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.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 μg/dose or 15 μg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

The emergence of potentially pandemic viruses has resulted in preparedness efforts to develop candidate vaccines and adjuvant formulations. We evaluated the dose-sparing effect and safety of two distinct squalene-based oil-in-water adjuvant emulsion formulations (IB160 and SE) with influenza A/H7N9 antigen. This phase I, randomized, double-blind, placebo-controlled, dose-finding trial (NCT03330899), enrolled 432 healthy volunteers aged 18 to 59. Participants were randomly allocated to 8 groups: 1A) IB160 + 15μg H7N9, 1B) IB160 + 7.5μg H7N9, 1C) IB160 + 3.75μg H7N9, 2A) SE + 15μg H7N9, 2B) SE + 7.5μg H7N9, 2C) SE + 3.75μg H7N9, 3) unadjuvanted vaccine 15μg H7N9 and 4) placebo. Immunogenicity was evaluated through haemagglutination inhibition (HI) and microneutralization (MN) tests. Safety was evaluated by monitoring local and systemic, solicited and unsolicited adverse events (AE) and reactions (AR) 7 and 28 days after each study injection, respectively, whereas serious adverse events (SAE) were monitored up to 194 days post-second dose. A greater increase in antibody geometric mean titers (GMT) was observed in groups receiving adjuvanted vaccines. Vaccinees receiving IB160-adjuvanted formulations showed the greatest response in group 1B, which induced an HI GMT increase of 4.7 times, HI titers ≥40 in 45.2% of participants (MN titers ≥40 in 80.8%). Vaccinees receiving SE-adjuvanted vaccines showed the greatest response in group 2A, with an HI GMT increase of 2.5 times, HI titers ≥40 in 22.9% of participants (MN titers ≥40 in 65.7%). Frequencies of AE and AR were similar among groups. Pain at the administration site and headache were the most frequent local and systemic solicited ARs. The vaccine candidates were safe and the adjuvanted formulations have a potential dose-sparing effect on immunogenicity against influenza A/H7N9. The magnitude of this effect could be further explored.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.