FIG 2.

Vaccine α1α1 fragment. (A) Schematic overview of TIGR4 PspA α1α2, adapted from Vaccine vol. 33, K. Kuipers et al., Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization, p. 2022–2029, Copyright 2015, with permission from Elsevier (17). The slightly overlapping α1 and α2 fragments (overlap of 9 amino acids [aa]) were selected to cover the α-helical coiled-coil domain. The lactoferrin binding domain (LFBD), the proline-rich region (PRR), and the choline binding domain (CBD) of PspA were not included in the vaccine. SS, signal sequence. (B) Alignment of the α1α2 domains derived from 345 sequenced pneumococcal invasive isolates. Based on amino acid identity, MIME analysis subdivided the α1α2 vaccine region into six variable regions (VR1 to VR6). Some VRs appeared more conserved and were alternately expressed by the clinical isolates. Variable domains are numbered according to their sequence conservation, as determined by the E value. The percentage of VR expression among pneumococcal isolates was calculated. The presence of the VR is shown as percentages. VRs 3, 4, 5, 2, and 1 were present in TIGR4; VR3, VR4, and VR5 are part of α1; and VR2 and VR1 are part of α2, and the positions of the amino acids are indicated. (C) NetMHCII was used to predict T-cell epitopes present in TIGR4 α1α2. The binding affinity is expressed as IC₅₀ values (y axis). The different 15-mer peptides with a 1-amino-acid overlap are indicated on the x axis from amino acids 32 to 244, spanning the whole α1α2 domain. The region within the TIGR4 α1α2 domain showed a predicted IC₅₀ value of around or lower than 100 nm, indicating strong binding affinity, including amino acids 41 to 61 (blue), 151 to 171 (red), and 180 to 199 (green), with the lowest values being measured for EVRAVVVPEPNALAE (dashed red bar) and AKRKYDYATLKVALA (dashed green bar).