Fig 1.
Design of RSV F head-only immunogens.
(A) Genetic construct of RSV F illustrating the location of the head region. The signal peptide is colored yellow, F2 is colored blue, F1 is colored green, the transmembrane region is colored cyan and the cytoplasmic domain is colored gray. (B) Pre-F form of the RSV F trimer. One protomer of RSV F is depicted as a ribbon diagram and colored as in A. The other two protomers are gray surface representations. Antigenic site Ø and site II are red and orange respectively. Domains I, II and III are labeled as defined previously [9, 35]. (C) Models of head-only immunogens i-273, i-693 (upper panels), i-210 and i-447 (lower panels). For each immunogen a cartoon of the genetic construct is depicted (top) and a ribbon diagram (bottom), color-coded as in A and B with brown coloring for purification tags. PDB entries 4JHW [9], 1RFO [36] and 1GCM [37] were used to depict RSV F, the foldon and the coiled coil respectively. RSV F residue numbering follows the numbering in PDB entry 4JHW [9].
Fig 2.
Characterization of head-only immunogens i-273, i-693, i-210 and i-447.
(A-D) Size exclusion chromatography (SEC) analysis of all four immunogens after purification by affinity chromatography. For each immunogen an asterisk indicates the peak that was chosen for antigenic, physical, structural and immunization studies. The calculated molecular weight (MW) in kDa based on elution volume and the predicted MW based on the sequence and predicted glycan content (2.5 kDa/glycan) are also indicated. Immunogens i-273, i-693, i-210 and i-447 were predicted to have two, four, three and three glycans respectively based on the occurrence of NXT/S sequons in the sequence. (E) Reduced SDS-PAGE analysis (3μg/well) of immunogens (1) i-273, (2) i-693, (3) i-210 and (4) i-447 pooled from the asterisk-indicated peaks in (A-D). Doublet bands likely arise from differential glycosylation.
Table 1.
Physical Characterization of Head-only RSV F Immunogens.
Fig 3.
Negative-stain electron microscopy of head-only immunogens.
Panels (A-P) show typical 2D averaged classes of i-273,i-693, i-210 and i-447 alone (A, E, I and M), in complex with D25 (in blue; B, F, J and N), in complex with motavizumab (in green; C, G, K and O) and in complex with both D25 and motavizumab (in blue, green; D, H, L and P). Yellow indicates Fabs of ambiguous identity. Two separate averages are shown for each panel. White scale bars are 50 Å long.
Fig 4.
Head-only RSV F immunogen boost elicits neutralization titers surpassing DS-Cav1.
(A) All four immunogens were used to immunize mice two times, resulting in high neutralization titers for i-693 and i-447 above the protective threshold of 100 (dotted line). P values between i-447 and i-210 or i-273 were 0.001 and <0.0001 respectively and those between i-693 and i-210 or i-273 were 0.0014 and <0.0001 respectively. (B) Immunogens i-693 and i-447 were used to boost DS-Cav1 seven weeks later resulting in titers higher than a DS-Cav1 homologous boost. Scatter plots show the geometric mean (numerical value below) with error bars representing the 95% confidence level. P values were determined by two-tailed Mann-Whitney tests. Each group included 10 mice.
Fig 5.
Boost by head-only RSV F immunogen can focus the RSV-neutralizing response to antigenic sites Ø and II.
(A) Recognition of DS-Cav1, DS-Cav1 site Ø knockout (KO) and DS-Cav1 site II KO probes by sera from 2×DS-Cav1 immunized mice boosted with DS-Cav1, i-447 or i-693, respectively or unimmunized mice. (B) Neutralization competition of sera from 2×DS-Cav1 immunized mice boosted with DS-Cav1, i-447 or i-693, respectively. P values for differences between DS-Cav1 and DS-Cav1 site Ø or site II KO probes were all <0.0001. P values were determined by two-tailed Mann-Whitney test.