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Molecular vaccine linking intercellular spreading protein to an antigen

a technology of intercellular spreading protein and molecular vaccine, which is applied in the field of molecular biology, immunology and medicine, can solve the problems of achieve limited potency of naked dna vaccine, enhance vaccine potency, and enhance spreading and mhc class i presentation of antigens

Inactive Publication Date: 2008-11-20
WU TZYY CHOOU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about improving the potency of DNA vaccines by enhancing their ability to spread and be presented by MHC class I proteins. This is achieved by fusing a nucleic acid sequence encoding a protein called VP22, which has the ability to intercellular transport and be presented by MHC class I proteins, with a nucleic acid sequence encoding an antigen. This fusion results in a much more effective vaccine, as it increases the number of antigen-specific CD8+ T cells and converts a less effective vaccine into a highly effective one. The invention also includes a nucleic acid molecule that encodes a fusion polypeptide and a vector for expressing the nucleic acid molecule in a cell. The invention also provides a particle comprising the nucleic acid molecule. Overall, the invention enhances the potency of DNA vaccines and makes them more effective against tumors and other disease-causing organisms.

Problems solved by technology

The potency of naked DNA vaccines is limited by their inability to amplify and spread in vivo.

Method used

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  • Molecular vaccine linking intercellular spreading protein to an antigen
  • Molecular vaccine linking intercellular spreading protein to an antigen
  • Molecular vaccine linking intercellular spreading protein to an antigen

Examples

Experimental program
Comparison scheme
Effect test

example i

Materials and Methods for HSV-VP22 DNA Study

Plasmid DNA Construction

[0189]The pcDNA3 expression vector and pcDNA3-E7 have been described (Chen, C H et al., 1999, Gene Ther 6:1972-81; Ji, H. et al., 1999, Human Gene Therapy 10:2727-2740). pcDNA3 has been used successfully in DNA vaccine induced immune responses and antitumor effects (Chen, C H et al., 2000, Cancer Res 60:1035-42; co-pending, commonly assigned U.S. patent applications U.S. Ser. No. 09 / 421,608, filed 20 Oct. 1999, U.S. Ser. No. 0911,097, filed 9 Feb. 2000 and U.S. Ser. No. 09 / 693,450; filed 20 Oct. 2000, which are incorporated by reference). For the generation of pcDNA3-VP22, VP22 was subcloned from pVP22 / myc-His (Invitrogen, Carlsbad, Calif.) into the unique EcoRV and BamHI cloning sites of the pcDNA3.1(−) expression vector (Invitrogen, Carlsbad, Calif.) downstream of the CMV promoter. The generation of pcDNA3-E7 has been described previously (Chen et al., supra). For the generation of pcDNA3-VP22 / E7, VP22 was subclon...

example ii

Enhanced Intercellular Spreading of VP22 Fusion Proteins

[0207]We initially generated several DNA constructs (E7, VP22, VP22 / E7, VP22(1-267) / E7, E7 / GFP, VP22 / GFP, VP22 / E7 / GFP, and VP22(1-267) / E7 / GFP) using a mammalian cell expression vector (pcDNA3). To demonstrate if VP22 / E7 protein generated enhanced intercellular spreading of E7 in 293 DbKb cells, we used green fluorescent protein (GFP) as a marker protein and examined green fluorescence. 293 DbKb cells were transfected with E7 / GFP, VP22(1-267) / E7 / GFP, or VP22 / E7 / GFP DNA. We performed fluorescent microscopic examination of 293 DbKb cells to investigate the topological distribution of GFP protein. We observed significant spread of GFP protein in cells transfected with VP22 / E7 / GFP DNA but not in cells transfected with E7 / GFP or VP22(1-267) / E7 / GFP DNA (FIG. 1A).

[0208]We also administered VP22 / GFP or GFP intradermally into C57BL / 6 mice via gene gun. To demonstrate if the linkage of VP22 to protein led to enhanced intercellular spreadi...

example iii

Enhanced T Cell Activities Induced by VP22 Linked to Antigen

[0209]The observed increase in intercellular spreading of the marker protein within the epidermis raises the possibility of generating an increased number of antigen presenting cells (APCs) that present the linked protein since the epidermis is rich with Langerhans' cells, the professional APC precursors. To further investigate if such increased spreading can lead to enhanced antigen-specific T cell activities, we linked VP22 to a model antigen, HPV-16 E7, which is associated with a majority of cervical cancers. E7 is important in the induction and maintenance of cellular transformation and co-expressed in most HPV-containing cervical cancers and their precursor lesions and therefore represents an ideal target for vaccine development (21).

[0210]The importance of CD8+ cytotoxic T cells for the control of viral infections and neoplasms has been demonstrated in several pre-clinical models (9, 22). To determine whether vaccinat...

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Abstract

Superior molecular vaccines comprise nucleic acids, including naked DNA and replicon RNA, that encode a fusion polypeptide that includes an antigenic peptide or polypeptide against which an immune response is desired. Fused to the antigenic peptide is an intercellular spreading protein, in particular a herpes virus protein VP22 or a homologue or functional derivative thereof. Preferred spreading proteins are VP22 from HSV-1 and Marek's disease virus. The nucleic acid can encode any antigenic epitope of interest, preferably an epitope that is processed and presented by MHC class I proteins. Antigens of pathogenic organisms and cells such as tumor cells are preferred. Vaccines comprising HPV-16 E7 oncoprotein are exemplified. Also disclosed are methods of using the vaccines to induce heightened T cell mediated immunity, in particular by cytotoxic T lymphocytes, leading to protection from or treatment of a tumor.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention in the fields of molecular biology, immunology and medicine relates to a chimeric nucleic acid, including DNA and viral RNA encoding a fusion protein and its use as a vaccine to enhance immune responses, primarily cytotoxic T lymphocyte (CTL) responses to specific antigens such as tumor or viral antigens. The fusion protein comprises an antigenic polypeptide fused to a protein that promotes intercellular transport and processing via the MHC class I pathway, such as the VP22 protein from herpes simples virus and related herpes viruses.[0003]2. Description of the Background Art[0004]Naked DNA vaccines have emerged as attractive approaches for vaccine development (for review, see (1-3)). Intradermal administration of DNA vaccines via gene gun represents a convenient way of delivering DNA vaccines into professional antigen presenting cells (APCs) in vivo. Professional APCs are a superior candidate for ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C07H21/04C12N15/63C12N15/87A61K35/12C12N5/06C07K2/00A61K35/76A61K39/12A61P35/00C07K14/025C07K14/035C07K14/055C12N15/86
CPCA61K39/12A61K2039/6075C07K14/005C07K2319/00C12N7/00C12N15/86C12N2710/16322C12N2710/16622C12N2710/20022C12N2710/20032C12N2710/20034A61K39/385A61K2039/53A61K2039/585A61P35/00
Inventor WU, TZYY-CHOOUHUNG, CHIEN-FU
Owner WU TZYY CHOOU
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