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Therapeutic DNA vaccines targeting viral oncoproteins represent a promising strategy to induce antitumor immune responses. However, the impact of antigen combinations in bicistronic vectors and the immunization sequence on therapeutic efficacy remains poorly understood. Thus, this study aimed to characterize in silico previously described E5CP and E7CP vaccines, develop a new bicistronic construct, and evaluate its antitumor efficacy under different immunization regimens in vivo. The constructs were initially characterized regarding predicted physicochemical, immunological, and structural properties, followed by molecular confirmation of plasmid construction and qualitative assessment of transcript detection after transient transfection. In vivo, C57BL\/6 mice bearing C3 tumors were immunized with five different regimens combining the monocistronic or bicistronic constructs. At the endpoint, tumors and spleens were collected for histopathological and immunological analyses. The results showed that regimens using the bicistronic construct or including E5 in the first dose were associated with lower mean tumor volumes or delayed tumor growth compared to the control groups. Histopathological and immunophenotypic changes were variable among regimens, and an exploratory analysis suggested an association between lower nitric oxide levels and smaller tumor volumes. Therefore, the order of antigen administration and the use of bicistronic constructs warrant further investigation as strategies that could be optimized to improve the therapeutic efficacy of HPV DNA vaccines.<\/jats:p>","DOI":"10.3390\/molecules31193442","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T11:35:59Z","timestamp":1790595359000},"page":"3442","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Heterologous Antigen Regimen and Bicistronic DNA Vaccination Targeting HPV16 E5 and E7 Oncoproteins: Immunoinformatics and Preclinical Antitumor Efficacy"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0715-8674","authenticated-orcid":false,"given":"Ingrid Andr\u00eassa de","family":"Moura","sequence":"first","affiliation":[{"name":"Laboratory of Molecular Studies and Experimental Therapy\u2014LEMTE, Department of Genetics, Federal 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Clin. Oncol."},{"key":"ref_2","first-page":"e70043","article-title":"Cancer Statistics, 2026","volume":"76","author":"Siegel","year":"2026","journal-title":"CA Cancer J. Clin."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5887","DOI":"10.1038\/s41467-025-60836-9","article-title":"Genomic Landscape of Virus-Associated Cancers","volume":"16","author":"Nam","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Torres, M.K.D.S., Pereira Neto, G.D.S., Cayres Vallinoto, I.M.V., Reis, L.O., and Vallinoto, A.C.R. (2025). The Impact of Oncogenic Viruses on Cancer Development: A Narrative Review. Biology, 14.","DOI":"10.3390\/biology14070797"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3698","DOI":"10.1021\/acs.molpharmaceut.2c01080","article-title":"Recent Updates on Viral Oncogenesis: Available Preventive and Therapeutic Entities","volume":"20","author":"Chowdhary","year":"2023","journal-title":"Mol. Pharm."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1038\/s41392-025-02197-9","article-title":"Viral Oncogenesis in Cancer: From Mechanisms to Therapeutics","volume":"10","author":"Xiao","year":"2025","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1007\/s10147-023-02340-y","article-title":"Papillomaviruses and Cancer: Commonalities and Differences in HPV Carcinogenesis at Different Sites of the Body","volume":"28","author":"Egawa","year":"2023","journal-title":"Int. J. Clin. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.virusres.2016.12.017","article-title":"The Low-Risk Papillomaviruses","volume":"231","author":"Egawa","year":"2017","journal-title":"Virus Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2121","DOI":"10.1001\/jama.2023.20370","article-title":"Primary HPV Screening vs Cotesting for Cervical Cancer","volume":"330","author":"Brakebill","year":"2023","journal-title":"JAMA"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Hu, M., Wang, B., Li, J., and Wu, C. (2024). Editorial: The Association between Viral Infection and Human Cancers. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1371581"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"42","DOI":"10.21873\/cdp.10283","article-title":"Prevalence of Multiple High Risk Human Papilloma Virus (HR-HPV) Infections in Cervical Cancer Screening in Lazio Region, Italy","volume":"4","author":"Pisani","year":"2024","journal-title":"Cancer Diagn. Progn."},{"key":"ref_12","first-page":"1543","article-title":"Prevalence of Human Papillomavirus Subtypes 16 and 18 among Yemeni Patients with Cervical Cancer","volume":"18","author":"Ahmed","year":"2017","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12556","DOI":"10.1038\/s41598-019-49060-w","article-title":"Carcinogenic Risk of Human Papillomavirus (HPV) Genotypes and Potential Effects of HPV Vaccines in Korea","volume":"9","author":"Park","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1186\/s13027-025-00709-4","article-title":"Clinical Study on the Pathogenic Risks of Different Genotypes of High-Risk HPV Infection and Multiple Infections","volume":"20","author":"Song","year":"2025","journal-title":"Infect. Agents Cancer"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"001540","DOI":"10.1099\/jgv.0.001540","article-title":"The Human Papillomavirus Oncoproteins: A Review of the Host Pathways Targeted on the Road to Transformation","volume":"102","author":"Scarth","year":"2021","journal-title":"J. General. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1186\/s13046-017-0541-1","article-title":"hrHPV E5 Oncoprotein: Immune Evasion and Related Immunotherapies","volume":"36","author":"Barros","year":"2017","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.jcv.2011.06.007","article-title":"In Vivo HPV 16 E5 mRNA: Expression Pattern in Patients with Squamous Intra-Epithelial Lesions of the Cervix","volume":"52","author":"Lorenzon","year":"2011","journal-title":"J. Clin. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"11451","DOI":"10.1128\/JVI.78.21.11451-11460.2004","article-title":"Mechanisms of Human Papillomavirus-Induced Oncogenesis","volume":"78","author":"Baldwin","year":"2004","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Boilesen, D.R., Nielsen, K.N., and Holst, P.J. (2021). Novel Antigenic Targets of HPV Therapeutic Vaccines. Vaccines, 9.","DOI":"10.3390\/vaccines9111262"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1340","DOI":"10.1056\/NEJMoa1917338","article-title":"HPV Vaccination and the Risk of Invasive Cervical Cancer","volume":"383","author":"Lei","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"101880","DOI":"10.1016\/j.gendis.2025.101880","article-title":"Recent Advances in Human Papillomavirus Vaccines and Therapeutic Strategies: Combating Cervical and Non-Cervical Cancers","volume":"13","author":"Islam","year":"2026","journal-title":"Genes Dis."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Mordzi\u0144ska-Rak, A., Telejko, I., Adamczuk, G., Trombik, T., Stepulak, A., and B\u0142aszczak, E. (2025). Advancing Head and Neck Cancer Therapies: From Conventional Treatments to Emerging Strategies. Biomedicines, 13.","DOI":"10.3390\/biomedicines13051046"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"F71","DOI":"10.1016\/j.vaccine.2012.05.091","article-title":"Therapy of Human Papillomavirus-Related Disease","volume":"30","author":"Stern","year":"2012","journal-title":"Vaccine"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1358\/dot.2018.54.5.2807864","article-title":"Plasmid DNA Vaccines: Where Are We Now?","volume":"54","author":"Ghaffarifar","year":"2018","journal-title":"Drugs Today"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Hobernik, D., and Bros, M. (2018). DNA Vaccines\u2014How Far From Clinical Use?. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19113605"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"de Moura, I.A., Silva, A.J.D., de Mac\u00eado, L.S., Inven\u00e7\u00e3o, M.d.C.V., de Sousa, M.M.G., and de Freitas, A.C. (2022). Enhancing the Effect of Nucleic Acid Vaccines in the Treatment of HPV-Related Cancers: An Overview of Delivery Systems. Pathogens, 11.","DOI":"10.3390\/pathogens11121444"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"105104","DOI":"10.1016\/j.micpath.2021.105104","article-title":"A Novel Bicistronic DNA Vaccine with Enhanced Protective Immune Response against Bacillus Anthracis through DNA Prime-Protein Boost Vaccination Approach","volume":"158","author":"Sumithra","year":"2021","journal-title":"Microb. Pathog."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1080\/21645515.2017.1264766","article-title":"A Plant Protein Signal Sequence Improved Humoral Immune Response to HPV Prophylactic and Therapeutic DNA Vaccines","volume":"13","author":"Massa","year":"2017","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1007\/s12033-009-9195-3","article-title":"Potato Virus X Coat Protein Fusion to Human Papillomavirus 16 E7 Oncoprotein Enhance Antigen Stability and Accumulation in Tobacco Chloroplast","volume":"43","author":"Morgenfeld","year":"2009","journal-title":"Mol. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1080\/21645515.2017.1264777","article-title":"HPV 16 E5 Oncoprotein Is Expressed in Early Stage Carcinogenesis and Can Be a Target of Immunotherapy","volume":"13","author":"Paolini","year":"2017","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Walker, J.M. (2005). Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook, Humana Press.","DOI":"10.1385\/1592598900"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate Structure Prediction of Biomolecular Interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"W425","DOI":"10.1093\/nar\/gkab321","article-title":"ModFOLD8: Accurate Global and Local Quality Estimates for 3D Protein Models","volume":"49","author":"McGuffin","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1002\/pro.3330","article-title":"MolProbity: More and Better Reference Data for Improved All-atom Structure Validation","volume":"27","author":"Williams","year":"2018","journal-title":"Protein Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"W589","DOI":"10.1093\/nar\/gkab300","article-title":"ReFOLD3: Refinement of 3D Protein Models with Gradual Restraints Based on Predicted Local Quality and Residue Contacts","volume":"49","author":"Adiyaman","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2869","DOI":"10.1128\/JVI.02256-06","article-title":"Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients","volume":"81","author":"Liu","year":"2007","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"869","DOI":"10.2147\/IJN.S269986","article-title":"Peptide-Based Nanovaccines in the Treatment of Cervical Cancer: A Review of Recent Advances","volume":"17","author":"Zhang","year":"2022","journal-title":"Int. J. Nanomed. (IJN)"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"104721","DOI":"10.1016\/j.critrevonc.2025.104721","article-title":"Novel Therapeutic Strategies for Targeting E6 and E7 Oncoproteins in Cervical Cancer","volume":"211","author":"Ranasinghe","year":"2025","journal-title":"Crit. Rev. Oncol.\/Hematol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.virusres.2016.12.002","article-title":"The Current State of Therapeutic and T Cell-Based Vaccines against Human Papillomaviruses","volume":"231","author":"Yang","year":"2017","journal-title":"Virus Res."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Mart\u00edn, E., Reina, G., and Carlos, S. (2025). Advances in Therapeutic Vaccines Against HPV: A Review of Human Clinical Trials. Curr. Oncol., 32.","DOI":"10.3390\/curroncol32110600"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e03224-20","DOI":"10.1128\/mBio.03224-20","article-title":"Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody","volume":"12","author":"Peng","year":"2021","journal-title":"mBio"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/sj.cgt.7700723","article-title":"DNA Vaccines against the Human Papillomavirus Type 16 E6 or E7 Oncoproteins","volume":"11","author":"Wlazlo","year":"2004","journal-title":"Cancer Gene Ther."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Pal, A., and Kundu, R. (2020). Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.03116"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Yeo-Teh, N., Ito, Y., and Jha, S. (2018). High-Risk Human Papillomaviral Oncogenes E6 and E7 Target Key Cellular Pathways to Achieve Oncogenesis. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19061706"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Pereira Santos, V.E., De Fran\u00e7a S\u00e3o Marcos, B., Fontes, P.H.B., Silva, M.E.D.S., Le\u00e3o, S.L., Da Silva, G.R.P., Ribeiro, D.E., Da Gama, M.A.T.M., De Oliveira Is\u00eddio, B.E., and De Moura, I.A. (2025). E5 Oncoprotein: A Key Player in Human Papillomavirus-Positive Head and Neck Cancer Pathogenesis and Therapy Resistance. Viruses, 17.","DOI":"10.3390\/v17040512"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"P\u00e9rez-Morales, A.C., Maldonado-Gama, M., M\u00e9ndez-Armenta, M., Esquivel-Guadarrama, F., and Gutierrez-Xicotencatl, L. (2026). Role of E5 from HPV16 in the Evasion of the Immune Response. Int. J. Mol. Sci., 27.","DOI":"10.3390\/ijms27041985"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"45","DOI":"10.4161\/hv.34303","article-title":"Anti-Tumor Effects of Genetic Vaccines against HPV Major Oncogenes","volume":"11","author":"Cordeiro","year":"2015","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1007\/s11596-013-1189-5","article-title":"HPV16 E5 Peptide Vaccine in Treatment of Cervical Cancer in Vitro and in Vivo","volume":"33","author":"Liao","year":"2013","journal-title":"J. Huazhong Univ. Sci. Technol. [Med. Sci.]"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"7693","DOI":"10.1016\/j.vaccine.2022.10.069","article-title":"A Novel Multi-Epitope Vaccine of HPV16 E5E6E7 Oncoprotein Delivered by HBc VLPs Induced Efficient Prophylactic and Therapeutic Antitumor Immunity in Tumor Mice Model","volume":"40","author":"Qi","year":"2022","journal-title":"Vaccine"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Badillo-Godinez, O., Pedroza-Saavedra, A., Valverde-Gardu\u00f1o, V., Bermudez-Morales, V., Maldonado-Gama, M., Leon-Letelier, R., Bonifaz, L.C., Esquivel-Guadarrama, F., and Gutierrez-Xicotencatl, L. (2021). Induction of Therapeutic Protection in an HPV16-Associated Mouse Tumor Model Through Targeting the Human Papillomavirus-16 E5 Protein to Dendritic Cells. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.593161"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1329","DOI":"10.1007\/s00262-015-1734-0","article-title":"Immunotherapy of HPV-Associated Cancer: DNA\/Plant-Derived Vaccines and New Orthotopic Mouse Models","volume":"64","author":"Venuti","year":"2015","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"241","DOI":"10.2174\/1874357901206010241","article-title":"The Immune Response to Papillomavirus During Infection Persistence and Regression","volume":"6","author":"Hibma","year":"2012","journal-title":"Open Virol. J."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1097\/MS9.0000000000004352","article-title":"Inflammation, Immunity, and Invasion: The Pivotal Role of Cytokine Storms in Cervical Carcinogenesis","volume":"88","author":"Obeagu","year":"2026","journal-title":"Ann. Med. Surg."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"883","DOI":"10.1007\/s00281-022-00970-4","article-title":"Human T Lymphocytes at Tumor Sites","volume":"44","author":"Notarbartolo","year":"2022","journal-title":"Semin. Immunopathol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"634","DOI":"10.1038\/s42003-022-03596-2","article-title":"Activated-Memory T Cells Influence Na\u00efve T Cell Fate: A Noncytotoxic Function of Human CD8 T Cells","volume":"5","author":"Sasaki","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Kartikasari, A.E.R., Prakash, M.D., Cox, M., Wilson, K., Boer, J.C., Cauchi, J.A., and Plebanski, M. (2019). Therapeutic Cancer Vaccines\u2014T Cell Responses and Epigenetic Modulation. Front. Immunol., 9.","DOI":"10.3389\/fimmu.2018.03109"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1186\/s13045-022-01247-x","article-title":"Cancer Vaccines as Promising Immuno-Therapeutics: Platforms and Current Progress","volume":"15","author":"Liu","year":"2022","journal-title":"J. Hematol. Oncol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1146","DOI":"10.1124\/pharmrev.121.000500","article-title":"Opportunities for Nitric Oxide in Potentiating Cancer Immunotherapy","volume":"74","author":"Kim","year":"2022","journal-title":"Pharmacol. Rev."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"My\u015bliwiec, A., Bartusik-Aebisher, D., and Aebisher, D. (2025). The Role of Nitric Oxide in Cancer Treatment: Ally or Foe?. Molecules, 30.","DOI":"10.3390\/molecules30132802"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.niox.2026.02.005","article-title":"The Role of Nitric Oxide in Inflammation, Tumor Microenvironment, and Cancer Therapy","volume":"162","author":"Nair","year":"2026","journal-title":"Nitric Oxide"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1016\/j.trecan.2017.07.005","article-title":"Nitric Oxide: The Forgotten Child of Tumor Metabolism","volume":"3","author":"Achreja","year":"2017","journal-title":"Trends Cancer"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1016\/S0042-6822(03)00296-4","article-title":"Oligomerization of the E5 Protein of Human Papillomavirus Type 16 Occurs through Multiple Hydrophobic Regions","volume":"313","author":"Gieswein","year":"2003","journal-title":"Virology"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"5341","DOI":"10.1128\/JVI.06243-11","article-title":"High-Risk Human Papillomavirus E5 Oncoprotein Displays Channel-Forming Activity Sensitive to Small-Molecule Inhibitors","volume":"86","author":"Wetherill","year":"2012","journal-title":"J. Virol."},{"key":"ref_64","doi-asserted-by":"crossref","unstructured":"Gupta, S., Kapoor, P., Chaudhary, K., Gautam, A., Kumar, R., and Raghava, G.P.S. (2013). In Silico Approach for Predicting Toxicity of Peptides and Proteins. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0073957"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"2278","DOI":"10.1007\/s00894-014-2278-5","article-title":"AllerTOP v.2\u2014A Server for in Silico Prediction of Allergens","volume":"20","author":"Dimitrov","year":"2014","journal-title":"J. Mol. Model."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"W70","DOI":"10.1093\/nar\/gkn188","article-title":"The Vienna RNA Websuite","volume":"36","author":"Gruber","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1016\/j.ijbiomac.2018.09.120","article-title":"Lignins Isolated from Prickly Pear Cladodes of the Species Opuntia F\u00edcus-Indica (Linnaeus) Miller and Opuntia Cochenillifera (Linnaeus) Miller Induces Mice Splenocytes Activation, Proliferation and Cytokines Production","volume":"123","author":"Navarro","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"100173","DOI":"10.1016\/j.jtauto.2022.100173","article-title":"Preservation of Antigen-Specific Responses in Cryopreserved CD4+ and CD8+ T Cells Expanded with IL-2 and IL-7","volume":"5","author":"Clarkson","year":"2022","journal-title":"J. Transl. Autoimmun."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"102700","DOI":"10.1016\/j.xpro.2023.102700","article-title":"Protocol for the Expansion of Mouse Immune Effector Cells for in Vitro and in Vivo Studies","volume":"4","author":"Look","year":"2023","journal-title":"STAR Protoc."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/0003-2697(82)90118-X","article-title":"Analysis of Nitrate, Nitrite, and [15N]Nitrate in Biological Fluids","volume":"126","author":"Green","year":"1982","journal-title":"Anal. Biochem."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"892","DOI":"10.1093\/clinchem\/41.6.892","article-title":"Nitrite and Nitrate Determinations in Plasma: A Critical Evaluation","volume":"41","author":"Moshage","year":"1995","journal-title":"Clin. Chem."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1186\/s13071-015-0940-0","article-title":"L-Arginine Supplementation in Mice Enhances NO Production in Spleen Cells and Inhibits Plasmodium Yoelii Transmission in Mosquitoes","volume":"8","author":"Zheng","year":"2015","journal-title":"Parasites Vectors"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3442\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T04:26:32Z","timestamp":1790828792000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/19\/3442"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,27]]},"references-count":72,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["molecules31193442"],"URL":"https:\/\/doi.org\/10.3390\/molecules31193442","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,27]]}}}