Xanthan gum triple emulsion adjuvant effect in an experimental vaccine with recombinant epsilon toxin (rETX) of Clostridium perfringens
Ana Muñoz Vianna, Neida Conrad, Francisco Denis Souza Santos, Pedro Machado Medeiros de Albuquerque, Fabricio Rochedo Conceição, Fábio Pereira Leivas Leite
Abstract
Subunit vaccines composed of recombinant proteins are safer and more defined than traditional formulations but often require potent adjuvants to achieve strong and durable immunity. Aluminum hydroxide, the most widely used adjuvant, has limited capacity to induce robust humoral and cellular responses. This study evaluated a novel xanthan gum–based waterin-oil-in-water (W/O/W) emulsion adjuvant (Xenoil) as a delivery system for recombinant Clostridium perfringens epsilon toxin (rETX). Heifers (n = 15) with no prior history of clostridial vaccination were included in the study and randomly allocated into three experimental groups (n = 5 per group) as follows: 1. inoculated with rETX formulated with the triple emulsion; 2. inoculated with rETX adjuvanted with aluminum hydroxide; and 3. animals receiving the triple emulsion combined with phosphate-buffered saline (PBS), as the control formulation. An in-house indirect ELISA was used to measure specific anti-rETX IgG. Transcription levels of key cytokines (il2, il15, il17) were analyzed in peripheral blood mononuclear cells (PBMCs) using quantitative PCR (qPCR). The W/O/W emulsion was stable for six months under refrigeration and safe for administration in mice, horse and cattle. Heifers immunized with the emulsion exhibited a 1.5-fold increase in IgG titers compared with aluminum hydroxide (p < 0.05). Significant upregulation of il2, il15, and il17 transcription was observed (p < 0.05), suggesting a modulation towards Th1/ Th17 pathways, activating T cells and B-cell differentiation. The xanthan gum–based W/O/W emulsion induced stronger humoral responses than aluminum hydroxide, demonstrating its immunomodulatory potential in cattle. Although neutralizing activity and long-term memory were not assessed, these results highlight Xenoil as a promising next-generation adjuvant candidate. Future studies should confirm antibody neutralization capacity, evaluate protective efficacy under field conditions, and determine the durability of immunity.
Keywords
References
Ananya, A., Holden, K. G., Gu, Z., Nettleton, D., Mallapragada, S. K., Wannemuehler, M. J., Kohut, M. L., & Narasimhan, B. (2023). “Just right” combinations of adjuvants with nanoscale carriers activate aged dendritic cells without overt inflammation. Immunity & Ageing, 20(1), 10. https://doi.org/10.1186/s12979- 023-00332-0. PMid:36895007.
Athman, R., & Philpott, D. (2004). Innate immunity via Toll-like receptors and Nod proteins. Current Opinion in Microbiology, 7(1), 25-32. https://doi.org/10.1016/j.mib.2003.12.013. PMid:15036136.
Aucouturier, J., Dupuis, L., & Ganne, V. (2001). Adjuvants designed for veterinary and human vaccines. Vaccine, 19(17–19), 2666-2672. https://doi.org/10.1016/S0264-410X(00)00498-9. PMid:11257407.
Aucouturier, J., Dupuis, L., Deville, S., Ascarateil, S., & Ganne, V. (2002). Montanide ISA 720 and 51: A new generation of water in oil emulsions as adjuvants for human vaccines. Expert Review of Vaccines, 1(1), 111-118. https:// doi.org/10.1586/14760584.1.1.111. PMid:12908518.
Awate, S., Wilson, H. L., Lai, K., Babiuk, L. A., & Mutwiri, G. (2012). Activation of adjuvant core response genes by the novel adjuvant PCEP. Molecular Immunology, 51(3–4), 292-303. https:// doi.org/10.1016/j.molimm.2012.03.026. PMid:22521769.
Bachmann, M. F., & Oxenius, A. (2007). Interleukin 2: From immunostimulation to immunoregulation and back again. EMBO Reports, 8(12), 1142-1148. https://doi.org/10.1038/ sj.embor.7401099. PMid:18059313.
Becker, A., Katzen, F., Pühler, A., & Ielpi, L. (1998). Xanthan gum biosynthesis and application: A biochemical/genetic perspective. Applied Microbiology and Biotechnology, 50(2), 145-152. https:// doi.org/10.1007/s002530051269. PMid:9763683.
Borges, C. D., & Vendruscolo, C. T. (2008). Goma Xantana: Características e condições operacionais de produção. Semina. Ciências Biológicas e da Saúde, 29(2), 171. https:// doi.org/10.5433/1679-0367.2008v29n2p171.
Boyman, O., & Sprent, J. (2012). The role of interleukin-2 during homeostasis and activation of the immune system. Nature Reviews. Immunology, 12(3), 180-190. https://doi.org/10.1038/nri3156. PMid:22343569.
Bystrom, J., Al-Adhoubi, N., Al-Bogami, M., Jawad, A. S., & Mageed, R. A. (2013). Th17 lymphocytes in respiratory syncytial virus infection. Viruses, 5(3), 777-791. https://doi.org/10.3390/ v5030777. PMid:23462708.
Ciabattini, A., Pettini, E., Fiorino, F., Pastore, G., Andersen, P., Pozzi, G., & Medaglini, D. (2016). Modulation of primary immune response by different vaccine adjuvants. Frontiers in Immunology, 7, 427. https://doi.org/10.3389/fimmu.2016.00427. PMid:27781036.
Coffman, R. L., Sher, A., & Seder, R. A. (2010). Vaccine adjuvants: Putting innate immunity to work. Immunity, 33(4), 492-503. https://doi.org/10.1016/j.immuni.2010.10.002. PMid:21029960.
Cui, X., Xiang, Q., Huang, Y., Ji, Q., Hu, Z., Shi, T., Bao, G., & Liu, Y. (2024). Mixed Th1/Th2/Th17 Responses Induced by plant oil adjuvant-based b. bronchiseptica vaccine in mice, with mechanisms unraveled by RNA-Seq, 16S rRNA and Metabolomics. Vaccines, 12(10), 1182. https://doi.org/10.3390/ vaccines12101182. PMid:39460348.
Dar, A., Lai, K., Dent, D., Potter, A., Gerdts, V., Babiuk, L. A., & Mutwiri, G. K. (2012). Administration of poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) as adjuvant activated mixed Th1/Th2 immune responses in pigs. Veterinary Immunology and Immunopathology, 146(3–4), 289-295. https:// doi.org/10.1016/j.vetimm.2012.01.021. PMid:22377627.
Di Pasquale, A., Preiss, S., Da Silva, F. T., & Garçon, N. (2015). Vaccine adjuvants: From 1920 to 2015 and beyond. Vaccines, 3(2), 320- 343. https://doi.org/10.3390/vaccines3020320. PMid:26343190.
Dummer, L. A., Araujo, I. L., Finger, P. F., Santos Junior, A. G., da Rosa, M. C., Conceição, F. R., Fischer, G., van Drunen Littel-van den Hurk, S., & Leite, F. P. (2014). Immune responses of mice against recombinant bovine herpesvirus 5 glycoprotein D. Vaccine, 32(21), 2413-2419. https://doi.org/10.1016/j.vaccine.2014.03.011. PMid:24657716.
Eisenbarth, S. C., Colegio, O. R., O’Connor, W., Sutterwala, F. S., & Flavell, R. A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature, 453(7198), 1122-1126. https://doi.org/10.1038/nature06939. PMid:18496530.
Ferreira, M. R. A., Moreira, G. M. S. G., Da Cunha, C. E. P., Mendonça, M., Salvarani, F. M., Moreira, Â. N., & Conceição, F. R. (2016). Recombinant Alpha, Beta, and Epsilon toxins of Clostridium perfringens: Production strategies and applications as veterinary vaccines. Toxins, 8(11), 340. https://doi.org/10.3390/ toxins8110340. PMid:27879630.
Freitas, N. F. Q. R., Otaka, D. Y., Galvão, C. C., Almeida, D. M., Ferreira, M. R. A., Moreira Júnior, C. M., Hidalgo, M. M. M. H., Conceição, F. R., & Salvarani, F. M. (2021). Humoral immune response evaluation in horses vaccinated with recombinant Clostridium perfringens toxoids alpha and beta for 12 months. Toxins, 13(8), 566. https://doi.org/10.3390/toxins13080566. PMid:34437437.
Gaffen, S. L., & Liu, K. D. (2004). Overview of interleukin-2 function, production and clinical applications. Cytokine, 28(3), 109-123. https://doi.org/10.1016/j.cyto.2004.06.010. PMid:15473953.
Garg, R., Babiuk, L., van Drunen Littel-van den Hurk, S., & Gerdts, V. (2017). A novel combination adjuvant platform for human and animal vaccines. Vaccine, 35(Pt A), 4486-4489. https:// doi.org/10.1016/j.vaccine.2017.05.067. PMid:28599794.
Habjanec, L., Halassy, B., & Tomašić, J. (2008). Immunomodulatory activity of novel adjuvant formulations based on montanide ISA oil-based adjuvants and peptidoglycan monomer. International Immunopharmacology, 8(5), 717-724. https:// doi.org/10.1016/j.intimp.2008.01.017. PMid:18387514.
Ishizaka, S., Sugawara, I., Hasuma, T., Morisawa, S., & Möller, G. (1983). Immune responses to xanthan gum I. The characteristics of lymphocyte activation by xanthan gum. European Journal of Immunology, 13(3), 225-231. https://doi.org/10.1002/ eji.1830130309. PMid:6832212.
Leite, F., Kuckleburg, C., Atapattu, D., Schultz, R., & Czuprynski, C. J. (2004). BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood mononuclear cells in vitro. Veterinary Immunology and Immunopathology, 99(3–4), 193-202. https:// doi.org/10.1016/j.vetimm.2004.02.004. PMid:15135985.
Levast, B., Awate, S., Babiuk, L., Mutwiri, G., Gerdts, V., & van Drunen Littel-van den Hurk, S. (2014). Vaccine potentiation by combination adjuvants. Vaccines, 2(2), 297-322. https:// doi.org/10.3390/vaccines2020297. PMid:26344621.
Li, H., & Pauza, C. D. (2015). CD25+Bcl6low T follicular helper cells provide help to maturing B cells in germinal centers of human tonsil. European Journal of Immunology, 45(1), 298-308. https:// doi.org/10.1002/eji.201444911. PMid:25263533.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods : A Companion to Methods in Enzymology, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262. PMid:11846609.
Mitsdoerffer, M., Lee, Y., Jäger, A., Kim, H. J., Korn, T., Kolls, J. K., Cantor, H., Bettelli, E., & Kuchroo, V. K. (2010). Proinflammatory T helper type 17 cells are effective B-cell helpers. Proceedings of the National Academy of Sciences of the United States of America, 107(32), 14292-14297. https://doi.org/10.1073/ pnas.1009234107. PMid:20660725.
Moreira, G. M. S. G., Salvarani, F. M., Da Cunha, C. E. P., Mendonça, M., Moreira, Â. N., Gonçalves, L. A., Pires, P. S., Lobato, F. C., & Conceição, F. R. (2016). Immunogenicity of a trivalent recombinant vaccine against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants. Scientific Reports, 6(1), 22816. https:// doi.org/10.1038/srep22816. PMid:27004612.
Mutwiri, G., Benjamin, P., Soita, H., Townsend, H., Yost, R., Roberts, B., Andrianov, A. K., & Babiuk, L. A. (2007). Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens. Vaccine, 25(7), 1204-1213. https:// doi.org/10.1016/j.vaccine.2006.10.011. PMid:17140708.
O’Hagan, D. T., & Valiante, N. M. (2003). Recent advances in the discovery and delivery of vaccine adjuvants. Nature Reviews. Drug Discovery, 2(9), 727-735. https://doi.org/10.1038/nrd1176. PMid:12951579.
Oliveira, R. C., Oliveira Júnior, C. A., Alves, G. G., Assis, R. A., Silva, R. O. S., Xavier, M. A. S., & Lobato, F. C. F. (2021). Cattle and goats’ humoral response to vaccination with Clostridium perfringens type D purified epsilon toxoids. Anaerobe, 72, 102465. https:// doi.org/10.1016/j.anaerobe.2021.102465. PMid:34662696.
Poon, L. L. M., Leung, Y. H. C., Nicholls, J. M., Perera, P.-Y., Lichy, J. H., Yamamoto, M., Waldmann, T. A., Peiris, J. S., & Perera, L. P. (2009). Vaccinia virus-based multivalent H5N1 avian influenza vaccines adjuvanted with il-15 confer sterile cross-clade protection in mice. The Journal of Immunology : Official Journal of the American Association of Immunologists, 182(5), 3063-3071. https://doi.org/10.4049/jimmunol.0803467. PMid:19234203.
Pulendran, B. S., Arunachalam, P., & O’Hagan, D. T. (2021). Emerging concepts in the science of vaccine adjuvants. Nature Reviews. Drug Discovery, 20(6), 454-475. https://doi.org/10.1038/s41573- 021-00163-y. PMid:33824489.
Reed, S. G., Bertholet, S., Coler, R. N., & Friede, M. (2009). New horizons in adjuvants for vaccine development. Trends in Immunology, 30(1), 23-32. https://doi.org/10.1016/j.it.2008.09.006. PMid:19059004.
Reed, S. G., Orr, M. T., & Fox, C. B. (2013). Key roles of adjuvants in modern vaccines. Nature Medicine, 19(12), 1597-1608. https:// doi.org/10.1038/nm.3409. PMid:24309663.
Rocha, P. H., Assis, R. A., Lobato, F. C. F., Cardoso, V. N., & Heneine, L. G. D. (2008). Stability and toxicity of Clostridium perfringens type D epsilon prototoxin treated by iodine. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 60(4), 821-824. https:// doi.org/10.1590/S0102-09352008000400007.
Saikh, K. U., Kissner, T. L., Nystrom, S., Ruthel, G., & Ulrich, R. G. (2008). Interleukin-15 increases vaccine efficacy through a mechanism linked to dendritic cell maturation and enhanced antibody titers. Clinical and Vaccine Immunology; CVI, 15(1), 131-137. https://doi.org/10.1128/CVI.00320-07. PMid:18045883.
Songer, J. G. (1996). Clostridial enteric diseases of domestic animals. Clinical Microbiology Reviews, 9(2), 216-234. https:// doi.org/10.1128/CMR.9.2.216. PMid:8964036.
Stoppelenburg, A. J., De Roock, S., Hennus, M. P., Bont, L., & Boes, M. (2014). Elevated Th17 response in infants undergoing respiratory viral infection. The American Journal of Pathology, 184(5), 1274-1279. https://doi.org/10.1016/j.ajpath.2014.01.033. PMid:24650560.
Sutherland, I. W. (2001). Microbial polysaccharides from Gramnegative bacteria. International Dairy Journal, 11(9), 663-674. https://doi.org/10.1016/S0958-6946(01)00112-1.
Takeuchi, A., Kamiryou, Y., Yamada, H., Eto, M., Shibata, K., Haruna, K., Naito, S., & Yoshikai, Y. (2009). Oral administration of xanthan gum enhances antitumor activity through Toll-like receptor 4. International Immunopharmacology, 9(13–14), 1562-1567. https:// doi.org/10.1016/j.intimp.2009.09.012. PMid:19788935.
Takeuchi, O., & Akira, S. (2010). Review Pattern Recognition Receptors and Inflammation. Cell, 140(6), 805-820. https:// doi.org/10.1016/j.cell.2010.01.022. PMid:20303872.
Yao, W., Kang, J., Kang, L., Gao, S., Yang, H., Ji, B., Li, P., Liu, J., Xin, W., & Wang, J. (2016). Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX Y196E-C confers strong protection in mice. Scientific Reports, 6(1), 24162. https:// doi.org/10.1038/srep24162. PMid:27048879.
Zarandi, P. K., Zinatizadeh, M. R., Ghiasi, M., Rukerd, M. R. Z., Mirkamali, H., & Shokri, E. (2025). Efficacy of Immunostimulatory adjuvants and nano-adjuvants in current SARS-CoV-2 vaccines: a comprehensive review. Health Science Reports, 8(11), e71405. https://doi.org/10.1002/hsr2.71405. PMid:41255382.
Submitted date:
11/11/2025
Accepted date:
03/29/2026
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