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

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.2009年甲型H1N1流感大流行的应对实践凸显出，亟需开发无需提前获取流感毒株的新型干预策略。本研究从免疫羊驼噬菌体展示文库（phage displayed library）中，分离得到18株针对2009年甲型H1N1流感血凝素（hemagglutinin, HA）的单结构域VHH抗体（VHH antibody）。这些抗体可被划分为三类：(i) 非中和性抗体；(ii) 仅针对H1N1的中和性抗体；(iii) 具备广泛跨亚型中和活性的抗体。能够中和包括高致病性禽流感H5N1在内的多种病毒亚型的特性，与以下特征显著相关：不具备血凝抑制活性、在酸性pH环境下丧失与HA的结合能力，以及无法结合包含受体结合位点的HA头部结构域。上述数据支持这些抗体结合HA茎区表位，且其作用机制并非阻断病毒附着于细胞表面受体。将跨中和抗体R1a-B6与R1a-A5改造为二价格式后，针对甲型H1N1和甲型H5N1病毒的中和活性未出现显著提升。然而，二价化的R1a-B6对甲型H9N2病毒的效价提升了18倍，且意外获得了中和甲型H2N2病毒的能力。这表明，与差异更大的HA亚型膜近端茎区结合亲和力较低的跨中和抗体，可通过二价化策略进行优化，从而拓宽其抗病毒活性谱。这类单结构域抗体兼具广泛中和活性与多项优良特性，例如高稳定性、易于工程化改造为二价分子、生产成本低廉，因此可作为大流行性流感诊断与免疫治疗的极具潜力的候选制剂。