{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:00:30Z","timestamp":1785416430770,"version":"3.56.0"},"reference-count":43,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T00:00:00Z","timestamp":1779926400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Gates Foundation","award":["INV-038051"],"award-info":[{"award-number":["INV-038051"]}]},{"award":["INV-038051"],"award-info":[{"award-number":["INV-038051"]}],"id":[{"id":"https:\/\/ror.org\/0456r8d26","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Recombinant poliovirus (PV) virus-like particle (VLP) antigens mimic the conformation of the surface proteins in native PVs (i.e., serotype-specific D-antigen epitopes). Since they lack genomes and are non-infectious, PV-VLPs offer the promise of a safer, next-generation polio vaccine compared to traditional inactivated (IPV) or attenuated live (OPV) vaccines. Sandwich D-antigen ELISA formats are commonly used to measure the in vitro potency values (relative D-antigen content, DU\/mL) of unadjuvanted trivalent IPV antigens. If IPV is formulated with aluminum-salt adjuvants, however, a pretreatment step (i.e., adjuvant dissolution or antigen desorption) is required, which may compromise antigen integrity during sample handling. Methods: This work describes the development of three competitive ELISAs to measure the relative D-antigen content of aluminum-salt adjuvanted PV-VLPs (Types 1, 2, 3) without the need for pretreatment. Results: First, key assay parameters were established, including specificity, accuracy, precision, linearity, limit of quantification, and stability-indication. Next, preformulation characterization studies were performed with these methods including (1) rank-ordering the inherent thermal stability profiles of the PV-VLPs (Types 1 &gt; 3 &gt; 2) in-solution and adsorbed to an aluminum phosphate adjuvant (AdjuPhos\u2122, AP) and (2) determining the effect of formulation variables on the thermal stability profiles of AP-adsorbed PV-VLPs including antimicrobial preservatives (thimerosal, 2-PE) and five different antigens present in pediatric combination vaccines (D, T, wP, Hib, Hep B). Conclusions: The development and application of three competitive D-antigen ELISAs were demonstrated, and future use in formulation and storage stability studies with the AP-adjuvanted, trivalent PV-VLPs (Types 1, 2, 3) is discussed with the long-term goal to develop a stable, efficacious, multi-dose, hexavalent combination vaccine presentation.<\/jats:p>","DOI":"10.3390\/vaccines14060479","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T09:57:43Z","timestamp":1779962263000},"page":"479","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Three Competitive ELISAs to Quantify the D-Antigen Content of Aluminum-Salt Adjuvanted Recombinant Polio VLPs (Types 1, 2, 3) to Enable Preformulation Characterization Studies"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6717-9485","authenticated-orcid":false,"given":"Yanli","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John M.","family":"Hickey","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0479-6873","authenticated-orcid":false,"given":"Geetha Satya Sainaga Jyothi","family":"Vaskuri","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brandy","family":"Dotson","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sangeeta B.","family":"Joshi","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1448-1998","authenticated-orcid":false,"given":"David B.","family":"Volkin","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2106","DOI":"10.4161\/21645515.2014.981115","article-title":"The polio endgame","volume":"10","author":"Minor","year":"2014","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.virol.2005.09.015","article-title":"One hundred years of poliovirus pathogenesis","volume":"344","author":"Racaniello","year":"2006","journal-title":"Virology"},{"key":"ref_3","unstructured":"Global Polio Eradication Initiative (2025, September 30). Update from the Global Polio Eradication Initiative on Programmatic Challenges and Accountability. Available online: https:\/\/polioeradication.org\/."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e207","DOI":"10.1097\/INF.0000000000004330","article-title":"Polio Eradication: Status, Struggles and Strategies","volume":"43","author":"Bandyopadhyay","year":"2024","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Liang, J., Zhang, Q., Li, Y., and Wang, L. (2025). Advances and challenges in poliomyelitis vaccines: A comprehensive review of development, production, and global deployment. Front. Public Health, 13.","DOI":"10.3389\/fpubh.2025.1611028"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2154100","DOI":"10.1080\/21645515.2022.2154100","article-title":"Current and next-generation formulation strategies for inactivated polio vaccines to lower costs, increase coverage, and facilitate polio eradication","volume":"18","author":"Kumar","year":"2022","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Hird, T.R., and Grassly, N.C. (2012). Systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002599"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1080\/14760584.2025.2508517","article-title":"Virus-like particles: A versatile and effective vaccine platform","volume":"24","author":"Bachmann","year":"2025","journal-title":"Expert Rev. Vaccines"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1038\/s41423-022-00897-8","article-title":"Virus-like particle vaccinology, from bench to bedside","volume":"19","author":"Mohsen","year":"2022","journal-title":"Cell. Mol. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1038\/s41541-020-00267-3","article-title":"Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines","volume":"6","author":"Bahar","year":"2021","journal-title":"npj Vaccines"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Hong, Q., Wang, S., Wang, X., Han, W., Chen, T., Liu, Y., Cheng, F., Qin, S., Zhao, S., and Liu, Q. (2024). Vaccine Potency and Structure of Yeast-Produced Polio Type 2 Stabilized Virus-like Particles. Vaccines, 12.","DOI":"10.3390\/vaccines12091077"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1038\/s41467-017-00090-w","article-title":"Plant-made polio type 3 stabilized VLPs-a candidate synthetic polio vaccine","volume":"8","author":"Marsian","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2340","DOI":"10.1016\/j.vaccine.2019.03.031","article-title":"Virus-like particle vaccines for poliovirus types 1, 2, and 3 with enhanced thermostability expressed in insect cells","volume":"37","author":"Xu","year":"2019","journal-title":"Vaccine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1038\/s41467-025-56118-z","article-title":"Recombinant expression systems for production of stabilised virus-like particles as next-generation polio vaccines","volume":"16","author":"Sherry","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Han, T., Xiao, J., Zhang, S., Su, T., Liu, Y., and Zhang, Y. (2026). Research Progress Towards Poliovirus Virus-like Particle Vaccines: A Review. Vaccines, 14.","DOI":"10.3390\/vaccines14030216"},{"key":"ref_16","first-page":"1013","article-title":"The modification of poliovirus antigens by heat and ultraviolet light","volume":"269","year":"1955","journal-title":"Lancet"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Sherry, L., Grehan, K., Swanson, J.J., Bahar, M.W., Porta, C., Fry, E.E., Stuart, D.I., Rowlands, D.J., and Stonehouse, N.J. (2022). Production and Characterisation of Stabilised PV-3 Virus-like Particles Using Pichia pastoris. Viruses, 14.","DOI":"10.1101\/2022.09.16.508282"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1007\/978-1-4939-3292-4_15","article-title":"Methods for the Quality Control of Inactivated Poliovirus Vaccines","volume":"Volume 1387","year":"2016","journal-title":"Poliovirus. Methods in Molecular Biology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/1045-1056(95)90017-9","article-title":"A WHO Collaborative study on assays of the antigenic content of inactivated poliovirus vaccines","volume":"23","author":"Wood","year":"1995","journal-title":"Biologicals"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1006\/biol.1995.0049","article-title":"A WHO collaborative study of immunogenicity assays of inactivated poliovirus vaccines","volume":"23","author":"Wood","year":"1995","journal-title":"Biologicals"},{"key":"ref_21","unstructured":"(2015). Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines (inactivated) Replacement of Annex 2 of WHO Technical Report Series, No. 910. WHO Tech. Rep. Ser., 993, 89\u2013174. Available online: https:\/\/cdn.who.int\/media\/docs\/default-source\/biologicals\/vaccine-standardization\/poliomyelitis\/annex3_ipv_recommendations_eng.pdf?sfvrsn=a1606f58_3&download=true."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"113785","DOI":"10.1016\/j.jviromet.2019.113785","article-title":"Universal ELISA for quantification of D-antigen in inactivated poliovirus vaccines","volume":"276","author":"Kouiavskaia","year":"2020","journal-title":"J. Virol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1006\/jcis.1998.5648","article-title":"Effect of pH on the Elution of Model Antigens from Aluminum-Containing Adjuvants","volume":"205","author":"Rinella","year":"1998","journal-title":"J. Colloid Interface Sci."},{"key":"ref_24","first-page":"156","article-title":"Solubilization of aluminum-containing adjuvants by constituents of interstitial fluid","volume":"45","author":"Seeber","year":"1991","journal-title":"PDA J. Pharm. Sci. Technol."},{"key":"ref_25","first-page":"9","article-title":"Desorption Kinetics Characterization of Virus-Like Particles in Polyvalent HPV Vaccine Containing Aluminum Phosphate Adjuvant","volume":"24","author":"Zhou","year":"2024","journal-title":"Am. J. Biomed. Sci. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1038\/s41541-018-0089-x","article-title":"Optimizing the utilization of aluminum adjuvants in vaccines: You might just get what you want","volume":"3","author":"HogenEsch","year":"2018","journal-title":"npj Vaccines"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1186\/s13036-025-00562-4","article-title":"Insect cell expression system: Advances in applications, engineering strategies, and bioprocess development","volume":"19","author":"Huang","year":"2025","journal-title":"J. Biol. Eng."},{"key":"ref_28","unstructured":"(2022). Report on the WHO Collaborative Study to Establish Universal Reagents for the D-Antigen Potency Testing of Inactivated Polio Vaccines (Standard No. WHO\/BS\/2022.2432)."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3078","DOI":"10.1002\/jps.23175","article-title":"Preformulation characterization of an aluminum salt-adjuvanted trivalent recombinant protein-based vaccine candidate against Streptococcus pneumoniae","volume":"101","author":"Iyer","year":"2012","journal-title":"J. Pharm. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3600","DOI":"10.1016\/j.vaccine.2020.02.001","article-title":"Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation","volume":"38","author":"Colaprico","year":"2020","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"34528","DOI":"10.1021\/acsomega.2c04266","article-title":"Improved Aluminum Adjuvants Eliciting Stronger Immune Response When Mixed with Hepatitis B Virus Surface Antigens","volume":"7","author":"Wang","year":"2022","journal-title":"Acs Omega"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2079346","DOI":"10.1080\/21645515.2022.2079346","article-title":"Antigen-adjuvant interactions, stability, and immunogenicity profiles of a SARS-CoV-2 receptor-binding domain (RBD) antigen formulated with aluminum salt and CpG adjuvants","volume":"18","author":"Bajoria","year":"2022","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"13406","DOI":"10.1074\/jbc.M500687200","article-title":"Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens","volume":"280","author":"Jones","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Fox, H., Knowlson, S., Minor, P.D., and Macadam, A.J. (2017). Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006117"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1866","DOI":"10.1080\/21645515.2019.1709352","article-title":"Potency of the Sabin inactivated poliovirus vaccine (sIPV) after exposure to freezing temperatures in cold chains","volume":"16","author":"Cai","year":"2020","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.biologicals.2016.06.005","article-title":"Determination of critical quality attributes for monoclonal antibodies using quality by design principles","volume":"44","author":"Alt","year":"2016","journal-title":"Biologicals"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/j.xphs.2023.12.011","article-title":"An Industry Perspective on the use of Forced Degradation Studies to Assess Comparability of Biopharmaceuticals","volume":"113","author":"Campbell","year":"2024","journal-title":"J. Pharm. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2059310","DOI":"10.1080\/21645515.2022.2059310","article-title":"Multi-dose vials versus single-dose vials for vaccination: Perspectives from lower-middle income countries","volume":"18","author":"Basu","year":"2022","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"4572","DOI":"10.1016\/j.vaccine.2016.07.030","article-title":"Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine","volume":"34","author":"Kraan","year":"2016","journal-title":"Vaccine"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"851","DOI":"10.1016\/0264-410X(94)90296-8","article-title":"Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine","volume":"12","author":"Sawyer","year":"1994","journal-title":"Vaccine"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3155","DOI":"10.1002\/jps.20976","article-title":"Antimicrobial preservative use in parenteral products: Past and present","volume":"96","author":"Meyer","year":"2007","journal-title":"J. Pharm. Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1894","DOI":"10.4161\/hv.25407","article-title":"Hexavalent IPV-based combination vaccines for public-sector markets of low-resource countries","volume":"9","author":"Mahmood","year":"2013","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2132","DOI":"10.1080\/21645515.2015.1044180","article-title":"The value of childhood combination vaccines: From beliefs to evidence","volume":"11","author":"Maman","year":"2015","journal-title":"Hum. Vaccines Immunother."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/6\/479\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T04:24:54Z","timestamp":1780115094000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/6\/479"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,28]]},"references-count":43,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["vaccines14060479"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14060479","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,28]]}}}