H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.</p

Varying Viral Replication and Disease Profiles of H2N2 Influenza in Ferrets Is Associated with Virus Isolate and Inoculation Route

H2N2 influenza virus, the causative agent of the 1957 “Asian flu” pandemic, has disappeared from circulation. However, H2-influenza viruses are still circulating in avian reservoirs. Combined with the waning of H2N2-specific immunity in the human population, there is a risk of reintroduction of H2N2 influenza virus. Vaccines could help in preventing a future pandemic, but to assess their efficacy animal models are required. We therefore set out to expand the ferret model for H2N2 influenza disease by infecting ferrets intranasally or intratracheally with four different H2N2 viruses to investigate their influence on the severity of disease. The H2N2 viruses were collected either during the pandemic or near the end of H2N2 circulation and covered both clade I and clade II viruses. Infection of ferrets with the different viruses showed that viral replication, disease, and pathology differed markedly between virus isolates and infection routes. Intranasal inoculation induced a severe to mild rhinitis, depending on the virus isolate, and did not lead to lung infection or pathology. When administered intratracheally, isolates that successfully replicated in the lower respiratory tract (LRT) induced a nonlethal disease that resembles that of a moderate pneumonia in humans. Differences in viral replication and disease between viruses could be associated with their binding preference for a2,3- and a2,6-sialic acid. The model presented here could facilitate the development of a new generation of H2N2 influenza vaccines

H7N9 live attenuated influenza vaccine is highly immunogenic, prevents virus replication, and protects against severe bronchopneumonia in ferrets

Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virus-neutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model

Varying Viral Replication and Disease Profiles of H2N2 Influenza in Ferrets Is Associated with Virus Isolate and Inoculation Route.

H2N2 influenza virus, the causative agent of the 1957 "Asian flu" pandemic, has disappeared from circulation. However, H2-influenza viruses are still circulating in avian reservoirs. Combined with the waning of H2N2-specific immunity in the human population, there is a risk of reintroduction of H2N2 influenza virus. Vaccines could help in preventing a future pandemic, but to assess their efficacy animal models are required. We therefore set out to expand the ferret model for H2N2 influenza disease by infecting ferrets intranasally or intratracheally with four different H2N2 viruses to investigate their influence on the severity of disease. The H2N2 viruses were collected either during the pandemic or near the end of H2N2 circulation and covered both clade I and clade II viruses. Infection of ferrets with the different viruses showed that viral replication, disease, and pathology differed markedly between virus isolates and infection routes. Intranasal inoculation induced a severe to mild rhinitis, depending on the virus isolate, and did not lead to lung infection or pathology. When administered intratracheally, isolates that successfully replicated in the lower respiratory tract (LRT) induced a nonlethal disease that resembles that of a moderate pneumonia in humans. Differences in viral replication and disease between viruses could be associated with their binding preference for α2,3- and α2,6-sialic acid. The model presented here could facilitate the development of a new generation of H2N2 influenza vaccines. IMPORTANCE In 1957 the world was subjected to a pandemic caused by an influenza A virus of the subtype H2N2. Although the virus disappeared in 1968, H2 viruses continue to circulate in avian reservoirs. It is therefore possible that the H2N2 influenza virus will be reintroduced into the human population, which can lead to another pandemic. The impact of a new H2N2 influenza pandemic can be mitigated by vaccination. However, these vaccines first need to be developed and tested in animal models. In preparation for this, we expanded the ferret model to mimic the different facets of human H2N2 influenza infection and disease. This model can be used for the development and evaluation of new H2N2 influenza vaccines

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation

Replication of H2N2 influenza viruses in respiratory organs and brain of mice.

<p>Groups of 6 female CBA mice were infected i.n. with the corresponding viruses at a dose of 100 MID₅₀ in a volume of 50 µl. Three days after inoculation, mice were euthanized and lungs and nasal turbinateswere harvested. Virus titers in the organs were determined by end-point titration in eggs and expressed as log₁₀EID₅₀/ml. Significant lower titers with respect to the donor viruses are indicated by * (p<0,05) or ** (p<0,005). Bars indicate the average titer.</p

Reproductive capacity at different temperatures in eggs and median mouse infection dose (MID₅₀) of H2N2 LAIV candidates and their parental viruses.

<p>n/t, not tested.</p

Replication of H2N2 challenge viruses in organs of immunized mice.

<p>The animals were challenged with 100 MID₅₀ of either A/California/1/66 (left panel) or A/Tokyo/3/67 (right panel) wild-type virus on day 42 after the first immunization. Three days after challenge, respiratory (lung and nasal turbinates) and systemic (spleen and brain – not shown) organs were collected from four mice in each group. Virus titers in the organs were determined by end-point titration in eggs and expressed as log₁₀EID₅₀/ml. The depicted vaccine groups represent the low (100MID₅₀) and high (1000MID₅₀) dose as these were all below detection limit.</p

Immunogenicity and cross-reactivity of H2N2 LAIV candidates in a mouse model.

<p>Homologous and cross-reactive antibody responses in mice 21 days after 1^st vaccination (hatched boxes) and 21 days after 2^nd vaccination (day 42; open boxes) as measured by haemagglutination inhibition. Titers induced by the 17/Cal/395 (blue) and 17/Tok/326 (green) vaccines (either at a dose of 100 MID₅₀ or 1000 MID₅₀) are grouped by the respective antigens indicated at the top of the panels. Serum was titrated against 4HAU of the respective wild type viruses using 0.5% chicken erythrocytes. In sera of placebo vaccinated animals and sera prior to vaccination no titers above detection limit were detected (not shown). Presented are box plots of log₂ transformed HI-titers. Bars indicate the 5–95 percentile and black dots indicate the average titer.</p

Virus titers in the throat swabs of ferrets challenged with wt H2N2 viruses.

<p>Ferrets vaccinated with either placebo (squares), 17/Cal/395 (triangles), 17/Tok/326 (inverted triangles) or Len/17 (diamonds) were challenged 21 days after vaccination with either A/Tok/67 wt (upper panel) or A/Cal/66 wt (lower panel) virus. Throat swabs were taken prior to the challenge and 2, 3 and 5 days after challenge. The virus titer (TCID₅₀) in the transport buffer was determined by end-point titration on MDCK cells using a 5 fold serial dilution. Presented are the average log₁₀ transformed titers. Bars represent the standard deviation and the dotted line indicates limit of detection.</p