title: The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency.
abstract: 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.
authors.affiliation: Biotherapeutics Group, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Simon E
authors.initials: SE
authors.lastname: Hufton
authors.affiliation: Biotherapeutics Group, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Risley
authors.affiliation: Technology Development and Infrastructure, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Christina R
authors.initials: CR
authors.lastname: Ball
authors.affiliation: Division of Virology, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Diane
authors.initials: D
authors.lastname: Major
authors.affiliation: Division of Virology, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Othmar G
authors.initials: OG
authors.lastname: Engelhardt
authors.affiliation: Biotherapeutics Group, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Poole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002161:Camelids, New World; D061505:Cell Surface Display Techniques; D003429:Cross Reactions / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D006801:Humans; D009980:Influenza A virus / Q000145:classification / Q000276:immunology; D007251:Influenza, Human / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D061905:Single-Domain Antibodies
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