Anti-Influenza A Virus H1N1 Hemagglutinin HA1 subunit Recombinant Antibody (MRO-234LC-VHH) (CAT#: MRO-234LC-VHH)

Figure 1 Display of hemagglutinin on yeast and evaluation of binding of single domain antibodies.

FACS plots of seven HA specific single domain antibodies (sdAbs) R1a-F5, R1a-G6, R2b-E8, R2b-D9, R1a-A5, R1a-B6, R2a-G8 binding to yeast displayed HA0. Negative controls sdAb R1a-G2 and no sdAb control are shown. The vertical arrow indicates absence of binding.

Gaiotto, T., & Hufton, S. E. (2016). Cross-neutralising sdAbs bind to a conserved pocket in the hemagglutinin stem region identified using yeast display and deep mutational scanning. PloS one, 11(10), e0164296.

Figure 2 Specificity of single domain antibodies to different influenza antigen reference reagents.

ELISA comparing binding of purified VHH antibodies at 30 mg/ml against H1N1, seasonal H1N1 and H5N1.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 3 Antibody affinity on recombinant HA by surface plasmon resonance.

Affinity on recombinant H1-HA, H1N1.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 4 Viral neutralisation activity of cross-reactive sdAbs.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 5 Characterisation of antibody epitopes.

ELISA showing reactivity of purified antibodies at 30 mg/ml to HA antigen standard H1N1 either treated with low pH or neutral pH.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 6 Characterisation of antibody epitopes.

ELISA showing binding of phage displayed HA gene fragments to purified sdAbs.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 7 Grouping of antibody epitopes.

SPR co-injection experiments were used to determine if pairs of VHH antibodies can bind to recombinant H1-HA simultaneously. The antibodies R1a-A5, R1a-B6, R2b-D9, R2b-E8, R1a-C5 and R2a-G9 were tested in combination with each other. Example series of sensorgrams of R2b-D9 injected as the first antibody followed by injection of each of the other antibodies as indicated R2b-D9/R1a-A5.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 8 Grouping of antibody epitopes.

SPR co-injection experiments were used to determine if pairs of VHH antibodies can bind to recombinant H1-HA simultaneously. The four antibodies R2b-D9, R1a-B6, R2b-E8 and R1a-A5 predicted to share overlapping epitopes were further analysed by injecting each VHH on their own or as equimolar mixture of all four antibodies (Mixture).

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 9 Grouping of antibody epitopes.

The sensorgrams indicate the Rmax value for each of the individual antibodies with no significant increase in Rmax following injection of the antibody mixture, which suggests these antibodies recognise an overlapping epitope or an epitope that hinders binding of a second antibody. If the antibodies recognised non-overlapping or non-hindering sites the Rmax would be expected to be the sum of the individual Rmax values. This was confirmed when R1a-C5 which was predicted to recognise a non-overlapping epitope was included in a equimolar mixture of five antibodies and the response was seen to increase by an amount equivalent to R1a-C5 binding individually.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 10 Evaluation of competition with C179.

The antibody C179 was injected first and the test VHH antibody injected as the second antibody species C179/R1a-A5. The solid line represents co-injection of a first antibody and then a second antibody whereas a dotted line represents injection of a first antibody followed by injection of buffer.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.

Figure 11 Comparison of viral neutralisation activity with monovalent and bivalent antibody formats.

Monovalent and bivalent versions of R1a-A5 were compared in neutralisation assays with laboratory adapted X-181 strain [corresponding to H1N1, H5N1, H9N2, H7N2 and H2N2.

Hufton, S. E., Risley, P., Ball, C. R., Major, D., Engelhardt, O. G., & Poole, S. (2014). The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency. PLoS One, 9(8), e103294.