Methods and compositions for producing a virus

An adenovirus, recombinant adenovirus technology, applied in the direction of viruses, biochemical equipment and methods, microorganisms, etc., can solve the problem of not being suitable for rapid production of recombinant adenovirus, and achieve the effect of saving a lot of time and enhancing HIV infection

Pending Publication Date: 2021-04-09
OXFORD UNIV INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These people do not yet have access to methods for producing cloned single recombinant viruses where clonal selection methods do not have to be performed after isolation of recombinant viruses
[0005] Therefore, there is no prior art method suitable for the rapid production of recombinant adenoviruses for use as vaccines

Method used

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  • Methods and compositions for producing a virus
  • Methods and compositions for producing a virus
  • Methods and compositions for producing a virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 - Purification of adenovirus terminal protein complex viral gDNA (TBC-gDNA) by cesium chloride density gradient ultracentrifugation.

[0064] A 55 kDa terminal protein (TP) is covalently linked to the 5' end of each strand of adenoviral genomic DNA to generate terminal protein complex viral gDNA (TPC-Ad gDNA). Both serotype-matched ("autologous") and mismatched ("heterologous") TPs can be used in the present invention. TP protects viral gDNA from digestion by cellular exonucleases, acts as a primer to initiate DNA replication, and forms a heterodimer with DNA polymerase. Through subtle changes in the origin of replication, TP enhances replication by increasing template activity by more than 20-fold compared to protein-free templates, enabling the incorporation of other replication factors. TPC-Ad gDNA was isolated from disrupted purified virus particles using guanidine hydrochloride and purified by cesium chloride density gradient ultracentrifugation.

[006...

Embodiment 2

[0069] Example 2 - Preparation of TPC-Ad gDNA for Recombination by Unique Restriction Enzyme Digestion

[0070] Parental adenoviral genome, such as ChAdOx1-Bi-GFP, such as image 3 As shown, contains the GFP coding sequence at El, flanked by the long tetracycline-regulated CMV promoter (LPTOS) and the bovine growth hormone (BGH) polyadenylation signal (poly A). The GFP ORF is flanked by a pair of unique restriction sites recognized by PsiI restriction endonucleases, which can be excised using PsiI to generate 3 fragments: the left arm of the adenovirus genome, the GFP ORF and the right arm genome of the adenovirus. The parental virus can also be digested with AsiSI to excise the entire GFP expression cassette including LPTOS and poly A. This parental virus can also be digested by RsrII to prepare gDNA for insertion of the expression cassette at the S14 (E4) locus.

[0071] Incubate 120 ng of TPC-Ad gDNA overnight in a 37 °C incubator with 10 U of PsiI in the recommended reac...

Embodiment 3

[0072] Example 3 - Rapid Production of Adenovirus: Recombination and Transfection

[0073] The antigen sequence of interest or the expression cassette of interest was introduced into TPC-Ad gDNA by in vitro recombination, and then the recombination reaction product was directly transfected into complementary HEK293 cells for virus rescue. Transfection was performed to obtain single viral clones.

[0074] NEBuilder (NEB) and In-fusion (Takara) are commercially available systems that seamlessly assemble multiple DNA fragments regardless of fragment length or end compatibility. These products can be used to insert the antigen / expression cassette into TPC-Ad gDNA prepared appropriately from Example 2. The recombination reaction mixture includes exonucleases, polymerases, and in the case of NEBuilder, DNA ligases that work together to produce double-stranded DNA molecules. Exonucleases generate single-stranded 3' overhangs that promote annealing of fragments whose ends share comp...

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Abstract

The invention relates to methods for generating a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest for use as a vaccine comprising the steps of inserting the heterologous gene of interest into the adenovirus genome by recombining terminal protein complexed adenovirus genomic DNA (TPC-Ad gDNA) with a polynucleotide comprising a nucleotide sequence encoding the gene of interest and having 5' and 3' ends that are homologous to the insertion site sequence of the adenovirus genomic DNA in an in vitro recombination reaction, transfecting cells growing in individual vessels with a dilution of the in vitro recombination reaction mixture from (i) such that a number of such individual vessels contain a single cell that is infected by a recombinant adenovirus comprising the nucleotide sequence encoding the heterologous gene of interest, and identifying those individual vessels in which a single cell has been infected by the recombinant adenovirus comprising the nucleotide sequence encoding the heterologous gene of interest. Suitably said TPC-Ad gDNA comprises serotype-matched terminal protein and adenovirus genome, and said gene of interest codes for a single epitope, a string of epitopes, a segment of an antigen or a complete antigen protein. The invention also relates to recombinant adenoviruses and compositions made using these methods.

Description

technical field [0001] The present invention relates to the rapid production of recombinant adenoviruses for inducing an immune response, suitably a protective immune response, against heterologous antigens, including infectious pathogen antigens and cancer-associated tumor antigens. Background technique [0002] Replication-poor adenoviral vectors derived from human adenovirus serotype 5 (HAdV-C5) or other human or simian adenoviruses have been used as vaccine vectors to deliver infectious pathogen antigens in multiple clinical trials and cancer antigens (Ewer et al. (2017) Hum Vaccin Immunother.13(12):3020-3032 and Cappuccini et al. (2016) Cancer Immunol Immunother.65(6):701-13.). These vectors offer many advantages for vaccine development. They are not replication competent in humans and are therefore safer than replicating vectors; they infect replicating and non-replicating cells; they have broad tissue tropism and elicit a hyperimmune response, including particularly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12C12N7/00C12N15/86
CPCA61K39/12C12N7/00C12N15/86C12N2710/10343C12N2710/10351A61K39/00C12Q1/70A61K2039/5256C12Q2563/107C12N15/66
Inventor S·吉尔伯特S·J·莫里斯
Owner OXFORD UNIV INNOVATION LTD
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