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Vaccines

A technology of nucleotide sequences and fragments, used in vaccine preparations, administering vaccines by particle-mediated release, application of preparations in medicines, and the field of DNA vaccines

Inactive Publication Date: 2008-09-03
GLAXO GROUP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some HIV isolates have mutations in this region, resulting in non-coding functional proteins that seriously affect their replication and pathogenicity in vivo

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0207] Example 1: Optimization of p55gag (p17, p24, p13) to resemble codon utilization of highly expressed human genes

[0208] gene of interest

[0209] An optimized synthetic gene encoding the p55gag antigen of HIV-1 clade B strain HXB2 for expression in mammalian cells (GenBank Accession No. K03455) was assembled using PCR amplified overlapping oligonucleotides.

[0210] Optimization involves altering the codon usage patterns of viral genes to obtain codon frequencies close to those found in highly expressed human genes. Codons were determined using a statistical Visual Basic program called Syngene (latest version of Calcgene by R.S. Hale and G. Thompson, Protein Expression and Purification, Vol. 12, pp. 185-188, 1998).

[0211] clone:

[0212] The 1528 bp gag PCR product was gel purified, digested with restriction endonucleases Not I and Bam HI, and ligated into NotI / BamHI digested vector WRG7077. That is, the gene was placed between the CMV promoter / intron A and the bo...

Embodiment 2

[0214] Example 2: Preparation of p17 / p24 truncated Nef fusion gene

[0215] gene of interest

[0216] by PCR from plasmid pHXB? Pr (B. Maschera, E Furfine and E.D. Blair 1995 J. Virol 695431-5436) amplifies the p17 and p24 portions of the p55gag gene derived from HIV-1 clade B strain HXB2. pHXB from the 3' end of the HXB2nef gene was amplified from this plasmid? Pr. 426bp. Since the HXB2nef gene contains a premature stop codon, the codon was repaired with two overlapping PCRs (TGA[stop] to TGG[Trp]).

[0217] The PCR products of the p17 / p24 linker and the trNEF linker were joined by one PCR reaction to form the p17p24trNEF fusion gene ( image 3 )(antonym).

[0218] The 1542 bp product was gel purified, digested with restriction endonucleases NotI and BamHI, and cloned into the NotI BamHI site of vector WRG7077. That is, the gene was placed between the CMV promoter / intron A and the bovine auxin polyadenylation signal.

Embodiment 3

[0219] Example 3: Preparation of Gag p17 / 24opt / trNef1 ('Gagopt / Nef') fusion gene

[0220] gene of interest

[0221] The plasmid pGagOPTrpr2 was used as a template for PCR amplification to obtain codon-optimized p55gag gene p17 and p24 parts derived from HIV-1 clade B strain HXB2. A truncated HXB2Nef gene with a repaired premature stop codon (TGA[stop] to TGG[Trp]) was amplified by PCR from plasmid 7077trNef20. The two PCR products were designed to have overlapping ends so that the two genes were joined in the second round of PCR.

[0222] The 1544 bp product was gel purified, digested with restriction endonucleases NotI and BamHI, and cloned (see figure) into the NotI BamHI site of vector WRG7077. That is, the gene was placed between the CMV promoter / intron and the bovine auxin polyadenylation signal.

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Abstract

The invention provides a nucleotide sequence that encodes an HIV-1 gag protein or fragment thereof containing a gag epitope and a second HIV antigen or a fragment encoding an epitope of said second HIV antigen, operably linked to a heterologous promoter. Preferred polynucleotide sequences further encodes nef or a fragment thereof and RT or a fragment thereof.

Description

[0001] This application is a divisional application of the Chinese patent application 02823087.6 "vaccine" filed on September 18, 2002. technical field [0002] The present invention relates to nucleic acid constructs, host cells containing such constructs and their use in nucleic acid vaccines. The invention further relates to vaccine formulations containing such constructs and to the use of such formulations in medicine. The invention particularly relates to DNA vaccines for the prevention and treatment of HIV infection, and more particularly to vaccines administered by particle-mediated release. Background technique [0003] HIV-1 is the leading cause of Acquired Immunodeficiency Syndrome (AIDS), which is considered one of the major global health problems. Despite extensive research worldwide to produce a relevant vaccine, these efforts have so far been unsuccessful. [0004] The non-envelope proteins of HIV-1 have been described, including, for example, internal struct...

Claims

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Application Information

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IPC IPC(8): C12N15/49C12N15/63A61K31/7088A61K48/00A61K39/39A61P31/18C07D471/04A61K39/00A61PA61P31/00A61P37/04C07D215/22C07D215/42C12N
Inventor A·贝顿P·F·埃尔特尔G·W·古A·利尔J·P·蒂特C·A·范维利
Owner GLAXO GROUP LTD
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