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.

Background and aimThe majority of seasonal influenza vaccines are trivalent, containing two A virus strains (H1N1 and H3N2) and one B virus strain. The co-circulation of two distinct lineages of B viruses can lead to mismatch between the influenza B virus strain recommended for the trivalent seasonal vaccine and the circulating B virus. This has led some manufacturers to produce quadrivalent influenza vaccines containing one strain from each B lineage in addition to H1N1 and H3N2 strains. However, it is also important to know whether vaccines containing a single influenza B strain can provide cross-protectivity against viruses of the antigenically distinct lineage. The aim of this study was to assess in naïve ferrets the potential cross-protective activity of trivalent live attenuated influenza vaccine (T-LAIV) against challenge with a heterologous wild-type influenza B virus belonging to the genetically different lineage and to compare this activity with effectiveness of quadrivalent LAIV (Q-LAIV) in the ferret model.Methods and resultsFerrets were vaccinated with either one dose of trivalent LAIV containing B/Victoria or B/Yamagata lineage virus, or quadrivalent LAIV (containing both B lineages), or placebo. They were then challenged with B/Victoria or B/Yamagata lineage wild-type virus 28 days after vaccination. The ferrets were monitored for clinical signs and morbidity. Nasal swabs and lung tissue samples were analyzed for the presence of challenge virus. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay. All LAIVs tested were found to be safe and effective against wild-type influenza B viruses based on clinical signs, and virological and histological data. The absence of interference between vaccine strains in trivalent and quadrivalent vaccine formulations was confirmed. Trivalent LAIVs were shown to have the potential to be cross-protective against infection with genetically different influenza B/Victoria and B/Yamagata lineages.ConclusionsIn this ferret model, quadrivalent vaccine provided higher protection to challenge against both B/Victoria and B/Yamagata lineage viruses. However, T-LAIV provided some cross-protection in the case of a mismatch between circulating and vaccine type B strains. Notably, B/Victoria-based T-LAIV was more protective compared to B/Yamagata-based T-LAIV.

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

The development of universal influenza vaccines, i.e. vaccines that can provide broad protection against seasonal and potentially pandemic influenza viruses, has been a priority for more than 20 years. Several approaches have been proposed that redirect the adaptive immune responses from immunodominant hypervariable regions to low-immunogenic but highly conserved regions of viral proteins. Here we induced broadly reactive anti-hemagglutinin (HA) stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HA (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. In addition, the source of the viral nucleoprotein (NP) of the LAIV strains was changed from A/Leningrad/17 master donor virus (MDV) to wild-type (WT) H1N1pdm09 virus, in order to induce CD8 T-cell immune responses more relevant to current infections. 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 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)). 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 LAIVs that combine the two approaches of inducing anti-HA stalk antibody and more relevant CD8 T-cell immune responses are highly promising candidates for further clinical development.

Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.