Attenuated SARS and use as a vaccine

A virus, virus titer technology, used in DNA/RNA vaccination, vaccines, inactivation/attenuation, etc.

Inactive Publication Date: 2008-08-20
CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0019] This conundrum is now solved by nucle

Method used

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  • Attenuated SARS and use as a vaccine
  • Attenuated SARS and use as a vaccine
  • Attenuated SARS and use as a vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Construction of SARS-CoV Replicon cDNA

[0162] A cDNA containing the untranslated 5' and 3' ends of the Urbani genome and the replicase and N genes was cloned as a BAC under the control of the CMV promoter.

[0163] In addition, a multiple cloning site containing unique restriction sites PacI, AscI and BamHI was cloned downstream of the replicase gene to allow cloning of heterologous genes (Fig. 3). This method uses a two-step amplification system that couples replicon RNA transcription in the nucleus from the CMV promoter with a second amplification step in the cytoplasm driven by a viral polymerase. The plasmid pBAC-SARS-CoV-REP encoding the SARS-CoV replicon was stable for at least 180 passages during proliferation in DH10B cells as determined by restriction enzyme analysis.

[0164] In a first step, suitable restriction sites in the viral genome that could be used to engineer replicons and full-length cDNA clones of SARS-CoV were identified (Fig. 1).

[0165] In ...

Embodiment 2

[0168] Analysis of cloned dDNA stability

[0169] The stability of the viral sequences cloned into pBeloBAC 11 was analyzed by studying the restriction enzyme patterns at different passages. Bacteria transformed with recombinant plasmids were grown at 37°C in 10 ml LB containing 12.5 μg / ml chloramphenicol. Cells from these primary cultures (considering passage 0) were diluted by daily dilution of 10 6 Doubling to multiply continuously. Each passage is considered to represent about 20 generations.

Embodiment 3

[0171] Sequence analysis

[0172] DNA was sequenced using an automated 373 DNA sequencer (Applied Biosystem) using fluorescent dye-labeled dideoxynucleotides and temperature resistant DNA polymerase (Perkin Elmer).

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Abstract

The present invention relates to nucleic acids encoding attenuated SARS-CoV viruses which are capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 2 when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture. According to a further aspect of the present invention, the nucleic acids encoding an attenuated SARS-CoV virus, are obtainable by a method comprising steps, wherein the genome of a SARS-CoV virus is modified by amending the sequence of the gene encoding the SARS-CoV E protein so that the nucleic acid cannot express a functional E protein. The present invention further relates to the viruses encoded by these nucleic acids as well as the medical use of the nucleic acids and of the viruses.

Description

[0001] The present invention relates to a nucleic acid encoding an attenuated SARS-CoV virus capable of growing in a cell culture when compared to the maximum viral titer of a wild-type SARS-CoV virus in the same cell culture The maximum virus titer produced in the culture was reduced. technical background [0002] Treatment methods that involve the insertion of functional genes into cells to achieve a therapeutic effect are also known as gene therapy methods because genes act as drugs. Gene therapy is a technique primarily used to correct defective genes responsible for the development of diseases. [0003] Carrier molecules, also known as vectors, are used to deliver therapeutic genes to target cells in a patient. Currently, the most common vectors are viruses, which have been genetically altered to carry human or animal genes. Viruses have naturally evolved to package and deliver their genes into human and animal cells in a pathogenic manner. At the same time, however, s...

Claims

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Application Information

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IPC IPC(8): C12N7/04A61K39/215A61P11/00
CPCC12N2770/20034C12N2770/20061A61K2039/5254A61K2039/53C07K14/005C12N7/00A61K39/215C12N2770/20022A61K39/12A61P11/00
Inventor L·恩朱阿尼斯桑切斯M·洛佩茨德迪戈E·阿尔瓦雷茨戈梅茨F·阿尔马詹托拉尔
Owner CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC)
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