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.
Highlights
Pandemic influenza generally occurs when a new virus emerges and infects the global human population which has little or no preexisting immunity [1]
To investigate further which antibodies might interact with the HA stem region we evaluated binding after exposure of HA to low pH
With this in mind we have chosen to use single domain antibodies (sdAbs) derived from alpacas as a route to high affinity cross-neutralising antibodies which are naturally devoid of a paired light chain
Summary
Pandemic influenza generally occurs when a new virus emerges and infects the global human population which has little or no preexisting immunity [1]. The answer may, at least partially, be in the virus striving to keep its most important and conserved determinants of pathogenicity hidden, combined with the challenges the human immune system has in accessing these parts of the viral coat structure Antibodies to these conserved epitopes are likely to occur rarely and it is only through the advent of sophisticated antibody engineering techniques such as phage display that it has become easier to isolate such monoclonal antibodies. Two recent examples are the human monoclonal antibodies F10 [16] and CR6261 [17] which have both been shown to bind to a highly conserved binding pocket in the membrane proximal stem region using only their heavy chain for antigen binding, with no antigen contact being made by their light chains This suggests that ‘heavychain only’ recognition may be a preferred mode of binding for broadly neutralising antibodies to influenza as has been suggested for cross-neutralising antibodies to HIV [33,34]. These findings and the potential applications of these antibodies are discussed
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