Extended Data Fig. 3: Controllable co-display of multiple antigenic variants on two-component nanoparticle immunogens.

a, Sandwich BLI comparing qsCocktail-I53_dn5 and qsMosaic-I53_dn5. Biotinylated 5J8 immobilized on streptavidin probes was used to capture H1-containing nanoparticles from each sample. The captured particles were then exposed to antibodies specific to H3 (CR8020; left) or influenza B HA (CR8071; right). b, Numerical approximation of the H1 HA content of individual qsMosaic-I53_dn5 nanoparticles assuming an equimolar quadrivalent in vitro assembly reaction (that is, 25% of the input HA-I53_dn5B trimers bear H1 HA) and random incorporation of each HA-I53_dn5B trimer at each of the 20 trimeric positions into the nanoparticle. A distribution centred on 25% valency (5 H1 HA trimers per nanoparticle) is observed. c, Calculation of the fraction of individual mosaic nanoparticles displaying at least one H1 HA trimer as a function of the fractional concentration of H1-I53-dn5B in the in vitro assembly reaction ([H1]), expressed as: 1 − (1 − [H1])²⁰. At the 25% fractional concentration used to assemble qsMosaic-I53_dn5, 99.7% of the individual nanoparticles are expected to display at least one H1 HA trimer. d, Quantification of HA antigen content by peptide mass spectrometry in three distinct qsMosaic-I53_dn5 nanoparticles with various antigen ratios before and after preparative SEC. Dashed lines represent the fractional concentration of each HA in the in vitro assembly reactions used to prepare the mosaic nanoparticle immunogens, main bars represent the mean values of four unique peptides from each HA, and error bars represent the standard deviation of measurements across the four unique peptides from each HA. The peptides used to quantify each HA are provided in Supplementary Table 3.