AUTHOR CONTRIBUTIONS : Conceptualization: Kamogelo M. Sepotokele, Martha M. O’Kennedy.
Data curation: Kamogelo M. Sepotokele, Celia Abolnik.
Formal analysis: Kamogelo M. Sepotokele, Celia Abolnik.
Funding acquisition: Celia Abolnik.
Investigation: Kamogelo M. Sepotokele, Martha M. O’Kennedy, Daniel B. R. Wandrag, Celia
Abolnik.
Methodology: Kamogelo M. Sepotokele, Martha M. O’Kennedy, Celia Abolnik.
Resources: Martha M. O’Kennedy, Celia Abolnik.
Supervision: Martha M. O’Kennedy, Celia Abolnik.
Writing – original draft: Kamogelo M. Sepotokele, Celia Abolnik.
Writing – review & editing: Kamogelo M. Sepotokele, Martha M. O’Kennedy, Daniel B. R.
Wandrag, Celia Abolnik.
SUPPORTING INFORMATION : FIGURE S1. Protein sequence of the synthetic gene mIBV-S2P. The murine signal peptide is highlighted in blue, the linker in magenta, and the S2 domain in grey with heptad repeat 1 in yellow, the central helix in green, and the two stabilizing proline substitutions in boldface and underlined. FIGURE S2. Protein confirmation using LC-MS/MS-based peptide sequencing of the modified IBV spike protein constructs compared in this study (A) mIBV-S2P, (B) mIBV-- S2P-IAV-H6TM/CT, and (C) mIBV-S2P-NDV-FTM/CT. The percentage sequence coverage is indicated above with several unique peptides identified with > 90% confidence. Peptides with > 95% confidence are highlighted in green, those with 50–95% confidence in yellow, and those with <50% confidence in red. No peptides were identified for the non-highlighted regions of the sequence (grey). FIGURE S3. Densitometric analysis by SDS-PAGE of partially-purified mIBV-S2P-NDV-FTM/ CT VLPs. Lane 1: SeeBluePlus2 protein ladder; Lane 2: BSA Standard 100 ng/μl; Lane 3: BSA Standard 150 ng/μl; Lane 4: BSA Standard 200 ng/μl; Lane 5: BSA Standard 250 ng/μl; Lane 6: BSA Standard 300 ng/μl; Lane 7: Dialysed VLP sample (25 μl); Lane 8: Dialysed VLP sample (10 μl); Lane 9: PageRuler Prestained protein ladder; Lane 10: Positive control (Live QX-like IBV); Lane 11: Negative control (pEAQ-HT-empty). FIGURE S4. Original uncropped, unedited SDS-PAGE of partially-purified plant-produced IBV S protein Lane 1: molecular weight marker; Lane 2: plant-expressed empty pEAQ-HT vector; Lane 3: purified live QX-like IBV strain ck/ZA/3665/11; Lanes 4–7: mIBV-S2P:M:E:N fractions 2 (lanes 4 and 6) and 3 (lanes 5 and 7) extracted in either PBS or bicine as indicated; Lanes 8–11: mIBV-S2P-IAV-H6TM/CT:M2 fractions 2 (lanes 8 and 10) and 3 (lanes 9 and 11) extracted in either PBS or bicine as indicated; Lanes 12–15: mIBV-S2P-NDV-FTM/ CT:NDV Matrix fractions 2 (lanes 12 and 14) and 3 (lanes 13 and 15) extracted in either PBS or bicine as indicated. FIGURE S5. Original uncropped, unedited Western blot (B) of partially-purified plant-produced IBV S protein (Primary antibody–IBV antisera, secondary antibody—Goat-α- Chicken IgY HRP). Lane 1: molecular weight marker; Lane 2: plant-expressed empty pEAQ-HT vector; Lane 3: purified live QX-like IBV strain ck/ZA/3665/11; Lanes 4–7: mIBV-S2P:M:E:N fractions 2 (lanes 4 and 6) and 3 (lanes 5 and 7) extracted in either PBS or bicine as indicated; Lanes 8–11: mIBV-S2P-IAV-H6TM/CT:M2 fractions 2 (lanes 8 and 10) and 3 (lanes 9 and 11) extracted in either PBS or bicine as indicated; Lanes 12–15: mIBV-S2P-NDV-FTM/CT:NDV Matrix fractions 2 (lanes 12 and 14) and 3 (lanes 13 and 15) extracted in either PBS or bicine as indicated. RAW IMAGES S1.