Introduction H5N1 is an avian influenza. It was detected in humans for the first time in 1997 in Hong Kong. Since then the spread to humans has been limited where 228 humans has been infected of whom 130 has died (1). Evidence suggests that the transmission so far has been bird-to-human (2). Influenza binds to host cells via binding of the viral surface protein hemagglutinin (HA) to receptors containing a terminal sialic acid. Human influenza preferentially binds to α-2,6 sialic acid-galactose-linkages whereas avian influenza preferentially binds to α-2,3 sialic acid-galactose-linkages (3). So far there has not been an effective inter-human transmission which potentially can lead to a pandemic (1, 4).To obtain inter-human transmission, H5N1 must adapt so it can bind to α-2,6 sialic acid-galactose-linkages. In vitro studies has shown that only two mutations in HA at residue 226 and 228 is needed (2). Because of this knowledge we have designed a vaccine directed against the mutated H5 to prevent a future H5N1 pandemic. The vaccine is intended as a recombinant protein vaccine to obtain a B-cell response. Furthermore we have designed a polytope vaccine against H5N1, where a MHC class I and class II response is obtained. This polytope vaccine is intended as a DNA-vaccine or a peptide vaccine.

Preventing a future H5N1 flu pandemic by vaccination

Anna Irene Vedel Sørensen, Thilde Warrer Jensen, Lisbeth Elvira de Vries and Michael Back Dalgaard

Immunological Bioinformatics 2006, CBS, DTU

Figure 1. Alignment with ClustalW (5) showing the proposed introduction of mutations in aa position Q226L and G228S (HA1/1-334) in the sialic acid terminal receptor binding domain (RBD) of H5N1 avian influenza. This confers a switch from HA found in avian species preferring a binding to sialic acid in an α2,3-linkage to galactose to a HA recognising sialic acid in α2,6-linkage which is found in humans.The four top sequences are obtained from human cases; the bottom sequence is the H5N1 reference strain. C

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B Cell epitopeWe applied the following strategy in the pursue of defining an appropriate recombinant protein vaccine candidate eliciting a humeral response which could neutralise an invading mutated avian H5N1 virus. CPHmodels (6) was used to obtain a protein structure from pdb (8) for the H5N1 reference strain (YP_308669.1_HA, 9) resulting in a nearly perfect match with crystal structure found in pdb (id:1JSM, 8). Substitutions were introduced at aa position Q226L and G228S inferring a HA protein with affinity for the human α2,6-sialic acid-galactose (2). Detecting areas of conservation 66 human H5N1 isolates and approximately 80 avian isolates were aligned using ClustalW (5). We used BepiPred for the prediction of possible linear B-cell epitopes (6), and both DiscoTope (6) and CEP (8) for the prediction of conformational B-cell epitopes. Furthermore we also created a consensus model combining the predictions from DiscoTope and CEP (6, 8). PyMol (10) was used as protein visualization tool. Further we investigated the protein for MHC DR4 II binding using SYFPEITHI (11), EasyGibbs (6) and ProPred (12) and found some good binders e.g. CIGYHANNSTEQVDT.

Figure 2. B-cell epitope predictions (red sticks) and protein structures for the receptor hemagglutinin (HA) from a H5N1 influenza virus. The mature HA protein is composed of two subunits HA1/chain a (green) and HA2/chain b (yellow) with HA1 containing the sialic acid terminal receptor binding domain (RBD) (blue). The very distal part of the HA2 as to the RBD composes the transmembrane structure. A. Linear B-cell epitope prediction using BepiPred (6). B. Conformational B-cell epitope prediction using DiscoTope (6). C. Conformational B-cell epitope prediction using CEP (7). D. Conformational B-cell epitope prediction using consensus prediction from DiscoTope (6) and CEP (7).

Polytope vaccineWe have designed a polytope vaccine directed against H5N1 containing three-five MHC class I epitopes and one class II epitope. The epitopes were selected by the principal: wisdom of the crowd, using several prediction servers (NetCTL, NetMHC, NetChop, EasyGibbs (6), SYFPEITHI (11), and ProPred MHC class-II binding peptide prediction server (12)). A criteria for selecting the epitopes was that they had to be conserved in all the H5N1 human isolates (9) and in the H5N1 reference sequence (YP_308669, 9, Figure 3). Finally, the polytopes were optimized considering processing in the MHC class I pathway (13, Figure 4). The selected epitopes were found in the polymerase subunits PB1 and PB2 and the nucleoprotein NP (Figure 3). The polytope shown in figure 4.a contains epitopes for MHC class I A1, A2, A3, B7, B44 and class II DR4. In addition, the A2 epitope also appears to be an epitope for A24 and the B7 epitope may also be an epitope for B8. Thus in theory this polytope should cover 98,1-100% of the worlds population (14). As shown in figure 4.a the polytope has strong internal proteasomal cleavage sites in the A3 epitope. Furthermore a new epitope is appearing in the DR4 epitope. However, it was not possible to find an A3 epitope without internal cleavages in both the native context and in the polytope. Therefore, we have also optimized a smaller polytope containing only the A2, A3, B7 and DR4 epitopes. This should cover 83 - 88,5 and 5 % of the world’s population (14). As shown in figure 4.b this has a lower probability of internal cleavage and has no new epitopes. Thus we believe that this polytope (figure 4.a) is the best candidate for a polytope vaccine against H5N1.

References1. WHO: http://www.who.int 2. Stevens, J. et al., 2006, Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus, Science vol. 312

(404-10) 3. Brown, E. G., 2000, Influenza virus genetics, Biomed & Pharmacother, 54 (196-209)4. Center For Infectious Disease Research & Policy, University of Minnesota: http://www.cidrap.umn.edu5. ClustalW: http://www.ebi.ac.uk/clustalw6. BepiPred, DiscoTope, CPHmode, EasyGibbs, NetCTL, NetChop, NetMHC http://www.cbs.dtu.dk/services7. Conformational Epitope onformational Server (CEP): http://202.41.70.74:8080/cgi-bin/cep.pl 8. pdb: http://www.rcsb.org/9. Influenza Virus Resource, NCBI: http://www.ncbi.nih.gov/genomes/FLU/FLU.html10. Pymol: http://pymol.sourceforge.net/ 11. SYFPEITHI: http://www.syfpeithi.de 12. ProPred MHC class-II binding peptide prediction server: http://www.imtech.res.in/raghava/propred/index.html13. polytope_cont3, Morten Nielsen, CBS, DTU14. Lund, O. et al., 2005, Immunological Bioinformatics, The MIT Press, Cambridge, Massachusetts, London, England

Figure 3. Multiple alignment of four human isolates and the reference strain for H5N1 avian influenza (9) in ClustalW (5)The number of the first amino acid in the segments they are derived from (PB2, PB1and NP respectively) are indicated above the epitopes covering the MHC class I supertypes A2, B7 and A3; respectively. Accession numbers:AF144300, AF144301, AF144303, AB212051, AB212052, AB212055, DQ492896, DQ493422, DQ493160, AY818126, AY818129, AY818138, DQ372598, DQ372597, DQ372594.

ConclusionIn the present study a potential human recombinant protein vaccine candidate against the highly virulent H5N1 avian influenza was designed using the Hemagglutinin as a template. In the recombinant HA1 subunit the amino acids 226 and 228 were replaced with leucine and serine which in-vivo would enable the virus to bind to receptors on human cells. Potential linear and conformational B-cell epitopes exposed on the surface of the protein were identified. Using linear B-cell epitope prediction does not seem to make much sense as these only comprise app. 10% of the total number of epitopes and the linear prediction method does predict epitopes internally in the protein which obviously not are surface exposed. When we used two different models for predicting conformational B-cell epitopes we observed quite some differences in the predictions. However surface areas exhibiting consensus between the two models were identified including the sialic acid terminal receptor binding domain.Furthermore potential T-cell epitopes for the MHC class II supertype DR4 were identified. The proposed recombinant protein vaccine needs “trimming”, cutting of the trans-membrane region and confirmation that the protein has folded and been glycosylated correctly in our production organism e.g. yeast or E. coli. In addition vaccination methods and adjuvants must be considered.Two polytopes were constructed selecting epitopes from the full genome of influenza H5N1. All epitopes included in the polytopes were conserved in all public available sequences of human isolates of H5N1 and in the reference sequence of H5N1. Unfortunately it was not possible to avoid strong internal cleavages in the epitope covering A3 thus reducing the efficacy for this vaccination polytope.The polytope is intended for use as a DNA vector vaccine, but needs quite a lot of optimization, and several other factors such as promotors, adjuvants and vector type have to be taken into consideration in the next step.

A.

B.

Figure 4. Proteasomeatlases of two optimized polytopes using polytope_cont3, Morten Nielsen, CBS, DTU . A. Shows the proteasome cleavage (red and black bars) of a polytope consisting of 6 epitopes (blue squeres) separated by linker regions. The red squre illustrates a new A2 epitope arising in the optimized polytop. The polytope contains 5 MHC class I epitopes and 1 MHC class II epitope (DR4). The MHC class I epitopes cover superclass: B7, A2, A3, B44 and A1. The amino acid sequence of the polytope with epitope sequences in upper case letters and linker sequences i lower case letters: mysdIPKRNRSILarVLTGNLQTLyyenrGVFELSDEKntdakaFEDLRVSSFfvSSDDFALIViLVGIDPFRLLQNSQVFSLiv. B. Shows the proteasome cleavage of a polytope containing the B7, A2, A3 and DR4 epitopes as shown in A. The amino acid sequences: mrmIPKRNRSILrarVLTGNLQTLarrrGVFELSDEKrlvkLVGIDPFRLLQNSQVFSLiltp

PB2(344-353) pb1 (443-452) np (462-471)

A/Goose/Guangdong/1/96 (H5N1) KKEEEVLTGNLQTLKIRV...HSWIPKRNRSILNTS...QGRGVFELSDEKATNP

A/Human/Hong Kong/213/2003 (H5N1) KKEEEVLTGNLQTLKIRV...HSWIPKRNRSILNTS...QGRGVFELSDEKATNP

A/Human/Viet Nam/CL26/2004 (H5N1) KKEEEVLTGNLQTLKIRV...HSWIPKRNRSILNTS...QGRGVFELSDEKATNP

A/Viet Nam/1203/2004 (H5N1) KKEEEVLTGNLQTLKIRV...HSWIPKRNRSILNTS...QGRGVFELSDEKATNP

A/Human/Thailand/NK165/2005 (H5N1) KKEEEVLTGNLQTLKIRV...HSWIPKRNRSILNTS...QGRGVFELSDEKATNP

A2 B7 A3