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Optimization of gene sequences of virus-like particles for expression in insect cells

a technology of viruslike particles and gene sequences, applied in the field of viruslike particle gene sequence optimization for insect cell expression, can solve the problems of reducing antigen and immunogenity, reducing the number of recombinant peptides, and affecting the normal gene regulation of host cells, so as to reduce the number of dna structures in the further-modified nucleotide sequence, the effect of minimizing the number of transcription and post-transcription repressor elements and reducing the number of r

Inactive Publication Date: 2004-06-24
NOVAVAX
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021] In yet another aspect, the invention provides a method for preparing a codon-optimized polynucleotide comprising one or more of the following steps: (a) replacing codons that are underutilized in insect cells with codons that are utilized at high levels in insect cells, to create an initially-modified nucleotide sequence; (b) modifying the initially-modified nucleotide sequence by choosing a preferred codon for the initially modified sequence, wherein: (i) the ratio of GC nucleotide pairs to AT nucleotide pairs in the further-modified nucleotide sequence trends toward approximately 1:1; (ii) the number of palindromic and stem-loop DNA structures in the further-modified nucleotide sequence is minimized; and (iii) the number of transcription and post-transcription repressor elements are minimized.

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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  • Optimization of gene sequences of virus-like particles for expression in insect cells
  • Optimization of gene sequences of virus-like particles for expression in insect cells
  • Optimization of gene sequences of virus-like particles for expression in insect cells

Examples

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example 1

ESTABLISHMENT OF SERUM-FREE SF-9 INSECT CELL LINE

[0204] 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.

[0205] Initially, cell clones capable of growing in commercial serum-free media as suspen...

example 2

ESTABLISHMENT OF TRANSFORMED SF-9S CELL LINE

[0207] 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.

[0208] 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

[0211] 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.

[0212] 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

Codon optimized polynucleotides for optimal expression of recombinant proteins in eukaryotic cells are provided. The codon optimized polynucleotides encode a viral capsid protein that self assembles into a virus-like particle. The virus-like particle is expressed extracellularly and exhibits conformational antigenic epitopes capable of raising neutralizing antibodies. Pharmaceutical compositions, vaccines, and diagnostic test kits containing the gene products of the codon-optimized polynucleotides are also provided.

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.
Owner NOVAVAX
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