20.109:Final Project

T/R Team Purple Han Zhu and Mathangi Radhakrishnan

A brief project overview
To find an antibody-antigen match which can be inserted into a virus. The virus can then be used to infect a specific bacteria. The bacteria will then synthesize the antigen and present it on its cell surface. The system will be presented with antibodies and the white blood cells will be able to kill designed bacteria.

Background Information
White Blood Cell Information:

- different types of leukocytes - Agranulocytes: leukocytes characterized by the absence of granules in their cytoplasm. These include lymphocytes, monocytes, and macrophages (the other type is membrane bound) - Lymphocytes - B-cells (make antibodies to bind to pathogens) - Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an animal these memory cells will “remember” each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. - The two basic types of immunological interaction include (i) binding of an antibody to an antigen and (ii) binding of a T cell receptor (TCR) to a major histocompatibility complex (MHC)-presented antigen-derived peptide. - Over the past decade, approaches to efficiently select phage libraries on cultured cells have evolved [31, 222, 223].

Statement of Research Problem
Identify a peptide sequence, or a transmembrane protein, that can efficiently bind (this antigen), and can be effectively inserted into (this virus’s) vector. (extra goal) Identify a link between bacteria and virus which causes the virus to specifically target this bacteria and form a plasmid, in which this parameter can be manipulated.

Project Details and Methods
Problems: 1.	identify virus – must be a bacteriophage and nontoxic 2.	Viruses only infect specific bacteria. Must develop a way to make a connection between viruses and bacteria 3.	identify antigen- (maybe made into a transmembrane protein) do a library screen of peptide sequences that bind any antibody we want. The antibody will later be presented to the system. Different peptides can be expressed on yeast cells and washed on a plate containing antibodies.

Additional Problems: - Viruses must be able to be delivered efficiently and be able to access target bacteria. - Do we need to modify the virus to make it safe for humans? - What effect will this method have on the immune system? Can we measure this?

Questions that will be tested Where will the most efficient location be to insert the antigen peptide sequence in the virus? Is this antigen expressed efficiently in the bacteria cells? How many copies are presented on the surface of a bacteria. (tested though fluorescent techniques) Design a fluorescent indicator that binds to the presented antigen. (In vivo or vitro testing?)

Clinical Testing: Test this on different animals to see what the immune response to it is.

Predicted Outcomes
If the antigen can be expressed properly, the antibodies presented to the system should efficiently bind it. This will then alert the T-cells? To engulf/lyce the target bacteria.

Resources
Ditzel HJ, Masaki Y, Nielsen H, Farnaes L and Burton DR: Cloning and expression of a novel human antibody-antigen pair associated with Felty's syndrome. Proc Natl Acad Sci USA 97: 9234-9239, 2000.

Griffiths, A. D., Malmqvist, M., Marks, J. D., Bye, J. M., Embleton, M. J., McCafferty, J., Baier, M., Holliger, K. P., Gorick, B. D., Hughes-Jones, N. C., Hoogenboom, H. R., and Winter, G. (1993) Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12, 725 –734[Medline]

Reiersen, H., Lobersli, I., Loset, G.A., Hvattum, E., Simonsen, B., Stacy, J.E., McGregor, D., FitzGerald, K., Welschof, M., Brekke, O.H., Marvik, O.J. (2005), Covalent antibody display—an in vitro antibody-DNA library selection system., Nucleic Acids Research. 33 (1).

Sergeeva, A., Kolonin, M.G., Molldrem, J.J., Pasqualini, R., Arap, W. (2006), Display technologies: application for the discovery of drug and gene delivery agents. Advanced Drug Delivery Reviews. 58 (15): 1622-1654.