Ricardo das Neves Oliveira

Background/Objectives: Adjuvanted influenza vaccines are critical to enhance immune responses in high-risk populations, such as the elderly. 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 °C ± 2.0 °C 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 µg 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 < 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 µg 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.

Background/Objectives:H5N1 influenza viruses are spreading worldwide and threaten global public health. Preparedness is necessary to mitigate the worst-case scenario should an H5N1 influenza pandemic occur and justify the development of vaccines against circulating H5N1 viruses of concern. Methods: The production and characterization of egg-based split and inactivated H5Nx of three distinct monovalent antigens from clades 2.3.4.4b, 2.3.2.1c, and 2.3.4 were performed at an industrial scale. These antigens were formulated and their immune responses, when combined or not with IB160 squalene-based oil-in-water emulsion adjuvant in a rat model, were evaluated in a one- or two-dose immunization schedule. IgG antibodies, hemagglutination inhibitions, and microneutralization titers were measured for vaccine-induced immunity and cross-reactivity. Results: Three monovalent vaccines from clades 2.3.4.4b, 2.3.2.1c, and 2.3.4 were produced at an industrial scale and characterized. The immune responses against the monovalent vaccines showed a clade-specific antibody response and the need to combine with IB160 adjuvant for a required immune response. Conclusions: Considering the candidate vaccine viruses (CVVs) with the testing potency reagents available and that the antibody response obtained against the CVVs produced was clade-specific, IDCDC RG-71A is the indicated CVV for the predominant currently circulating H5N1 influenza virus of clade 2.3.4.4b and must be combined with adjuvant to induce a higher and efficacious immune response in a two-dose immunization protocol.

The prevalence of the highly pathogenic avian influenza virus H5N1 in wild birds that migrate all over the world has resulted in the dissemination of this virus across Asia, Europe, Africa, North and South America, the Arctic continent, and Antarctica. So far, H5N1 clade 2.3.4.4.b has reached an almost global distribution, with the exception of Australia and New Zealand for autochthonous cases. H5N1 clade 2.3.4.4.b, derived from the broad-host-range A/Goose/Guangdong/1/96 (H5N1) lineage, has evolved, adapted, and spread to species other than birds, with potential mammal-to-mammal transmission. Many public health agencies consider H5N1 influenza a real pandemic threat. In this sense, we analyzed H5N1 hemagglutinin sequences from recent outbreaks in animals, clinical samples, antigenic prototypes of candidate vaccine viruses, and licensed human vaccines for H5N1 with the aim of shedding light on the development of an H5N1 vaccine suitable for a pandemic response, should one occur in the near future.