Project The Wellcome Trust - Research Paper Example

Registered charity no.210183. 183 Euston Rd, London, NW1 2BE. Tel: +44 (0)20 7611 8888 Fax: +44 (0)20 7611 8545 www.wellcome.ac.uk APPLICATION FOR APROJECT GRANT Q1 Applicants Principal Applicant Coapplicant (1) Surname You  Forenames   Title   Position   Coapplicant (2) Coapplicant (3) Coapplicant (4) Surname    Forenames    Title    Position    Q2 Name and address of employing institution:  Q3 Type of grant requested:  Q4 Period for which support is sought (state in months): 24 months Q5 Proposed start date (dd/mm/yy): 01/09/2009 Q6 Title of project (no more than 220 characters): Functional characterization and determination of binding specificity of putative chemokine binding protein B29R of Vaccinia virus Principal applicant: Khalid Alquthami Title of project: Functional characterization and determination of binding specificity of putative chemokine binding protein B29R of Vaccinia virus UNDERTAKINGS (i) In signing the application form where shown below, and in consideration of the receipt of this application by the Trust, all applicants (principal applicant, coapplicant, sponsors) UNDERTAKE that the information provided in the application form and otherwise in connection with this application is to the best of their knowledge and belief accurate and complete and that, in relation to any Award of Grant resulting from the application, they will: 1. Take all reasonable actions to ensure that the Trust’s contribution to the funding of the research is suitably acknowledged. 2. Ensure that all research papers (whether based wholly or partly upon the research to be funded by the grant) are forwarded to the Trust upon publication. 3. Comply as Trust-funded researchers with the Trust’s principles and policies on relationships between Trust-funded researchers and commercial entities as set out in Annex A to the Trust’s grant conditions. 4. Consult with Wellcome Technology Transfer, prior to entering into an arrangement with any enterprise that will provide for the exploitation of any results arising from any activity funded under a Trust award. 5. Promptly inform the Trust of any material changes during the period of the Grant to any of the details provided in this application. I have read the conditions under which grants are awarded and the undertakings detailed above and, if a grant is made, I agree to abide by them. I shall be actively engaged in the day to day control of the project. Signature of Applicant Date: Signature of Coapplicant (1) Date: Signature of Coapplicant (2) Date: Signature of Coapplicant (3) Date: Signature of Coapplicant (4) Date: (ii) In signing the application form where shown below, and in consideration of the receipt of this application by the Trust, the Head of Department UNDERTAKES that the information provided in the application form and otherwise in connection with this application is to the best of his/her knowledge and belief accurate and complete and that, in relation to any Award of Grant resulting from the application, he/she will: 1. Ensure compliance with the Trust’s principles and policies on relationships between Trust-funded researchers and commercial entities as set out in Annex A to the Trust’s grant conditions. 2. Consult with Wellcome Technology Transfer, prior to entering into an arrangement with any enterprise that will provide for the exploitation of any results arising from any activity funded under a Trust award. 3. Promptly inform the Trust of any material changes during the period of the Grant to any of the details provided in this application. I have read the conditions under which grants are awarded and the undertakings detailed above and, if a grant is made, I agree to abide by them. I confirm that I have read and support this application, that I agree to this research being carried out in my department, and that all necessary licences and approvals have been or are being obtained. Signature of Head of Department Date: (iii) In signing the application form where shown below, and in consideration of the receipt of this application by the Trust, the Institution UNDERTAKES that the information provided in the application form and otherwise in connection with this application is to the best of its knowledge and belief accurate and complete, and that it will: 4. Ensure compliance with the Trust’s principles and policies on relationships between Trust-funded researchers and commercial entities as set out in Annex A to the Trust’s grant conditions. 5. Consult with Wellcome Technology Transfer, prior to entering into an arrangement with any enterprise that will provide for the exploitation of any results arising from any activity funded under a Trust award. 6. Obtain from all individuals, subsequently funded as a result of the application, the equivalent undertakings as required from the applicants ab initio (i.e. before funding takes place). 7. Apply with full rigour all relevant arrangements for the protection of any patentable intellectual property rights arising from any research funded as a result of this application, as may from time to time be mutually agreed between Wellcome Technology Transfer and the Institution.. However, if the Institution decides not to proceed with the protection of any patentable intellectual property rights, it will co-operate fully (and ensure that its employees, students, contractors, and representatives co-operate) with Wellcome Technology Transfer such that Wellcome Technology Transfer will have an unreserved and unrestricted right, but not a duty, to seek patent protection. 8. Take full responsibility for the management, monitoring and control (including the requirements of all regulatory authorities governing the use of radioactive isotopes, animals, pathogenic organisms, genetically manipulated organisms (GMOs), toxic and hazardous substances, and research on human subjects and human embryos) of all the research work funded as the result of the application and all those staff (permanent, temporary and students) employed in or involved in any research funded as a result of the application. 9. Ensure that all permanent and temporary staff and students employed in or involved in the research receive training appropriate to their duties, in accordance with the regulations set down under the COSHH, ACDP and ACGM guidelines, the Health and Safety at Work regulations, and any other statutory or regulatory requirements as may apply from time to time. 10. Promptly inform the Trust of any material changes during the period of the Grant to any of the details provided in this application. If a grant is made I will ensure that the funds provided are used for the purpose for which they have been given. I confirm that it is this Institution’s intention to maintain its support for the department of the applicant[s] during the period for which this grant is requested. I also confirm that this Institution holds/is not required to hold* a Certificate of Designation under the Animals (Scientific Procedures) Act 1986. I also confirm that I have read and I accept for and on behalf of the Institution the conditions under which grants are awarded and these undertakings. *Delete as appropriate Signature of Secretary of Institution/Finance Officer: Date: Position: Institution: PRINCIPAL APPLICANT Name:  Title:  Post held:  Address:    Telephone Number:  Fax Number:  E-mail Address:  COAPPLICANT (1) Name:  Title:  Post held:  Address:    Telephone Number:  Fax Number:  E-mail Address:  COAPPLICANT (2) Name:  Title:  Post held:  Address:   Telephone Number:  Fax Number:  E-mail Address:  COAPPLICANT (3) Name:  Title:  Post held:  Address:    Telephone Number:  Fax Number:  E-mail Address:  COAPPLICANT (4) Name:  Title:  Post held:  Address:  Telephone Number:  Fax Number:  E-mail Address:  Q7 SUMMARIES OF PROPOSED RESEARCH (both parts combined should be no more than 400 words). (a) For scientifically qualified assessors: The proposed project aims to isolate, clone, and identify the specific ligands for B29R, a Vaccinia virus protein found at the C-terminal region of the genome. Initial analysis of this protein has identified it to be a 24 kDa protein with motifs similar to chemokine binding proteins that are implicated in virus entry and compromising the host response. The strategy proposed is to determine the gene sequence and perform in situ analysis to infer function. Using an expression vector, the protein will be expressed and its specific ligand will be identified by screening different human chemokines using surface plasmon resonance. Based on the chemokine that will be identified to be specific for B29R, knockout mice without the gene for the ligand will be given doses of the virus to induce infection and symptoms. This will validate the binding specificity of the protein and chemokine. A small-compound library will be screened to determine inhibitors of the specific binding in vitro, and results will be validated on mice with Vaccinia virus infection. (b) For readers who are not scientifically qualified: Vaccinia virus is used in vaccines against smallpox. Its use is expected to increase in the face of threats of bioterrorism. However, the danger of using live Vaccinia in vaccines is its known tendency to be transmitted to others who have not been vaccinated. Therefore, this study is proposed to understand the contribution of the Vaccinia protein B29R in mediating the infection and virus entry in human and mammalian cells. The protein will be isolated, characterized, and compared to other known proteins. Then it will be purified and used to identify the compounds with which it binds, the chemokines. Chemokines are responsible for summoning the immune system of the body to fight the infection. If bound, then the infection will progress. Knowing what binds B29R protein will allow the creation of genetically altered mice that has the identified compound in its system. Furthermore, different compounds will be tested to see if these can alter the binding of the chemokine and B29R. NAME OF APPLICANT:  Q8 Previous Applications to the wellcome trust None Q9 DETAILS OF OTHER CURRENT GRANTS HELD BY APPLICANT(S) None Q10 DETAILS OF RESEARCH PROJECT Include (a) Aims of the project, (b) Work that has led up to the project, (c) Experimental design and methods to be used in investigating this problem. A) The objectives of this project are: 1. To isolate, clone and characterize B29R gene in Vaccinia virus (VACV) Copenhagen strain. 2. To prepare B29R expression vectors 3. To identify human chemokine ligands which bind to the B29R 4. To produce knockout mice without the gene for the identified ligand and to study the effect of VACV on mice lacking the ligand gene 5. To screen for inhibitors of VACV and chemokine/ligand binding 6. Test the selected compounds on VACV infected mice B) Background of the Project The poxvirus family is a large group of dsDNA viruses that replicate in the cell cytoplasm. Variola virus, which causes smallpox, and monkeypox virus are significant human pathogens. Although smallpox has been eradicated, the latter is an emerging disease in Africa. The prototypic poxvirus is Vaccinia virus that was used live in the vaccine, which wiped out smallpox. Currently, it is the vector of choice for recombinant vaccines. The possible use of VACV vaccination is a cause for concern because secondary infection has been documented especially in children with skin diseases and hospital personnel who get the live virus from those who were vaccinated (Sepkowitz, 2003). Therefore it is necessary to characterize the proteins in VACV that are essential for virus transmission and infection, to enable the design of drugs that can counteract any adverse effects coming from secondary infection that will most probably rise in the event of widespread VACV vaccination. The genome of VACV has been fully sequenced and found to encode about 200 proteins (Goebel, Johnson, Perkus, Davis, Winslow, & Paoletti, 1990). The large number of proteins is necessary to meet the requirement virus replication in the cytoplasm which necessitates the use of its own machinery for replication. The virus also produces structural proteins for the formation of the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV) which are the infectious forms. Genes that are located near the end of the genome, aside from facilitating virus replication in vivo, are also involved in interfering with the host immune response such as inflammatory responses, the first host response against the invading organism. Interestingly, these specific genes belong to the category of the early expressed genes during the infection cycle (Assarsson, et al., 2008). Among these genes is B29R, identified as a major secreted protein, which is encoded towards both the C and N terminals of the protein. It has 244 amino acids, and a molecular weight of 24 kDa. The Prosite motif shows lists it under the prokaryotic membrane lipoprotein lipid attachment site profile, while the protein motif belongs to that of a chemokine binding protein (VBRC Gene Record for: VACV-COP:B29R, 2009). However, there is no work on B29R reported in the literature with respect to its specific function, ligand specificity, and essential role in the process of infection and virus transmissibility. Chemokines are proteins that the host cells express first when an invasion by a foreign agent is felt. These chemicals send out signals to recruit the components of cellular immune machinery (Luster, 1998). Viruses have exploited the principles of chemokine and chemokine receptors in gaining entry to the host cell; the best example would be HIV glycoprotein envelop sequentially interacting with chemokine receptors on the cell surface to gain entry into the cells (Lusso, 2006) (Doms, 2004). Chemokine binding proteins, are different from the host chemokine receptors, and block host chemokines from binding with cell surface elements like glycosaminoglycans and host chemokine receptors (Alcami, 2003). Several studies on chemokine binding proteins in poxviruses have identified genus-specific ligands (Seet, et al., 2003) (Najarro, Lee, Fox, Pease, & Smith, 2003). Moreover, the absence of phylogenetic analysis of the current data sequences does not allow for any inference on functional and sequential similarity. So far, no data has been presented on the essentiality of the gene B29R in virus replication, host entry, infectivity, and transmission. C. Design and methodology General Strategy (standard protocols will be used whenever possible) Cells are to be infected with the VACV, then the virus RNA will be extracted. Primers will be designed to amplify the full-length B29R gene. After PCR, products will be inserted into a linear vector and E. coli, for multiplication. When enough clones have been produced, the gene segment will be cut and transferred to an expression vector to produce the B29R protein in large amounts. The proteins will be used to screen different chemokines in a binding assay experiment. Knockout mice will be produced without the gene encoding for the identified chemokine. The VACV virus (concentration to be optimized) will be injected to the normal and knockout mice. Symptoms of VACV infection (skin rashes and lesions), will be monitored closely. Infection can also be visualized by tissue examination. It is expected that knockout mice will have no symptoms. After identification of the chemokine that has specific binding with B29R, screening will commence for compounds that disrupt the B29R-chemokine binding. The screening can be aided by using a small compound library similar to the one that was used in the search for the HIV integrase inhibitor. Details of Experiments 1. Virus culture As described elsewhere (Najarro, Lee, Fox, Pease, & Smith, 2003), monkey kidney BSC-1 and CV-1 cells, will be purchased from a Cell Bank, or a reputable supplier in the United Kingdom. Cells will be grown in MEM or minimal essential medium supplemented with ten percent foetal bovine serum. Cells will be infected with VACV strain Copenhagen, which is the strain used in vaccine production. Working stocks of virus are to be prepared by Dounce homogenization of infected BSC-1 cells, removal of nuclei by centrifugation, and sedimentation. Virus infectivity will be titrated by plaque assay on BSC-1 cells. 2. Gene isolation, cloning, sequencing and sequence analysis a. RNA collection Viral RNA will be extracted using commercial RNA extraction kits. First strand cDNA will be synthesized using commercial kits. The B29R gene will be amplified using specific and degenerate primers from gene homologues in the Genbank. The isolated B29R cDNA will be gel-purified and cloned into a cloning vector, and sent for sequencing. The sequences will be used to design specific primers for the B29R gene. After cloning and verification of the more accurate gene, the gene will be cut from the vector and transferred to another expression vector to produce the protein in sufficient amounts for the binding assays. Restriction enzymes will be used as recommended by the manufacturers of the cloning vectors. b. BLAST, sequence similarity alignments, phylogenetic analysis of the gene sequence will be performed to determine sequence and functional homologies with genes reported in the database. 3. Binding assays to determine specific host chemokine ligand Biomolecular interaction analysis will be performed by using surface plasmon resonance (SPR) imaging as described by Seet, et al. (2002). For ligand screening experiments, the B29R protein will be immobilized at high density onto a CM5 chip and a BiacoreX biosensor. All experiments will be performed at 25°C with HBS-EP (10mMHepes, pH 7.4 150 mM NaCl 3 mM EDTA 0.005% polysorbate 20) as the running buffer. Several human cytokines and chemokines (to be purchased from commercial suppliers) will be injected over the control flow cells while the B29R protein will be in the other flow cell. The interaction will be monitored as per manufacturer’s recommendation. Experiments will be replicated to ensure the reliability of results 4. After the identification of the chemokines/cytokines that bind with B29R, knockout mice, which will not have the gene for the specific chemokine, will be ordered from a mice production facility. This step will eliminate the added step of generating knockouts especially if the lab is not equipped to do the work. Alternatively, a B29R deletion mutant of the VACV will be produced to be injected in normal mice. Virus in the optimized concentration will be injected in the mice for observations of symptoms of infection, which are skin rashes or lesions. 5. When the binding activity in vivo has been established, then it is now possible to screen for inhibitors of the specific B29R/chemokine binding. Different compounds can be screened similar to the strategy that was used in finding the inhibitor for the HIV integrase gene (Pommier, Johnson, & Marchand, 2005). The screening can be done on pure protein extracts and chemokines in vitro, before proceeding to further mice trials. Milestones The project will take two years. In the first year, the following milestones and schedules will be achieved: Milestone Period 1. Gene sequence and full-length B29R clones 4th month 2. Purified proteins and identified chemokines 8th month 3. Response of knockout mice known 14th month 4. Identified binding inhibitor compounds 18th month 5. Known in vivo response to inhibitory action of 22th month identified compounds 6. Report 24th month Possible Pitfalls In every step of the experiment, there are possible pitfalls. Most of these are procedural and can be avoided by following protocols carefully. However, if no protocol has been developed for a certain step, then optimization of the method has to be performed. This could lead to the extension of the time required to achieve the milestones. Q11 REFERENCES (Research Project) This page may be duplicated to allow applicants to use more than one sheet if necessary. Please give citation in full, including title of paper and all authors. Alcami, A. (2003). Viral mimicry of cytokines, chemokines and their receptors. Nature Reviews Immunology , 3, 36-50. Assarsson, A., Greenbaum, J., Sundstrom, M., Schaffer, L., Hammond, J., Pasquetto, V, Oseroff, C., Hendrickson, RC, Lefkowitz, EJ, Tscharke, DC, Sidney, J, Grey, HM., Steven R., Head, SR, Peters, B., Sette,A. (2008). Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. Proceedings of the National Academy of Sciences USA , 105 (6), 2140-2145. Doms, R. (2004). Viral entry denied. New England Journal of Medicine , 351 (8), 743-744. Goebel, S. J., Johnson, G. P., Perkus, M., Davis, S. W., Winslow, J., & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus. Virology , 179, 247-266. Lusso, P. (2006). HIV and the chemokine system: 10 years later. The EMBO Journal , 25, 447-456. Luster, A. (1998). Chemokines, chemotactic cytokines that mediate inflammation. New England Journal of Medicine , 338 (7), 436-445. Najarro, P., Lee, H.-J., Fox, J., Pease, J., & Smith, G. (2003). Yaba-like disease virus protein 7L is a cell-surface receptor for chemokine CCL1. Journal of General Virology , 84, 3325-3336. Pommier, Y., Johnson, A., & Marchand, C. (2005). Integrase Inhibitors to treat HIV/AIDS. Nature Reviews , vol. 4, pp. 236-250. Seet, B., McCaughan, C., Handel, T., Mercer, A., Brunetti, C., McFadden, G., & Fleming, S. (2003). Analysis of an orf virus chemokine-binding protein:Shifting ligand specificities among a family of poxvirus viroceptors. Proceedings of the Nationa Academy of Sciences USA , 100 (25), 15137–15142. Sepkowitz, K. (2003). How contagious is vaccinia? New Englnd Journal of Medicine , 348 (5), 439-446. VBRC Gene Record for: VACV-COP:B29R . (2009). Retrieved May 5, 2009, from Viral Bioinformatics Resource Center: http://www.vbrc.org/protein_analysis.asp?gene_id=39790 Q12 RESEARCH ON HUMAN PARTICIPANTS OR HUMAN TISSUE (a) Does your project involve the use of human participants or human tissue? YES  NO  If yes, refer to notes. If the project includes studies on patients being cared for by the NHS, please also answer Q13. (b) Does your project involve the use of human participants or other human tissue, outside the UK? YES  NO  If yes, refer to notes. (c) Does your project involve the use of human embryos requiring a licence from the Human Fertilisation and Embryology Authority (HFEA)? YES  NO  If yes, refer to notes. (d) Does your proposal involve research on gene therapy which requires regulatory approval? YES  NO  If yes, refer to notes. Q13 RESEARCH USING NHS FACILITIES OR PATIENTS (a) In the course of your project, do you propose to use facilities within the National Health Service and/or does your research involve patients being cared for by the NHS? YES  NO  If yes, please confirm that your project is in accordance with the principles of the Statement of Partnership on Non-Commercial R&D in the NHS in England (or the corresponding statements in Northern Ireland, Scotland and Wales), distributed with Department of Health EL(97)77, dated 27 November 1997 (a link to this site can be found in the associated guidance notes).  (b) Which NHS provider(s) has agreed to facilitate this research?  Q14 EXPERIMENTS ON ANIMALS Do your proposals involve the use of animals or animal tissue? YES  NO  (a) If yes, do your proposals include procedures to be carried out on animals in the UK which require a Home Office licence? YES  NO  If yes, has the Home Secretary granted a Project licence, under the terms and the Animals (Scientific Procedures) Act 1986, authorizing the proposed experiments? YES  NO  If yes, state the name and address of the licensee, the Project Licence reference number, date of issue and end date. Yes, the research will be using tissues from monkey and mouse. Necessary permits will be processed from the proper authorities Do you, or any other researchers associated with the project, hold a Personal Licence under the Animals (Scientific Procedures) Act 1986, permitting the procedures required for the research to be carried out? YES  NO  If yes, give Personal Licence Reference Number and name of Licence Holder.  If no, has application been made for such a licence? YES  NO  Please give a brief explanation, including the date when an application will be made.  (b) Do your proposals involve the use of animals or animal tissue outside the UK? YES  NO  If yes, give details of the local ethics committee approval that has been sought, relating this approval to the permission which would be required if the research were to be conducted in the UK. The use of the tissues are essential to this research in consideration of the fact that the target beneficiaries of the research are humans. Knockout mice are essential to the study. All ethical considerations will be included in the handling of the animals. Read More