N1 NA selection, characterization of recombinant N1 NAs, and generation of immune sera. (a) Phylogenetic relatedness based on the amino acid sequence of 17 selected N1 NAs next to 5 randomly selected isolates from 2009-2020 (5 per year), and the reference A/California/07/2009 strain. Orange triangles: seasonal vaccine strains that were included here as N1 NA antigens; blue triangle: prototypic H1N1pdm. (b) The specific NA activity of the recombinant N1 NAs based on the MUNANA assay. (c) Ratio of protein to oseltamivir binding (mg/ml)/(mg/ml). (d) Groups of 5 female BALB/c mice were immunized in a prime-boost regimen in a 2 weeks interval with AF03-adjuvanted N1 NAs. Homologous endpoint ELISA titers were determined in pooled sera collected 4 weeks after the prime immunization.

Breadth of N1 NA inhibition. NAI against 15 different HxN1 strains (X-axis) by mouse sera raised by immunization with recombinant N1 NA (Y-axis). The sera are ordered on the Y-axis according to the phylogenetic relatedness of the N1 NA amino acid sequence, with amino acid substitutions depicted above the branches. The two phylogenetic groups are color coded and labeled as PG1 and PG2. Hierarchical clustering is depicted above the heat-map and the derived antigenic groups AG1, AG2, and NIBRG-14 are color coded. The red to blue scale represents the normalized NAI values as fold lower than the serum with the highest normalized NAI for each virus. The highest NAI value against NIBRG-14 was obtained with a homologous post-challenge as positive control serum (not presented).

Machine learning algorithm allows to predict the antigenic distance and identify antigenically important amino acid substitutions. (a) Sera to sera differences were used to calculate the average antigenic distances that are represented in the heat map. (b) Correlation of the antigenic distances predicted by the RF model and the values obtained based on the observed values. (c) The “Mean Squared Error Increase” and “Times in a root” for each amino acid substitution in the N1 NAs. (d) Distribution of amino acid substitutions on the surface of N1. The substitutions with the strongest impact according to the RF model are indicated in red. The NA catalytic pocket is indicated on one of the protomers on the top view of the structure. Top, side, and bottom views of tetrameric H1N1pdm 2009 NA (PDB: 3NSS) are shown.

NAI against NIBRG-14 and comparison of immune serum raised against two batches of A/Stockholm/25/2012. (a) NAI titers against NIBRG-14 of phylogenetic group 1 (2009-2013) and group 2 (2015-2020). (b) Paired t test and (c) Pearson correlation of ELISA normalized NAI of sera raised against batch 162 and 946 of recombinant N1 NA derived from A/Stockholm/25/2012. ****P<0.0001.

Antigenic map of NA of the H1N1 viruses. Sera are represented by squares and strains by dots. The phylogenetic and antigenic group 1 NAs are represented by orange and phylogenetic and antigenic group 2 NAs by green symbols. NA of NIBRG-14 is depicted in blue.