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Kit for treating gastrointestinal tract

a technology for the gastrointestinal tract and a kit is applied in the field of viral vaccines, therapeutics, and diagnostics, which can solve the problems of reducing antigen and immunogenity, affecting the normal regulation of host cell gene regulation, and affecting the detection and treatment of human papillomavirus infections

Inactive Publication Date: 2004-04-01
NOVAVAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wild type and intact versions of these viral genes and their gene products in the context of a vaccine may disrupt normal host cell gene regulation by increasing the levels of Rb and p53 proteins and facilitate cell transformation.
Thus proteins frequently are not stable in the presence of endogenous bacterial proteases, and tend to aggregate into inactive complexes.
Consequently, recombinant peptides often suffer from low yield and demonstrate reduced antigencity and immunogencity as compared with native peptides.

Method used

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  • Kit for treating gastrointestinal tract
  • Kit for treating gastrointestinal tract
  • Kit for treating gastrointestinal tract

Examples

Experimental program
Comparison scheme
Effect test

example 1

ESTABLISHMENT OF SERUM-FREE SF-9 INSECT CELL LINE

[0200] A new insect cell line designated Sf-9S was derived from the parent S. frugiperda Sf-9 cell line (ATCC CRL-1771) by several rounds of selective processes based on serum-independent growth and enhanced expression of secreted recombinant proteins from baculovirus vectors. Specifically, Sf-9 cells were cultivated to passage 38 in Grace's insect media (Life Technologies, Grand Island, N.Y. 14072) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, N.Y. 14072) as monolayer cultures in T-75 flasks (Corning, Inc., Corning, N.Y.). The master cell bank of Sf-9 cells was stored at passage 38 in serum-containing media at -70.degree. C. and in liquid nitrogen. A working cell bank was established from a single cryovial of the Sf-9 master cell bank and cultivated in serum-containing insect media for an additional five (5) passages.

[0201] Initially, cell clones capable of growing in commercial serum-free media as suspen...

example 2

ESTABLISHMENT OF TRANSFORMED SF-9S CELL LINE

[0203] In a second selection process, one of the serum-free cell clones developed in Example 1 was chosen to select cell clones that may produce enhanced levels of recombinant extracellular proteins and VLPs from several viruses including rotaviruses and human papillomaviruses by successive rounds of clonal selection of cells infected with recombinant baculoviruses and expressing extracellular self-assembled VLPs.

[0204] This process involved the plating of cell aliquots (200 .mu.l) from a cell suspension (one cell per 200 .mu.l) of the parent cell clone (#23) in serum-free media onto 96-well dishes at a ratio of 200 .mu.l per well. From wells containing a single cell in the original seeding, cells were grown to confluency and subcultured into six replica-plates (96-well). The first round of selection was performed when a total cell density of 2-4.times.10.sup.3 cells / well was obtained; the cells were infected with recombinant baculoviruses...

example 3

CLONING CODON-OPTIMIZED HPV-16 L1 GENES AND ESTABLISHMENT OF RECOMBINANT BACULOVIRUS STOCKS

[0207] A HPV-16 L1 prototype (GenBank Accession No. K02718) and modified in U.S. Pat. No. 5,985,610, was optimized for codon usage in insect cells of the Lepidopteran family. The HPV-16 L1 gene was optimized (FIG. 1A) in this embodiment of the present invention for codon usage based on the following criteria: (1) abundance of aminoacyl-tRNAs for a particular codon in Lepidopteran species of insect cells for a given amino acid as described by Levin and Whittome (2000); (2) maintenance of GC-AT ratio in L1 gene sequence at approximately 1:1; (3) minimal introduction of palindromic or stem-loop DNA structures, and (4) minimal introduction of transcription and post-transcription repressor element sequences.

[0208] The optimized gene sequence was synthesized in vitro as overlapping oligonucleotides, cloned into a subcloning plasmid vector, and then cloned into a bacmid transfer vector (i.e., Luckow ...

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Abstract

Methods for isolation and purification or recombinant gene products are disclosed. In particular, methods for isolation and purification of extracellular and intracellular viral gene products, including virus-like particles, are disclosed herein.

Description

[0001] This application claims benefit under 37 U.S.C. .sctn. 119(e) based on U.S. Provisional Application Nos. 60 / 356,119, 60 / 356,161, 60 / 356,118, 60 / 356,133, 60 / 356,157, 60 / 356,156, 60 / 356,123, 60 / 356,113, 60 / 356,154, 60 / 356,135, 60 / 356,126, 60 / 356,162, 60 / 356,150, 60 / 356,151, and 60 / 356,152, each filed Feb. 14, 2002, the entire contents of each of which are incorporated herein by reference.I. FIELD OF THE INVENTION[0002] The present invention relates to the field of viral vaccines, therapeutics, and diagnostics, compositions and methods for the detection, protection and treatment of human papillomavirus (HPV) infections and associated dysplasia. In particular, the invention relates to novel polynucleotide molecules encoding recombinant HPV gene products having increased antigenicity and immunogenicy in mammals.II. BACKGROUND OF THE INVENTION[0003] Cervical cancer results in over 200,000 deaths per year worldwide (Parkin et al., 1990; Pisani et al., 1990). The greatest burden of d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/12C07K14/025C07K14/14C12N5/06C12N7/00C12N7/01C12N7/04C12N15/86C12P21/04
CPCA61K2039/5258C07K14/005C12N7/00C12N2720/12322C12N2710/14143C12N2710/20022C12N2710/20023C12N2510/02
Inventor ROBINSON, ROBIN A.THOMPSON, MARK W.
Owner NOVAVAX
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