table 2). Non-immunized neg- ative control mice in all groups died after in- jecting one LD100 of rPLD or one LD100 of crude venom. From these results, it can be de- duced that the amount of vaccine (toxoid= 80 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 6 http://jad.tums.ac.ir Published Online: March 31, 2022 μg/mouse) which promoted the efficacy of the immune response was certainly adequate to pro- tect the mice against rPLD or crude venom of H. lepturus. As a result, toxoid vaccine devel- oped an effective cellular immunity against the scorpion venom of H. lepturus. Table 1. Hemiscorpius lepturus toxoid concentration of inflammatory IFN-γ and anti-inflammatory IL-4 in the mice vaccinated with toxoid -Freund /Alum adjuvants and control groups Variable Case group Control group P* Mean SD Mean SD IFN-γ (a) IFN-γ (b) 263.33 231.51 6.77 4.13 42.66 40.24 2.51 3.14 0.001 IL-4 (a) IL-4 (b) 53.55 50.21 5.26 3.42 38.17 35.82 2.31 4.14 0.16 (a): IFN-γ/ IL-4 in the mice vaccinated with toxoid- Freund adjuvant. The mice that only received Freund-Adjuvant + PBS (control group) (b): IFN-γ/ IL-4 in the mice vaccinated with toxoid- Alum adjuvant. The mice that only received Alum-adjuvant + PBS (control group). *Comparison based on unpaired t-test. P≤ 0.05 indicates a significant difference in all cases. The IFN-γ variable is statistically significant Table 2. In vivo vaccination trials in immunized mice Number of mice rPLD Challenge (LD100s)a Survival Rate (%) Number of mice Crude Venom Challenge (LD100s)(b) Survival Rate 6 10 100 6 1 100 6 20 100 6 2 100 6 50 100 6 5 100 6 100 100 6 7 100 6 200 100 6 10 100 Neg. Cont.(C) Neg. Cont.(C) 6 1 0 6 1 0 (a): One LD100 of rPLD is equal to 3.7μg. (b): One LD100 of Hemiscorpius lepturus scorpion crude venom is equal to 100µg (c): The mice that only received Adjuvant + PBS (negative controls) Fig. 1. Hemiscorpius lepturus toxoid Intracutaneous injection of 3µg of PLD toxoid into rabbit’s shaved skin. No der- monecrotic activity was observed in 96 hours after injection (A). Injection of 3µg of PLD as the positive control showed a specific dermonecrotic activity, and the necrosis site reached 0.72cm3 after 72 hours (B). PBS was used as the nega- tive control (C). Each experiment was conducted with two repeats A B C http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 7 http://jad.tums.ac.ir Published Online: March 31, 2022 0 50 100 150 200 250 300 350 S F C IFN-γ control INFγ IL-4 Control IL-4 * * A B Fig. 2. Hemiscorpius lepturus concentration of inflammatory IFN-γ from mice vaccinated by toxoid -Freund/Alum adjuvants and anti-inflammatory IL-4 from mice vaccinated by toxoid -Freund/Alum adjuvants and control groups by ELISpot method. (A) assay was performed on Freund-immunized mice. The mice that only received Freund-Adjuvant + PBS (negative control). (B) assay was performed on Alum-immunized mice. The mice that only received Alum- adjuvant + PBS (negative control). The mean ± S.E.M is shown in each column Discussion Given the high percentage of the annual mortality due to H. lepturus sting in the warm regions of Iran, especially in the southern are- as (Khuzestan Province) (3), the main solu- tion is prevention instead of treatment. Pre- vention occurs with vaccine production, and currently, the only effective treatment against H. lepturus sting is the use of the polyvalent antiserum obtained from horses immunized against the whole venoms of the six venomous scorpions of Iran (2). The main disadvantages of the common antisera on the market include their lack of specific activity against particular scorpion venoms and the complications they cause, especially in susceptible groups of the population (21). Various methods are used in the preparation of vaccines, and among them, formalin use is the most common (22) and basic (23) method. Toxoid vaccines obtained by using formalin on the venom of Cobra (24) and Habu (25) snakes are currently used in southern Asian countries, especially in India. When toxoid is used as a vaccinating agent, an augmented im- mune response ensues that forms an immuno- logical memory against the toxoid immunogen- ic epitopes (26). Based on previous reports, we used formalin for the first time to detoxify the most venomous component of H. lepturus ven- om (phospholipase D protein) to produce a tox- oid vaccine (10). Detoxification of this protein and its conversion into toxoid can provide a suitable candidate for vaccinating individuals against H. lepturus venom if the three-dimen- sional structure of the protein is preserved. The use of formalin modifies histidine residues and produces uncommon imidazole derivatives in the protein (27). The resulting toxoid can neu- tralize the deadly effect of the phospholipase D toxin and the complete H. lepturus venom (10). After developing this new molecule, in the present study, we assessed the toxicity of this toxoid and the active immunity type of the vaccine. Dermonecrotic activity assessment is the most important confirmatory test for tox- oids developed from toxins. It can be inferred from the results that the produced toxoid does http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 8 http://jad.tums.ac.ir Published Online: March 31, 2022 not have any enzymatic activity even to the slightest degree, while all toxins in the whole venom, such as phospholipase D toxin, and the whole venom contain this activity (9, 28). In agreement with our results, it has previous- ly been reported that recombinant PLD anti- sera inhibit the necrotic activity of the whole venom of Gadim scorpion (11). Other findings confirm that some antibodies against der- monecrotic toxins can fully neutralize the der- monecrotic activity of the scorpion’s whole venom. For instance, in the study conducted in Brazil, anti-dermonecrotic antibodies were pro- duced against the brown Loxosceles interme- dia spider venom, which were fully capable of neutralizing the spider’s venom (29). Thus, the antisera obtained by using the toxoid pro- duced in the previous study can be regarded as an effective agent in preventing necrotic wounds caused by H. lepturus venom (10). Studies have shown that inflammatory effects are observed following the scorpion sting (30). The toxicity of the toxoid and the effective- ness of the vaccine were assessed through der- monecrotic test and T-helper (Th) cell immune response, respectively. To transmit a message and affect other cells, the immune system cells must secrete the products that mediate the trans- mission of this message (26). In response to mi- crobes and other antigens, cells of the innate and specific immunity system secrete protein mol- ecules, generally known as cytokines, that pro- duce different effects (26, 31). Two subgroups of Th immune cells, Th1 and Th2, secrete different types of cytokines (26). Among the cytokines secreted by Th2 cells, the main interest is in interleukin-4 (IL-4), which has the key role in the synthesis of IgE (32). The IL-4 producing cells stimulate and differentiate lymphocytes and switch isotypes from IgM to IgE production in B lymphocytes and thus have a major role in inducing and maintaining allergies, inflammation, and necrosis (33). IL-4 stimulation can affect mast cell responses through IgE signaling (34). Studies have shown that the increase in serum IL-4 levels is di- rectly associated with allergic symptoms and inflammation (33, 34). IL-4 has a central role in type 1 allergic reactions and can have a role as a mediator in the T-cells differentiation (35). On the other hand, as the most important cy- tokine synthesized by Th1 cells, IFN-γ has a role in cell immunity and inhibits Th2-related immune response (36, 37). Studies have shown that IFN-γ has a highly important role in Th2 lymphocyte dependent allergic reactions (37, 38). IFN-γ can cause the release of pro inflam- matory cytokines during allergic reactions (39). Th1 and Th2 cytokines’ role in the immune re- sponse and their mutual balance have decisive effects on disease incidence and complications (26, 37). Differentiation of T-helper lympho- cytes can be induced through the effect of var- ious cytokines and antigens (40). IFN-γ cy- tokine can cause differentiation of Th0 toward Th1 (39). Activation of the Th1 immune re- sponse has pro inflammatory properties and activates B cells and macrophages and induc- es IgG toward IgG2β, IgG2α in mice, result- ing in the activation of the cellular acquired im- mune system (37, 39-40). IL-4 cytokine causes differentiation of Th0 toward Th2 (39). Acti- vation of the Th2 immune response, which has anti-inflammatory properties, results in the growth and differentiation of lymphocytes, production of immunoglobulin E, basophils, mastocytes, inactivation of macrophages, re- cruitment of eosinophils, and induction of IgG toward IgG1 in mice and causes inhibition of the immune system (26, 37). In the present study, to assess the inflam- matory effects of the vaccine and type of im- munity conferred by it, concentrations of in- flammatory IFN-γ and anti-inflammatory IL-4 cytokines in the supernatant of the spleen of the mice immunized with toxoid was determined and their relationship with cellular immunity was investigated. Given the sensitivity of this experiment, the immune response was meas- ured by ELISpot assay to evaluate the toxoid as a vaccine candidate. This test is 20 to 200 times more sensitive than the ELISA test and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 9 http://jad.tums.ac.ir Published Online: March 31, 2022 its results are almost 100% accurate (41). In addition to the increase in humoral immunity that was assessed using the ELISA test in our previous article (10), the present study provides evidence for an increase of 400–500 % in the concentration of the inflammatory cytokine in the immunized mice compared to control groups. This increase is significant and has noticeably increased the cellular immunity. However, the anti-inflammatory cytokine increased by 1.1– 1.33% compared to the control group, and this increase was not significant. To evaluate the potency of this toxoid protein as a vaccine can- didate and its effectiveness in neutralizing the lethality of crude venom of H. lepturus scor- pion, the following experiments were performed. The groups of mice vaccinated by toxoid using Freund or alum adjuvants, were challenged by whole venom of H. lepturus. Their survival rate was documented for two weeks’ post-injection and over longer time periods. This survival strongly affirms that all mice were completely protected against wide range of different le- thal dosages of crude venom (10 LD100, equal to 50mg/Kg), thus illustrating a successful and 100% immunization in the mice. The results obtained confirm that the developed vaccine, in addition to inducing humoral immunity, can in- crease the Th1 cytokine response within lym- phocytes and consequently increase cellular immunity. In agreement with the present study results, a study conducted by Borchani et al. showed that a type of PLD in H. lepturus called hemonecrolysin can increase the secre- tion of pro-inflammatory factors TNF-α and IL-6 on human monocytes by 3.5 times com- pared to the control. This PLD can also dou- ble the level of the anti-inflammatory factor IL-10 compared to the control (42). Further- more, in agreement with the present study re- sults, it has been reported that the recombinant phospholipase D obtained from the brown Lox- osceles recluse is also able to increase pro-in- flammatory cytokines TNF-α, IL-1β, IL-8 on human fibroblasts (43). These results show that the vaccine produced from toxoid has the same effect on T-cell as the whole venom, recombi- nant phospholipase D from the scorpion venom, or recombinant phospholipase D of the brown spider and can activate inflammatory process- es and that it can be used as a booster of the immunological memory against scorpion/spi- der stings. In a study conducted by Homayoon et al. (44), it was shown that Pasteurella mul- tocida vaccine, inactivated by formalin, can significantly increase pro-inflammatory cyto- kines, and significantly increase cellular im- munity in the immunized BALB/c mice com- pared to the control group. In agreement with the results obtained, the vaccines produced from Leishmania major also significantly increased the ability to produce IFN-γ and resulted in the Th1 response in mice, while the produc- tion of IL-4 and the Th2 cytokine response de- creased significantly (45). Overall, lack of clin- ical assessments was among the limitations of the present study and is proposed to be ad- dressed in future studies. Moreover, according to the present study results, this vaccine, as a protective agent in people exposed to H. lep- turus scorpion sting, can increase Th1 cytokines and thus increase the acquired immunity of the cellular kind. Conclusion The present study results showed for the first time that toxoid, as a vaccine, can increase the production of certain components of Th1 and Th2 cytokine response in the serum of mice immunized with rPLD toxoid, and this increase is significant in terms of the level of IFN-γ cy- tokine. Furthermore, it was shown that the sup- portive immunity produced in the mice was as- sociated with the activity of Th1 cytokines. Acknowledgements This article is a part the Ph.D. thesis of Narges Safari-Foroshani approved by the Pas- teur Institute of Iran, Tehran, Iran. This re- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 1–12 N Safari-Foroushani et al.: Cellular Immunity in … 10 http://jad.tums.ac.ir Published Online: March 31, 2022 search did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Ethical considerations Male BALB/c mice (18–20g) were pur- chased from Pasteur Institute of Iran. 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