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

<p>Monovalent and bivalent versions of R1a-B6 (A) and R1a-A5 (B) were compared in neutralisation assays with laboratory adapted X-181 strain [corresponding to A/California/07/2009(H1N1)pdm09], NIBRG-14 [reverse genetics reassortant of A/Vietnam/1194/2004(H5N1)], NIBRG-91 [reverse genetics reassortant of A/chicken/Hong Kong/G9/97(H9N2)], NIBRG-109 [reverse genetics reassortant of A/New York/107/03(H7N2)] and NIBRG-147 [reverse genetics reassortant of A/Singapore/1/57(H2N2)]. Representative curves are shown and are the mean of two replicates. Antibody concentrations are in nM.</p

Characterisation of antibody epitopes.

<p>(A) ELISA showing reactivity of purified antibodies at 30 µg/ml to HA antigen standard A/California/07/2009 (H1N1)pdm09 either treated with low pH or neutral pH. (B) ELISA showing binding of phage displayed HA gene fragments to purified sdAbs. Antibodies were coated onto an ELISA plate at 10 µg/ml and incubated with purified phage particles. The panel of 18 antibodies were tested for binding to the HA gene fragments L193-K225, P66-I282 and D363-G478 numbered according to Feshchenko <i>et al</i>., (2012)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103294#pone.0103294-Feshchenko1" target="_blank">[52]</a>.</p

The Breadth of Cross Sub-Type Neutralisation Activity of a Single Domain Antibody to Influenza Hemagglutinin Can Be Increased by Antibody Valency

<div><p>The response to the 2009 A(H1N1) influenza pandemic has highlighted the need for additional strategies for intervention which preclude the prior availability of the influenza strain. Here, 18 single domain VHH antibodies against the 2009 A(H1N1) hemagglutinin (HA) have been isolated from a immune alpaca phage displayed library. These antibodies have been grouped as having either (i) non-neutralising, (ii) H1N1 restricted neutralising or (iii) broad cross-subtype neutralising activity. The ability to neutralise different viral subtypes, including highly pathogenic avian influenza (H5N1), correlated with the absence of hemagglutination inhibition activity, loss of binding to HA at acid pH and the absence of binding to the head domain containing the receptor binding site. This data supports their binding to epitopes in the HA stem region and a mechanism of action other than blocking viral attachment to cell surface receptors. After conversion of cross-neutralising antibodies R1a-B6 and R1a-A5 into a bivalent format, no significant enhancement in neutralisation activity was seen against A(H1N1) and A(H5N1) viruses. However, bivalent R1a-B6 showed an 18 fold enhancement in potency against A(H9N2) virus and, surprisingly, gained the ability to neutralise an A(H2N2) virus. This demonstrates that cross-neutralising antibodies, which make lower affinity interactions with the membrane proximal stem region of more divergent HA sub-types, can be optimised by bivalency so increasing their breadth of anti-viral activity. The broad neutralising activity and favourable characteristics, such as high stability, simple engineering into bivalent molecules and low cost production make these single domain antibodies attractive candidates for diagnostics and immunotherapy of pandemic influenza.</p></div

Comparison of viral neutralisation activity of R1a-B6 and R1a-A5 in a monovalent and bivalent antibody format.

1<p>Neutralisation of laboratory adapted X-181 strain corresponding to A/California/07/2009 (H1N1)pdm09, NIBRG-14, a reverse genetics reassortant of A/Vietnam/1194/2004 (H5N1), NIBRG-147, a reverse genetics reassortant of A/Singapore/01/57 (H2N2), NIBRG-91 a reverse genetics reassortant of A/chicken/Hong Kong/G9/97 (H9N2), NIBRG-109 reverse genetics reassortant of A/New York/107/2003 (H7N2) and seasonal A(H3N2) virus A/Montana/05/2011.</p>2<p>IC50 is given in nM and is the concentration required to give 50% maximum neutralisation. Results are the mean of 3 or more independent assays.</p>3<p>– indicates no neutralisation was seen at antibody concentrations less than 1 µM.</p><p>NT is not tested.</p

Antibody classification.

1<p>Antibody neutralisation category, R is H1N1 restricted neutralisation activity, C refers to a cross-neutralising antibody, N is non-neutralising antibody</p>2<p>Hemagglutination inhibition (HI) titre was given as the minimum dilution of antibody at which inhibition of agglutination of turkey erythrocytes was observed (in µg/ml of purified antibody). Values are given as an average of three independent assays. Laboratory adapted A(H1N1) strain (X-181) was used.</p>3<p>Single cycle affinity determination on full length HA (D18-I530) and globular head HA1 domain (residues D18-R344) derived from A/California/06/2009 (H1N1pdm09). Affinity is given in nM.</p>4<p>Binding to phage displayed fragments of HA as indicated in ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103294#pone-0103294-g005" target="_blank">Figure 5B</a>).</p>5<p>ELISA on HA of A/California/07/2009 (H1N1pdm09) treated with low pH and neutral pH (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103294#pone-0103294-g005" target="_blank">Figure 5A</a>).</p

Grouping of antibody epitopes.

<p>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 (A) R2b-D9/R2b-D9 (B) R2b-D9/R2b-E8 (C) R2b-D9/R1a-A5 (D) R2b-D9/R1a-B6 (E)R2b-D9/R1a-C5 (F) R2b-D9/R2a-G9. 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. Antibodies R1a-B6, R1a-A5, R2b-D9, and R2b-E8 appeared to share overlapping epitopes as no significant increase in response was observed following injection of a second antibody. The antibodies R1a-C5 and R2a-G9 bound a distinct non-overlapping epitope as a significant increase in response was seen upon binding of these antibodies as the second antibody species. 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) (G). The sensorgrams indicate the Rmax value for each of the individual antibodies (∼150 RU's to 450 RU's) 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 (H). Analysis of R2a-G8 was not conclusive.</p

Specificity and affinity determination using surface plasmon resonance.

1<p>Association rate constant <i>kon</i>, dissociation rate constant <i>koff</i>, equilibrium dissociation constants K_D determined by single cycle kinetics on recombinant recombinant H1-HA, A/California/06/2009 (H1N1)pdm09, H5-HA, A/Vietnam/1203/2004 (H5N1), H2-HA, A/Japan/305/57 (H2N2), H9-HA, A/Hong Kong/1073/99 (H9N2). No binding to H3-HA, A/Brisbane/10/2007 (H3N2), H7-HA, A/Netherlands/219/2003 (H7N7) was seen (data not shown).</p><p>Specificity and affinity on recombinant H1-HA derived from. Affinity constant K_D is given in nM and to 2 decimal places. Control is a non-relevant antibody.</p>2<p>- No binding.</p>3<p>+ Some binding could be detected but was too low to be analysed using BIAevaluation software.</p

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

<p>ELISA showing binding of 18 purified single domain VHH antibodies to influenza reference reagents prepared from different viral subtypes (A) ELISA comparing binding of purified VHH antibodies at 30 µg/ml against A/California/07/2009 (H1N1pdm09), A/Brisbane/59/2007 (seasonal H1N1),A/Anhui/01/2005 (H5N1) (B) A/Vietnam/1194/2004 (H5N1) (C) A/Indonesia/05/2005 (H5N1) (D) A/HongKong/05/2005 (H5N1) (E) A/Singapore/1/57 (H2N2) (F) A/Hong Kong/1073/99 (H9N2). Antibodies R2b-E8, R2b-D9, R1a-A5, R1a-B6 and R2a-G8 were also positive on A/turkey/Turkey/01/2005 (H5N1), A/Duck/Sing-Q/119-3/97 (H5N3), A/mallard/Eng/727/2006 (H2N3) (data not shown). In addition all antibodies were negative on B virus control (B/Brisbane/60/2008) and on the group 2 strain A/Brisbane/10/07 (H3N2) (data not shown). Control represents a non-binding purified VHH antibody. Binding was measured in duplicate and the average OD 450 nM was plotted against a serial dilution of antibody from 30 µg/ml to 0.001 µg/ml (B-F).</p

Antibody mediated neutralisation of different influenza virus subtypes.

1<p>Neutralisation of laboratory adapted X-181 strain corresponding to A/California/07/2009 (H1N1)pdm09, NIBRG-14 a reverse genetics reassortant of A/Vietnam/1194/2004 (H5N1) with the polybasic cleavage site removed from the HA, NIBRG-147 reverse genetics reassortant of A/Singapore/01/57 (H2N2), NIBRG-91 reverse genetics reassortant of A/chicken/Hong Kong/G9/97 (H9N2), NIBRG-109 reverse genetics reassortant of A/New York/107/2003 (H7N7).</p>2<p>IC50 is given in nM and is the concentration required to give 50% maximum neutralisation. Data is given as the mean mean of a minimum of three independent assays ± SEM.</p>3<p>+ indicates neutralisation was seen but a IC50 could not be calculated as a maximum level of neutralisation activity was not reached. ⁴– indicates no neutralisation. ⁵ ND is not determined.</p

Antibody affinity on recombinant HA by surface plasmon resonance.

<p>The kinetic rate binding constants (<i>kon</i> and <i>koff</i>) of different antibodies were determined using SPR and single cycle kinetics <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103294#pone.0103294-Karlsson1" target="_blank">[49]</a>. Data are represented as rate plots with iso-affinity diagonals where the diagonals (dotted lines) are connecting the points of equal dissociation constant (K_D) ; (A) affinity on recombinant H1-HA, A/California/06/2009 (H1N1pdm09) and (B) affinity of the cross-reactive antibodies on recombinant H5-HA, A/Vietnam/1203/2004 (H5N1) recombinant H2-HA, A/Japan/305/57 (H2N2) and recombinant H9-HA, A/HongKong/1073/99 (H9N2). Fitting was with single cycle kinetics method and a 1∶1 Langmuir fitting model using BIAevaluation software. Equilibrium dissociation constants (K_D) are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103294#pone-0103294-t001" target="_blank">Table 1</a>.</p