Table 1.
Nanobody sequence, specificity and neutralisation activity.
| Nb | Sequence | Binding specificity (inactivated whole virus antigen) | Neutralisation | ||||||
|---|---|---|---|---|---|---|---|---|---|
| VHH CDR3 sequence (family) | TX/2012 (H3N2) | M/N/2000 (H7N3) | NY/2003 (H7N2) | AN/2013 (H7N9) | GD/2016 (H7N9) | HK/2017 (H7N9) | TX 2012 (H3N2) | SH 2013 (H7N9) | |
| NB7-01 | ALARSVDRLTYDY (1) | - | 2.83 | 2.53 | 2.26 | 2.59 | 1.94 | - | ++++ |
| NB7-02 | HASPRDVLVDYDY (2) | - | 1.98 | 2.44 | 1.63 | 2.48 | 0.26 | - | ++ |
| NB7-03 | AADFLTLQDMCVFSPITRYHD (3) | - | 2.04 | - | 1.30 | - | 0.52 | - | +++ |
| NB7-04 | AASPIPDLRDYDY (4) | - | 0.84 | 1.62 | 0.71 | 3.37 | 0.31 | - | + |
| NB7-05 | NVWIQFHNY | - | 0.17 | - | 0.19 | - | 0.38 | - | - |
| NB7-07 | AFGATLYGVRRDEYAY | - | 1.99 | 1.96 | 1.46 | 2.73 | 0.48 | - | +++ |
| NB7-08 | AADRYSGPYDLGTISPRQDHYDV | - | 0.91 | 1.52 | 0.91 | 2.65 | 2.05 | - | - |
| NB7-09 | ALARSVDRLEYDW (1) | - | 2.86 | 2.82 | 2.37 | 3.30 | 2.60 | - | ++++ + |
| NB7-10 | AADDLTLQDMCVMSPISRYHY (3) | - | 1.64 | - | 1.20 | - | 2.30 | - | +++ |
| NB7-11 | AVARSVDRLEYDY (1) | - | 1.03 | 1.23 | 1.24 | 3.12 | 2.53 | - | ++++ |
| NB7-12 | HASPRDVLVDYD (2) | - | 1.73 | 2.57 | 1.78 | 3.28 | 2.47 | - | ++++ |
| NB7-13 | AADFLTLQDMCVLSPITRYRS (3) | - | 1.14 | - | 0.72 | - | 2.37 | - | +++ |
| NB7-14 | ALARSVDRLTYDY (1) | - | 2.67 | 2.75 | 2.08 | 3.00 | 2.57 | - | +++++ |
| NB7-15 | AAGRTYCGLRGYEYDY | - | 2.23 | 0.20 | 1.92 | 2.97 | 2.56 | - | ++++ |
| NB7-16 | AASPIPTLRDYDY (4) | - | 1.44 | 1.71 | 1.08 | 2.86 | 2.42 | - | +++ |
| NB7-17 | AASPRRLMCVPAEFDLYY | - | 2.44 | 2.57 | 1.97 | 2.39 | 2.07 | - | ++++ |
| NB37X-01 | AQERERIGTTIRYY | 0.36 | 0.13 | 0.22 | 0.48 | 0.38 | 2.14 | - | - |
| NB37X-02 | AAPKTRYLAPRAMESEFDY (5) | 2.72 | - | 0.12 | - | 0.26 | 2.48 | - | - |
| NB37X-03 | AAPKTRYLAPRMTESDYDY (5) | 0.12 | - | - | - | 0.34 | - | - | - |
| NB37X-04 | NALCYGCTPSSY | 0.30 | 0.28 | - | 0.14 | 0.49 | 2.83 | - | - |
| NB37X-05 | AANPGTVVGRTRLSPRLRVPDEYDS | 0.40 | 0.14 | 0.17 | 0.26 | 0.31 | 2.32 | - | - |
| NB37X-06 | AADRVDYPGGGATCRTSSAAYDH | 0.26 | - | 0.20 | 0.20 | 0.36 | 2.55 | - | - |
VHH-CDR3 sequences and family grouping of related sequences. ELISA is given as the average OD450nm reading of two wells coated with whole virus antigen and Nbs added at a single concentration of 5 µg/ml. – indicates OD450 < 0.1 and represents no binding. Inactivated whole virus antigens used TX/2012(H3N2) [A/Texas/50/2012 (H3N2)], M/N/2000(H7N3) [A/Mallard/Netherlands/12/2000], NY/2003 (H7N2) [A/New York/107/2003], AN/2013 (H7N9) [A/Anhui/01/2013], GD/2016 (H7N9) [A/Guangdong/17SF003/2016], HK/2017 (H7N9) [A/HongKong/125/2017]. Pseudotype based micro-neutralisation assays using A/Texas/50/2012 (H3) and A/Shanghai/02/2013 (H7) HA bearing pseudoviruses. += 500-3000 nM, ++ < 500 nM, +++< 100 nM, ++++ < 50 nM, +++++< 10 nM, - no neutralisation.