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.

Slides:



Advertisements
Similar presentations
Bioinformatical design of a vaccine against influenza virus N1 subtype Bonaccorsi, Irene; Clausen, Martin Bau; Høj, Leif Howalt; Kjær, Jesper and Sayyad,
Advertisements

HeleneAndersenMayaBondeAndersenSimonCarlsen MortenAhlgreenGronemann&MadsChristianHjortsø Figure 4, Alignment of the NS3 protein: Alignment of NS3 performed.
Office of Infectious Diseases Computational Challenges for Infectious Diseases Michael Shaw, PhD OID/Office of the Director.
Prediction of B cell epitopes Pernille Haste Andersen Immunological Bioinformatics CBS, DTU
Influenza H5N1 By Steven Yi. Contents Contents 1. Overview 2. History 3. Attachment 4. Entry 5. Replication 6. Lytic Cycle 7. Diagnosis 8. Treatment.
80% Influenza A — H3N2 (some H1N1) 20 % influenza B (orthomyoxiviridae – seals and humans only) Deaths above epidemic threshold – 50% hospitalizations.
H1N1: “Swine Flu”. Why you should care… Every year between 5 and 20% of the population gets the flu. The CDC estimates that the flu kills 36,000 people.
Vaccines and Antivirals. Clinical Use of Interferon Therefore they have been used in the treatment of cancers of various types. Therefore they have been.
Influenza A Virus Pandemic Prediction and Simulation Through the Modeling of Reassortment Matthew Ingham Integrated Sciences Program University of British.
Epitope Selection Rational Vaccine design. Why? Therapeutic vaccines Therapeutic vaccines Treatment of viral infections (e.g., HIV, HCV), and resistant.
Bioinformatics Unit 1: Data Bases and Alignments Lecture 3: “Homology” Searches and Sequence Alignments (cont.) The Mechanics of Alignments.
INTRODUCTION TO INFLUENZA The (Ferret) Sneeze Heard Around The World: The Case Of The Bioengineered Bird Flu Case Study for AAC&U STIRS Project Jill M.
Avian flu H5N1 A bird adapted strain of Influenza A H5N1 is endemic in many areas of southeast Asia, and a highly pathogenic strain is now spreading globally.
Presented by Jan Haas Institute for Immunology.
The evolution of infectious disease. Influenza We generally think of the flu as nothing more than a minor annoyance In an average year, however, the flu.
Methods MHC class-I T cell epitope prediction for Nef Consensus and ancestral sequences of the Nef protein for the different HIV-1 subtypes were obtained.
Selection of T Cell Epitopes Using an Integrative Approach Mette Voldby Larsen cand. scient. in Biology PhD in Immunological Bioinformatics.
D-Influenza virus. Influenza epidemiology in humans Fields Virology, 2nd ed, Fields & Knipe, eds, Raven Press, 1990, Fig.40-1.
Prediction of avian Influenza A binding preference to human receptor using conformational analysis of receptor bound to hemagglutinin Wanwimon Mokmak,
Misconduct Case Study Our story so far: Peter:4 th -year grad. student makes mice lacking SLAM gene several cell types have abnormal function Sally:4 th.
Using Comparative Genomics to Explore the Genetic Code of Influenza Sangeeta Venkatachalam.
Evolution of influenza A Rachel Albert Craig Bland Evolution of influenza A.
THE QUESTION: SHOULD I GET A FLU SHOT EACH YEAR?.
REASSORTMENT OF INFLUENZA VIRUS
The Informatics Crystal Ball: Mining the Past to Predict the Species Jump Event 19 April 2011 Richard H. Scheuermann, Ph.D. Department of.
Evidence for Positive Epistasis in HIV-1 Sebastian Bonhoeffer, Colombe Chappe, Neil T. Parkin, Jeanette M. Whitcomb, Christos J. Petropoulos.
Influenza H1N1 A: A close insight Dr. Mustafa Ababneh Molecular Virologist.
Vaccines: A Molecular View
Lecture 1: Immunogenetics Dr ; Kwanama
Bioinformatics in Vaccine Design
P ANDEMICS T HROUGHOUT H ISTORY. A pandemic is defined as an unusually high outbreak of a new infectious disease that is spreading through the human population.
“Neutralizing Antibodies Derived from the B Cells of 1918 Influenza Pandemic Survivors” (Yu et. al) Daniel Greenberg.
Prediction of T cell epitopes using artificial neural networks Morten Nielsen, CBS, BioCentrum, DTU.
Lecture 13 Immunology and disease: parasite antigenic diversity.
CATEGORY: VACCINES & THERAPEUTICS HIV-1 Vaccines Shokouh Makvandi-Nejad, University of Oxford, UK HIV-1 Vaccines © The copyright for this work resides.
What is phage display? An in vitro selection technique using a peptide or protein genetically fused to the coat protein of a bacteriophage.
C5b6789 Terminal complex C3 C3b C5 C5b C9 C8 C7 C6 C5a C3a C4a + C2b C3 C3b C3bBb Factor B Factor D C1q + C1r + C1s C3bBb3b activated C1s MBL/Ficolins.
Higher Affinity Heptapeptides Against Influenza Hemagglutinin-Sialic Acid Identification for Treating Flu Virus Disease Ahmed ”e” Al Qaffas.
Tzachi Hagai, Ariel Azia, M. Madan Babu, Raul Andino  Cell Reports 
Influenza Virus: Evolution in real time
What makes H5N1 Avian influenza “Avian”
A Spring-loaded mechanism for the conformational change of Influenza Hemagglutinin Mani Foroohar.
Universal influenza virus vaccines and therapeutic antibodies
Volume 17, Issue 3, Pages (March 2015)
Epitopes in the Linker Subdomain Region of Envoplakin Recognized by Autoantibodies in Paraneoplastic Pemphigus Patients  Bingxin Zhang, Rui Zheng, Jing.
Rose-Anne Romano, Barbara Birkaya, Satrajit Sinha 
The Rational Design of an AIDS Vaccine
What Integration Sites Tell Us about HIV Persistence
Hung-Ta Chen, Steven Hahn  Cell 
Volume 39, Issue 5, Pages (May 2018)
J.A. McCullers  Clinical Microbiology and Infection 
Volume 3, Issue 1, Pages (January 2013)
Volume 13, Issue 12, Pages (December 2015)
Crystal Structure of the λ Repressor C-Terminal Domain Provides a Model for Cooperative Operator Binding  Charles E. Bell, Paolo Frescura, Ann Hochschild,
Volume 28, Issue 6, Pages (December 2007)
Volume 22, Issue 2, Pages (August 2017)
The Crystal Structure of the Costimulatory OX40-OX40L Complex
Structure, Exchange Determinants, and Family-Wide Rab Specificity of the Tandem Helical Bundle and Vps9 Domains of Rabex-5  Anna Delprato, Eric Merithew,
Volume 6, Issue 6, Pages (December 2000)
Tzachi Hagai, Ariel Azia, M. Madan Babu, Raul Andino  Cell Reports 
Meigang Gu, Kanagalaghatta R. Rajashankar, Christopher D. Lima 
Analyses of the Effects That Disease-Causing Missense Mutations Have on the Structure and Function of the Winged-Helix Protein FOXC1  Ramsey A. Saleem,
Mohammad Azam, Robert R. Latek, George Q. Daley  Cell 
Michael S. Kuhns, Mark M. Davis  Immunity 
NP sequence alignment of human H1N1 strains and phylogenetic analysis of representative NP from pandemic and seasonal H1N1 strains. NP sequence alignment.
Volume 17, Issue 3, Pages (March 2015)
Volume 153, Issue 7, Pages (June 2013)
Exchange of Regions between Bacterial Poly(A) Polymerase and the CCA-Adding Enzyme Generates Altered Specificities  Heike Betat, Christiane Rammelt, Georges.
Volume 25, Issue 4, Pages e3 (October 2018)
Volume 19, Issue 2, Pages (April 2017)
Presentation transcript:

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 A B C D B Cell epitope We 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_ _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 vaccine We 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 ,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. References 1.WHO: 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 ( ) 4.Center For Infectious Disease Research & Policy, University of Minnesota: 5.ClustalW: 6.BepiPred, DiscoTope, CPHmode, EasyGibbs, NetCTL, NetChop, NetMHC 7.Conformational Epitope onformational Server (CEP): 8.pdb: 9.Influenza Virus Resource, NCBI: 10.Pymol: 11.SYFPEITHI: 12.ProPred MHC class-II binding peptide prediction server: 13.polytope_cont3, Morten Nielsen, CBS, DTU 14.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, DQ Conclusion In 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( ) pb1 ( )np ( ) 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