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While initial studies relied on a two-vial system with antigen and adjuvant mixing prior to immunization, this study evaluated the feasibility, immunogenicity, and 12-month stability of a one-vial formulation combining a Trivalent Influenza Vaccine (TIV) with the locally produced squalene-based oil-in-water emulsion IB160. Methods: Formulations were evaluated by testing TIV:IB160 (v\/v) ratios (1:1, 2:1, 3:1) in an animal model, measuring HI antibodies as the primary immunogenicity metric, complemented by qualitative strain-specific total IgG screening by ELISA. One-vial and two-vial strategies were also compared regarding immune responses. Formulations stabilities were evaluated at 6.0 \u00b0C \u00b1 2.0 \u00b0C in one-and two-vial systems by monitoring physicochemical features and their antigenic responses, measuring hemagglutinin content of the formulated vaccines, and long-term HI antibody responses. Results: The 2:1 and 1:1 (v\/v) formulation ratios (15 \u00b5g TIV:IB160) induced significantly higher HI titers across all tested strains (A\/H3N2\/Thailand, A\/H1N1\/Victoria, and B\/Austria) when compared to non-adjuvanted TIV or adjuvanted TIV in the 3:1 ratio. Both the one and two-vial strategies showed significantly higher HI antibody responses than the non-adjuvanted control group (p &lt; 0.05), with no significant differences observed between the delivery formats. Indeed, the one-vial formulation with 1:1 and 2:1 ratios remained stable for 12 months, preserving TIV antigenicity, IB160 emulsion integrity, and robust immunogenicity. Conclusions: One- and two-vial formulations are viable, stable, and highly immunogenic alternatives for adjuvanted TIV in 2:1 and 1:1 (v\/v) formulation ratios (15 \u00b5g TIV:IB160). One-vial format simplifies immunization logistics, minimizes operational errors, and is more indicated for routine use, while the two-vial system remains a versatile asset for emergency responses.<\/jats:p>","DOI":"10.3390\/vaccines14090789","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T09:24:02Z","timestamp":1788945842000},"page":"789","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterization of Seasonal Trivalent Influenza Vaccine Formulated with Different Ratios (v\/v) of Squalene-Containing IB160 Adjuvant in One- or Two-Vial Presentations"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9234-1758","authenticated-orcid":false,"given":"Daniela","family":"Cajado-Carvalho","sequence":"first","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, 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Foundation, Sao Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6303-2144","authenticated-orcid":false,"given":"L\u00edvia Mendon\u00e7a Munhoz","family":"Dati","sequence":"additional","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Mahyumi","family":"Fujimori","sequence":"additional","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, 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Anselmo","family":"Sakihara","sequence":"additional","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Bianca Pereira Carvalho","family":"Holanda","sequence":"additional","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"given":"Douglas Gon\u00e7alves de","family":"Macedo","sequence":"additional","affiliation":[{"name":"Bioindustrial Center, Butantan Institute, and Butantan Foundation, Sao Paulo 05503-900, SP, 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