Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-09
2001-09-25
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Vector, per se
C530S333000, C514S04400A
Reexamination Certificate
active
06294378
ABSTRACT:
This application relates to improved reagents for use in “genetic immunization,” and to a method for genetic immunization which makes use of these reagents to elicit a more potent immune response.
The generation and regulation of immune response is a result of a complex system of interactions between B- and T-lymphocytes, circulating antibodies, and antigen presenting cells (APC). The induction of humoral and cell-mediated immune responses to protein antigens requires the recognition of the antigens by helper T (TH) cells. The reasons for this is that helper T cell are necessary for stimulating B-lymphocyte growth and differentiation, and for activating the effector cells of cell-mediated immunity, including macrophages and cytolytic T lymphocytes (CTLs). Briefly, foreign antigen is processed by APCs which result in the generation of antigen-derived peptide fragments bound to the major histocompatability complex (MHC) Class I and Class II molecules (referred to as human leukocyte antigens or HLA Class I and Class II proteins in humans). These complexes which are found on the cell surface of the APC are then presented to TH cells. Recognition of the peptide-MHC complex by T cells is the initiating stimulus for T cell activation. Thus, more efficient presentation of peptide-MHC complex can lead to more efficient T cell activation. Activation leads to the secretion of cytokines, proliferation, and regulatory or cytolytic effector functions which all lead to immunity, in part through the eradication of cells presenting antigen.
T cell-mediated eradication of cells expressing antigen can be accomplished in three ways. First, humoral responses occur when activated TH cells stimulate the proliferation and differentiation of specific B cell clones to produce antibodies which eventually eliminate cells expressing the antigen as well as extracellular antigen. Second, cell-mediated responses occur when cytokines activate T cells to differentiate into CTLs. The infected target cell is then lysed by the CTL. Endogenous antigens, such as viruses and tumor antigens, activate Class-I restricted CTLs, which lyse cells producing these intracellular antigens. Third, nonspecific responses occur when antigen-activated T cells secrete cytokines that recruit and activate inflammatory cells such as macrophages and natural killer cells that are not specific for the antigen. Overall, therefore, T cells play a central role in recruiting a broad immune response.
As used herein, the term “genetic immunization” refers to the use of DNA as a vaccine to produce an immune response to the protein or peptide antigen encoded by the DNA. Intramuscular administration of naked DNA has been shown to elicit both humoral and cellular immune response. The precise mechanism by which DNA vaccines elicit an immune response is not known, although several possibilities have been discussed. See Pardoll et al., “Exposing the Immunology of Naked DNA Vaccines”,
Immunity
3: 165-169 (1995). Regardless of the mechanism, however, the effectiveness of DNA vaccines to produce both humoral and cellular immunity indicates that naked DNA is expressed after administration, with the protein or peptide product being presented as an antigen in association with either Class I or Class II proteins.
The processing and presentation of antigens by Class I and Class II molecules occurs in different organelles within the cells. Specifically, the endoplasmic reticulum (ER) has been shown to be the site for loading peptide antigens derived from the cytoplasm onto Class I molecules, while the endosomes/lysosomes have been shown to be the site for loading peptide antigens onto Class II molecules. Thus, the type of immune response and the extent to which an immune response is generated may depend in significant measure on the amount of antigen reaching the ER and endosomal loading sites. It would therefore be highly advantageous to be able to direct and control the accumulation of antigen within a desired location within the cell to provide optimum immune response.
It is an object of the present invention to control the trafficking to and stability of selected antigens within specific cellular organelles, and to use this method to provide for enhanced genetic immunization.
It is a further object of the present invention to provide DNA vaccines which incorporate genetic sequences encoding sorting signals which direct the expressed antigen to a specific cellular organelle and facilitate loading of the antigen onto a Class II or Class II MHC molecule for immune presentation.
It is still a further object of the invention to provide a method for genetic immunization utilizing DNA vaccines which incorporate genetic sequences encoding sorting signals which direct the expressed antigen to a specific cellular organelle and facilitate loading of the antigen onto a Class I or Class II MHC molecule for immune presentation.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved through the construction of a genetic sequence encoding a protein or peptide antigen and a sorting signal which will direct expressed antigen to the ER or endosomal-lysosomal compartments within the cell. The resulting constructs are useful as DNA vaccines, and can be used as naked DNA, packaged in liposomes, or coated onto colloidal gold particles. The construct might also be delivered in an expression vector, for example a viral vector, which is expressed in cells of the organism being immunized.
REFERENCES:
Struck Vaccine R&D success rates and development times. Nature Biotechnology vol. 14 pp. 591-593, 1996.*
Ulmer et al. Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein. Science. 1993, pp. 1745-1749, vol. 259.
Vijayasaradhi et al. Intracellular Sorting and Targeting of Melanosomal Membrane Proteins: Identification of Signals for Sorting of the Human Brown Locus Protein, GP75. J. Cell Biology. 1995, pp. 807-820, vol. 130.
Tiff et al. The Folding and Cell Surface Expression of CD4 Requires Glycosylation. J. Biological Chemistry. 1992, pp. 3268-3273, vol. 267.
Krishnan et al. Paving the Way Towards DNA Vaccines. Nature Medicine. 1995, pp. 521-522, vol. 1, No. 6.
Nanda et al. Induction of Anti-Self-Immunity to Cure Cancer. Cell, 1995, 13-17, vol. 82.
Rowell et al. Lysosome-Associated Membrane Protein-1-Mediated Targeting of the HIV-1 Envelope Protein to an Endosomal/Lysosomal Compartment Enhances Its Presentation to MHC Class II-Restricted T Cells. J. of Immunology, 1995, 155:1818-1828.
Pardoll et al. Exposing the Immunology of Naked DNA Vaccines. Immunity. 1995, 165-169, vol. 3.
Bartido Shirley M.
Houghton Alan
Wang Siqun
Xu Yiquing
Brusca John S.
Oppedahl & Larson LLP
Sloan-Kettering Institute for Cancer Research
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