The development of universal influenza vaccines has been a priority for more than 20 years. We conducted a preclinical study in ferrets of two sets of live attenuated influenza vaccines (LAIVs) expressing chimeric hemagglutinin (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but had antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. The viral nucleoproteins (NPs) in the two sets of universal LAIV candidates were from different sources: one LAIV set contained NP from A/Leningrad/17 master donor virus (MDV), while in the other set this gene was from wild-type (WT) H1N1pdm09 virus, in order to better match the CD8 T-cell epitopes of currently circulating influenza A viruses. To avoid any difference in protective effect of the various anti-neuraminidase (NA) antibodies, all LAIVs were engineered to contain the NA gene of Len/17 MDV. Naïve ferrets were sequentially immunized with three doses of (i) classical LAIVs containing non-chimeric HA and NP from MDV (LAIVs (NP-MDV)); (ii) cHA-based LAIVs containing NP from MDV (cHA LAIVs (NP-MDV)); and (iii) cHA-based LAIVs containing NP from H1N1pdm09 virus (cHA LAIVs (NP-WT)). All vaccination regimens were safe, producing no significant increase in body temperature or weight loss, in comparison with the placebo group. The two groups of cHA-based vaccines induced a broadly reactive HA stalk-directed antibody, while classical LAIVs did not. A high-dose challenge with H1N1pdm09 virus induced significant pathology in the control, non-immunized ferrets, including high virus titers in respiratory tissues, clinical signs of disease and histopathological changes in nasal turbinates and lung tissues. All three vaccination regimens protected animals from clinical manifestations of disease: immunized ferrets did not lose weight or show clinical symptoms, and their fever was significantly lower than in the control group. Further analysis of virological and pathological data revealed the following hierarchy in the cross-protective efficacy of the vaccines: cHA LAIVs (NP-WT) > cHA LAIVs (NP-MDV) > LAIVs (NP-MDV). This ferret study showed that prototype universal cHA-based LAIVs are highly promising candidates for further clinical development.

The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, the S protein shows considerable sequence variations among variants of concern. The aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The vaccine, formulated as a squalane-based emulsion, was used to immunize Balb/c mice and NOD SCID gamma (NSG) mice engrafted with human PBMCs, rabbits and marmoset monkeys. Safety and immunogenicity of the vaccine was assessed via ELISA, cytokine titer assays and CFSE dilution assays. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Immunization induced sustainable N-specific IgG responses and an N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys, an N-specific CD4+/CD8+ T cell response was observed. Vaccinated Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower lung weight relative to the body, and faster body weight recovery. Convacell® thus is shown to be effective and may augment the existing armamentarium of vaccines against COVID-19.

Background The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, S and RBD from SARS-CoV-2 variants of concerns show considerable sequence variations and repeated injections for boosting specific immunity are necessary. Aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Methods Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The N protein vaccine was obtained by formulation of recombinant N as squalane-based emulsion and used to immunize Balb/c mice, NOD scid gamma (NSG) mice engrafted with human PBMC, rabbits and marmoset monkeys to study safety as well as antibody and cellular immunity using ELISA for antibodies, measurement of N-specific Th1 and Th2 cytokine secretion and carboxyfluorescein succinimidyl ester (CFSE) dilution assays for CD4 and CD8 T cell responses. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Results Immunization of mice, rabbits and Syrian hamsters with the recombinant N protein-based vaccine formulated as squalane-based emulsion (Convacell®) induced sustainable N-specific IgG responses and a N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys a N-specific CD4 as well as CD8 T cell response was observed. Vaccinated and then infected Syrian hamsters showed reduced lung histopathology, reduced virus was detected in lung tissue, lung weight relative to the body was not increased after challenge and body weight was regained faster than in non-vaccinated animals. Repeated dose toxicity studies in mice and rabbits showed that Convacell® was well tolerated and safe. Conclusions Convacell® induced a SARS-CoV-2-specific protective immune response in Syrian hamsters. It is a new vaccine targeting the nucleocapsid protein of SARS-CoV-2 and thus may augment the armamentarium of vaccines for COVID-19.

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

Influenza H7N9 virus is a potentially pandemic subtype to which most people are immunologically naïve. To be better prepared for the potential occurrence of an H7N9 pandemic, in 2017 the World Health Organization recommended developing candidate vaccine viruses from two new H7N9 viruses, A/Guangdong/17SF003/2016 (A/GD) and A/Hong Kong/125/2017 (A/HK). This report describes the development of live attenuated influenza vaccine (LAIV) candidates against A/GD and A/HK viruses and study of their safety and immunogenicity in the ferret model in order to choose the most promising one for a phase I clinical trial. The A/HK-based vaccine candidate (A/17/HK) was developed by classical reassortment in eggs. The A/GD-based vaccine candidate (A/17/GD) was generated by reverse genetics. Ferrets were vaccinated with two doses of LAIV or phosphate-buffered saline. Both H7N9 LAIVs tested were safe for ferrets, as shown by absence of clinical signs, and by virological and histological data; they were immunogenic after a single vaccination. These results provide a compelling argument for further testing of these vaccines in volunteers. Since the A/HK virus represents the cluster that has caused the majority of human cases, and because the A/HK-based LAIV candidate was developed by classical reassortment, this is the preferred candidate for a phase I clinical trial.