Fig 1.
Selection of naturally occurring CIDRα1 sequences for the design of CIDRα1-cVLP vaccines.
A. Structure of the HB3VAR03 CIDRα1.4 domain with close-up on the EPCR binding (EB) and EPCR binding supporting (EBS) helices [5]. Highlighted in red are the amino acids kept constant among CIDRα1 variants chosen for the mosaic-conserved-cVLP vaccine. B. Sequence conservation LOGO of 963 naturally occurring CIDRα1 sequences. Asterisks indicate the positions kept constant among CIDRα1 variants chosen for the mosaic-conserved-cVLP vaccine. Marked residues on the EB helix also interact directly with EPCR. C. Schematic representation of the selection process resulting in the identification of the CIDRα1 variants used in this study. D. Sequence LOGO of the 13 selected amino acid positions among the CIDRα1 variants selected for the three mosaic cVLP vaccines. E. Pairwise sequence identity of the 14 CIDRα1 variants selected for the mosaic cVLP vaccines. One variant (GA273) is shared between the conserved and semi-conserved cVLP vaccines.
Fig 2.
Formulation and characterization of vaccines.
A. Schematic representation of vaccine formulations. Coupling of individual CIDRα1 proteins on cVLPs gives rise to homotypic particles, which are mixed to obtain three different cVLP cocktails (cocktail-diverse, cocktail-semi-conserved, cocktail-conserved) matching composition of the mosaic cVLPs. Coupling of five variants on the same cVLP results in mosaic cVLP vaccines (mosaic-diverse, mosaic-semi-conserved, mosaic-conserved). Coupling of homotypic cVLPs with CIDRα1 variants highlighted by rectangles was unsuccessful thus, the equivalent homotypic cVLPs lack from the corresponding cocktails. B. SDS-PAGE of five homotypic cVLPs and mosaic-conserved-cVLPs. First lane (1) represents the samples pre-dialysis. The bands correspond to Tag-cVLP (16.5 kDa), unbound Catcher-CIDRα1 (~40 kDa) and coupled CIDRα1-cVLP (~56 kDa). The second lane (2) represents the samples post-dialysis and before spin test. The third lane (3) represents the samples post-dialysis and post-spin test. SDS-PAGE of the remaining samples is reported in S2 Fig. Gel images are uncropped but stitched together from multiple gels. C. Dynamic-Light-Scattering analysis of cocktail-conserved-cVLPs and mosaic-conserved-cVLPs. Similar data for the remaining cVLP vaccines is reported in S3 Fig. Naked SpyT-cVLPs (dashed line) predominant population shows 65.7 nm with 81.2% Pd. Cocktail-conserved-cVLPs indicate a population size of 69.3–162.9 nm with 38.9–72.4% Pd and mosaic-conserved-cVLPs show 170.6 nm and 58.7% Pd. D. Distribution of CIDRα1domains coupled to cVLPs as indicated by mass spectrometry analysis (details are reported in S1 File). Abbreviations: MC, mosaic-conserved.
Table 1.
Vaccination scheme.
Fig 3.
Reactivity and EPCR-binding inhibition of elicited antibodies.
A. Heat map showing antibody responses to 25 CIDRα1 protein variants (rows). For each vaccination group (columns), percentage of identity (ID) between vaccine and test protein, IgG’s reactivity (R) towards the test protein variant measured as mean fluorescence intensity (MFI) and the IgG’s EPRC-binding inhibitory ability (I) are reported. For each test antigen variant, the ID score refers to the highest pairwise identity observed between the test antigen and any of the five CIDRα1 variants in the given vaccine. Black rectangles identify CIDRα1 subgroups (CIDRα1.1–8) to which variants in the vaccine belong. B. Correlation of sequence identity (%ID) and IgG reactivity (MFI). Each symbol represents reactivity to and percentage of identity with one of the 25 CIDRα1 test proteins. Spearman’s rank correlation = 0,6447, p-value <0.0001, alpha = 0,05. C. Correlation of reactivity (MFI) and EPCR-binding inhibition ability of pooled IgG from each vaccination group. Each symbol corresponds to inhibition and reactivity against one of 25 test CIDRα1 proteins. Spearman’s rank correlation = 0.6626, p-value <0.0001, alpha = 0,05. CIDRα1 test variants showing 100% sequence identity to vaccine variants are circled. Abbreviations: CD, cocktail-diverse; MD, mosaic-diverse: CSC, cocktail-semi-conserved; MSC, mosaic-semi-conserved; CC, cocktail-conserved; MC, mosaic-conserved.