阅读说明：本技术 产生病毒的方法和组合物 (Methods and compositions for producing viruses ) 是由 S·吉尔伯特 S·J·莫里斯 于 2019-08-30 设计创作，主要内容包括：本发明涉及一种用于产生用作疫苗的重组腺病毒的方法,该重组腺病毒包含编码目的异源基因的核苷酸序列,该方法包括以下步骤：通过将末端蛋白复合腺病毒基因组DNA(TPC-Ad gDNA)与多核苷酸重组,将异源目的基因插入腺病毒基因组,该多核苷酸包含编码目的基因的核苷酸序列,并所述核苷酸序列的5’末端和3’末端与在体外重组反应中腺病毒基因组DNA的插入位点序列同源；用来自(i)的体外重组反应混合物的稀释液转染在单个容器中生长的细胞,使得许多这样的单个容器包含被重组腺病毒感染的单个细胞,所述重组腺病毒包含编码目的异源基因的核苷酸序列；鉴定其中单个细胞已被重组腺病毒感染的那些个容器,所述重组腺病毒包含编码目的异源基因的核苷酸序列。适当地,所述TPC-Ad gDNA包含血清型匹配的末端蛋白和腺病毒基因组,所述目的基因编码单个表位、表位串、抗原区段或完整抗原蛋白。本发明还涉及使用这些方法制备的重组腺病毒和组合物。(The present invention relates to a method for producing a recombinant adenovirus for use as a vaccine, the recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest, the method comprising the steps of: inserting a heterologous gene of interest into the adenovirus genome by recombining terminal protein complex adenovirus genomic DNA (TPC-Ad gDNA) with a polynucleotide comprising a nucleotide sequence encoding the gene of interest, and the 5 'end and the 3' end of said nucleotide sequence being homologous to the insertion site sequence of the adenovirus genomic DNA in an in vitro recombination reaction; (ii) transfecting cells grown in individual containers with a dilution of the in vitro recombination reaction mixture from (i) such that a plurality of such individual containers comprise individual cells infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest; identifying those containers in which the individual cells have been infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest. Suitably, the TPC-Ad gDNA comprises serotype-matched terminal proteins and an adenovirus genome, the gene of interest encoding a single epitope, an epibit string, an antigen segment or a complete antigenic protein. The invention also relates to recombinant adenoviruses and compositions made using these methods.)

1. A method for producing a recombinant adenovirus for use as a vaccine, the recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest, the method comprising the steps of:

(i) inserting a heterologous gene of interest into the adenovirus genome by recombining terminal protein complex adenovirus genomic DNA (TPC-Ad gDNA) with a polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the gene of interest, and the 5 'end and the 3' end of said nucleotide sequence being homologous to the insertion site sequence of the adenovirus genomic DNA in an in vitro recombination reaction;

(ii) (ii) transfecting cells grown in individual containers with a dilution of the in vitro recombination reaction mixture from (i) such that a plurality of such individual containers comprise individual cells infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest;

(iii) identifying those containers in which the individual cells have been infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest.

2. The method of claim 1, wherein the TPC-Ad gDNA comprises serotype-matched terminal proteins and an adenovirus genome.

3. The method of claim 1 or 2, wherein the gene of interest encodes a single epitope, an epibit string, an antigen segment, or a complete antigenic protein.

4. The method of any one of the preceding claims, wherein the polynucleotide is a synthetic DNA molecule, a purified DNA restriction fragment or a Polymerase Chain Reaction (PCR) product.

5. A method according to any one of the preceding claims wherein the polynucleotide has between 5 and 50bp bases at its 5 'end and between 5 and 50bp bases at its 3' end, which are homologous to the insertion site sequence of the adenoviral genomic DNA.

6. The method of claim 5, wherein the polynucleotide has between 10 and 20bp bases at its 5 'end and between 10 and 20bp bases at its 3' end, which are homologous to the insertion site sequence of the adenoviral genomic DNA.

7. The method of claim 6, wherein the polynucleotide has 15bp of bases at its 5 'end and 15bp of bases at its 3' end, which are homologous to the insertion site sequence of the adenoviral genomic DNA.

8. The method according to any one of the preceding claims, wherein the insertion site sequence of the adenoviral genomic DNA is located within the E1 locus.

9. The method of any one of the preceding claims, wherein the TPC-Ad gDNA is digested at a unique restriction site within the insertion site sequence of the adenovirus genomic DNA flanked at its 5 'end by a tetracycline regulated CMV promoter which drives expression of the gene of interest, and which is flanked at its 3' end by a bovine growth hormone polyadenylation sequence.

10. The method according to any of the preceding claims, wherein the in vitro recombination reaction comprises 40ng of digested TPC-Ad gDNA and 44fmol of the 3 'and 5' ends of the synthetic DNA encoding the gene of interest.

11. The method of any one of the preceding claims, wherein the cells to be transfected the day before transfection are at 3.75 x 10⁵The cells are seeded in a single container at a density of cells/ml.

12. The method of any one of the preceding claims, wherein cells are transfected while growing at about 80% confluence in a single vessel.

13. A method according to any one of the preceding claims, wherein the transfected cells stably express the tetracycline repressor.

14. The method according to any of the preceding claims, wherein the in vitro recombination reaction mixture is diluted and aliquoted in transfection medium in order to transfect cells grown in 60 separate containers.

15. A method according to any preceding claim, wherein the transfected cells are frozen and thawed to release cell-associated virus and the presence of recombinant adenovirus is identified by quantitative pcr (qpcr) using cell lysate of the transfected cells from each well, a set of primers and a probe designed to bind to the left end of the genome downstream of the adenovirus Inverted Terminal Repeat (ITR) and the genome upstream of the target gene insertion site in the non-coding region.

16. The method according to any of the preceding claims, wherein the adenoviral genome is derived from a simian adenovirus.

17. The method of claim 16, wherein the simian adenovirus is ChAdOx1 or ChAdOx2 or ChAd3 or ChAd 63.

18. The method of any one of claims 1 to 15, wherein the adenoviral genome is derived from a human adenovirus.

19. The method of claim 18, wherein the human adenovirus is not a human adenovirus serotype 5.

20. The method of any one of the preceding claims, wherein the single container is an individual well in a multi-well plate.

21. The method according to any of the preceding claims, wherein the TPC-Ad gDNA is provided from a pool of TPC-Ad gDNA raw materials that have been subjected to and passed through the necessary Quality Control (QC) assays to allow for production quality management practice (GMP) bio-manufacturing.

22. A recombinant adenovirus made according to the method of any preceding claim.

23. A composition comprising an adenovirus genome wherein the E1 gene is replaced by an expression cassette comprising a DNA sequence encoding a fluorescent marker protein flanked by a first pair of unique restriction sites that are absent anywhere else in the adenovirus genome for use in the method of any preceding claim.

24. The composition of claim 23, wherein the adenoviral genome is derived from a simian adenovirus.

25. The composition of claim 24, wherein the simian adenovirus is ChAdOx1 or ChAdOx2 or ChAd3 or ChAd 63.

26. The composition of claim 23, wherein the adenoviral genome is derived from a human adenovirus.

27. The composition of claim 26, wherein the human adenovirus is not a human adenovirus serotype 5.

28. The composition of any one of claims 23 to 27, wherein the fluorescently labeled protein is Green Fluorescent Protein (GFP).

29. The composition of any one of claims 23 to 28, wherein the first pair of unique restriction sites is selected from the group consisting of PsiI, AsiSi, or RsrII sites.

30. The composition of any one of claims 23-29, wherein the expression cassette further comprises a long tetracycline-regulated CMV promoter 5 'to the DNA sequence encoding the fluorescent marker protein and a bovine growth hormone polyadenylation sequence 3' to the DNA sequence encoding the fluorescent marker protein, wherein the first pair of unique restriction sites are located between the long tetracycline-regulated CMV promoter and the DNA sequence encoding the fluorescent marker protein and between the DNA sequence encoding the fluorescent marker protein and the bovine growth hormone polyadenylation sequence.

31. The composition of claim 30, wherein the expression cassette further comprises a second pair of unique restriction sites different from the first pair of unique restriction sites, the second pair of unique restriction sites located 5 'of the long tetracycline regulated CMV promoter and 3' of the bovine growth hormone polyadenylation sequence.

32. The composition of claim 31, wherein the second pair of unique restriction sites is selected from the group consisting of PsiI, AsiSi, and RsrII sites.

33. The composition of any one of claims 23 to 32, wherein the adenovirus genome is further engineered to comprise an additional unique restriction site at the S15/E4 locus.

34. The composition of claim 33, wherein the additional unique restriction site is selected from the group consisting of a PsiI, AsiSi, or RsrII site.

35. The composition of any one of claims 23 to 34, wherein the adenovirus genome is complexed with an autologous terminal protein.

36. The composition of any one of claims 23-34, wherein the adenoviral genome is complexed with a heterologous terminal protein.

37. The composition of any one of claims 23 to 36, wherein the adenoviral vector lacks Gateway recombination sequences.

38. A composition according to any one of claims 23 to 37, which has been subjected to and passed the necessary quality control assays to allow for GMP bio-manufacturing.

39. A recombinant adenoviral vector immunogen comprising any one of the compositions of claims 23-37, expressing a pathogen or tumor epitope or antigen against an immune response generated in a mammal.

Technical Field

The present invention relates to the rapid generation of recombinant adenoviruses for inducing an immune response, suitably a protective immune response, against heterologous antigens including infectious pathogen antigens and tumor antigens associated with cancer.

Background

Replication incompetent adenovirus vectors derived from human serum type 5 adenovirus (HAdV-C5) or other human or simian adenoviruses have been used as vaccine vectors to deliver infectious pathogen antigens and Cancer antigens in a number of clinical trials (Ewer et al (2017) Hum vaccine immunother.13(12): 3020-. These vectors offer many advantages for vaccine development. They do not have replication capacity in humans and are therefore safer than replicating vectors; they infect replicating and non-replicating cells; they have extensive tissue tropism and can elicit high immune responses, including particularly effective cellular immunity; they are furthermore easy to purify to high titers (Morris et al (2016) Future Virology 11(9), 649-. The advent of Bacterial Artificial Chromosome (BAC) technology in combination with bacteriophage lambda Red recombination (recombineering) has facilitated cloning and manipulation of the Adenovirus Genome (Ruzsics Z., Lemnitzer F., third C. (2014) Engineering Adenoviral Genome by Bacterial organism Chromosome Biology (BAC) technology in: Chill et al, Bosch A. (eds.) Adenoviral. Methods in Molecular Biology (Methods and Protocols), vol 1089.Humana Press, Totowa, NJ). This technique is combined with recombinant techniques for rapid insertion of expression cassettes, currently used for the production of adenoviral vectors. However, using this method and conventional manufacturing processes, the average time from antigen identification to clinical-grade adenoviral vectors was 33-44 weeks (FIG. 1). The preparation of recombinant viruses for use as vaccines involves starting materials produced prior to GMP (prior to quality management regulations for drug production) which require rescue (rescue) of the receptive cells after their initial infection before the recombinant virus can be cloned. This is a time consuming process, requiring up to 3 rounds of cloning, each round taking 5 weeks, since the chloramphenicol gene (for Bacterial Artificial Chromosome (BAC) selection during standard adenoviral genome manipulation) may need to be inserted into the adenoviral genome, and the viral vector produced from the BAC-derived adenoviral genome is heterogeneous. With BAC, any mutations that may be introduced in the genome of the gland during bacterial manipulation will be carried over into the adenovirus. This problem is solved in the method of the invention, wherein the adenoviral genomic DNA has been cloned and characterized before the start of the production of the recombinant adenovirus and is therefore known to be correct. Thus, it is not necessary to sequence the adenoviral genomic DNA, as has been described previously.

Hillgenberg and its partners (Journal of Virology 80(11) (2006) 5435-. However, these authors attempted to generate recombinant adenovirus populations expressing a large number of different heterologous genes and failed to provide rapid and simple cloning of individual recombinant viruses in their method of use as vaccines. According to regulations relating to GMP production and clinical use of such vaccines, single clones are required for clinical use of adenoviral vaccines.

Recently, micik and its collaborators (PLoS ONE 13(6) (2018) e0199563) described an in vitro assembly of the adenoviral genome from multiple fragments, which were then transfected into cells. These people have not obtained a method to produce cloned single recombinant viruses, wherein no clonal selection method is necessary after isolation of the recombinant virus.

Therefore, there is no method in the prior art suitable for the rapid production of recombinant adenoviruses for use as vaccines. The present invention seeks to address and overcome this challenge, and overcome one or more of the problems associated with the prior art methods, by providing a novel method for generating small clinical batches of incompletely replicated adenoviral vectors within 4 weeks (FIG. 2).

Disclosure of Invention

Adenoviruses are non-enveloped viruses with a linear double-stranded DNA (dsDNA) genome between 26-46kb in length. Adenovirus genomic DNA is infectious when transfected into recipient cells as naked DNA. However, it was reported that when human Ad-5(HAdV-C5) genomic DNA (gDNA) complexed with the 55kDa Terminal Protein (TP) from the same adenovirus was transfected into the recipient cells, viral plaques were produced 100-fold higher than naked DNA. TP protects viral gDNA from digestion by extracellular exonuclease, acts as a primer to initiate DNA replication, and forms heterodimers with DNA polymerase. The DNA polymerase covalently couples the first dCTP to Ser-580 of HAdV-C5 TP. Human adenovirus TP enhances human adenovirus replication by increasing template activity 20-fold compared to a template without protein. This is achieved by subtle changes in the origin of replication, allowing the incorporation of other replication factors. TP also promotes transcription by mediating HAdV-C5 genomic DNA-host nuclear matrix association.

The present inventors have attempted to use transfected TPC-adenovirus gDNA (TPC-Ad gDNA) in combination with existing recombinant technology to increase the plaque production characteristics to generate clinical batches of adenoviral vaccine vectors, particularly the currently preferred simian adenoviral vector.

TPC-Ad gDNA can be isolated and purified, tested for homogeneity, and stored prior to adenovirus production and manufacture. This approach eliminates the need to transmit adenoviral gDNA in bacteria, thus avoiding the possibility of inserting the chloramphenicol gene (for BAC selection) into the adenoviral genome and the possibility of heterogeneity of the viral genome after multiple rounds of amplification in bacterial hosts. When only a small amount of recombinant adenovirus genome is produced, the number of plaques produced after transfection of cells with TPC-Ad gDNA increases, recombinant virus can be successfully rescued, and furthermore, the resulting recombinant adenovirus can be cloned rapidly and easily at a very early stage in the manufacturing process. The present inventors have simplified the production and manufacturing process of viruses, and the obvious results have made it possible to produce and manufacture recombinant adenoviruses for use as vaccines in as short as 28 days. The reduction in vaccine production time will bring many advantages, including: i) allows the rapid generation of personalized cancer vaccines for more rapid treatment of malignancies through therapeutic immunization; ii) in the face of new epidemics, vaccines against new fulminant pathogens are produced more rapidly, so that a greater number of vaccines can be produced and produced more rapidly; iii) reduce the manufacturing cost of expensive GMP (production quality control Specification) manufacturing equipment by significantly reducing the equipment manufacturing time.

In a first aspect, the present invention provides a method for producing a recombinant adenovirus for use as a vaccine, said recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest, the method comprising the steps of: (i) inserting a heterologous gene of interest into the adenovirus genome by recombining terminal protein complex adenovirus genomic DNA (TPC-Ad gDNA) with a synthetic DNA comprising a nucleotide sequence encoding the gene of interest and having at least 15bp of bases at its 5 'end and at least 15bp of bases at its 3' end, which is homologous to the insertion site sequence of the adenovirus genomic DNA in an in vitro recombination reaction; (ii) (ii) transfecting cells grown in individual containers with a dilution of the in vitro recombination reaction mixture from (i) such that a plurality of such individual containers comprise individual cells infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest; (iii) identifying those containers in which the individual cells have been infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest.

The method of the first aspect may advantageously be used to produce recombinant adenoviruses with production times reduced from about 33-44 weeks to as low as 28 days for use as a vaccine.

Using current methods, viral stocks are produced by amplifying large numbers of transfections, which may contain many very low levels of small species that are difficult to detect. Therefore, 3 rounds of cloning are required to ensure that clonal stocks are generated using this method. The method of the invention starts with a characterised viral genome, so that only recombinant antigen sequences may be erroneous after recombination and transfection. The transfection is performed such that only one recombinant viral genome transfects each container, and therefore there is no mixture comprising many races. In rare cases, two viral genomes can transfect the same cell, but if they are not identical in the recombinant antigen coding sequence, they can be easily distinguished by sequencing the coding DNA sequence, since each virus will account for around 50% of the mixture and therefore we are no longer looking for a microspecies. Any virus samples that appear to contain a mixture of correct and incorrect sequences will be discarded, and only those that are correct will be selected as a vaccine.

The synthetic DNA encoding the heterologous gene of interest may contain small species that are not completely correct. The method of the present invention solves this problem by generating viral clones immediately after transfection and thus sequencing the gene coding sequence in each clone and selecting only the correct clone. This is advantageous over amplifying large amounts of virus stock that may represent a mixture of recombinant viruses, which are then cloned later.

In addition to providing instant cloning and rapid amplification of recombinant adenoviruses, the method of the invention provides an important improvement in relocating the mass Quality Control (QC) assay necessary for using recombinant viruses as vaccines before the start of production of any specific recombinant adenovirus. Such QC assays can be performed on a large number of starting materials and can save a lot of time when the method is used to generate recombinant adenoviruses for use as vaccines.

Another advantage of the novel methods is that they can be effectively used to generate simian adenovirus vectors as set forth herein. Most of the previous work on rapid generation of adenoviral vectors has used only human adenoviruses of one or several serotypes, especially human adenovirus serotype 5 (Ad-5). Simian adenoviruses are now preferred as immune vectors over human adenoviruses because: i) much less negatively affected by pre-existing anti-vector immunity caused by natural exposure to human adenovirus; ii) they were found to be safe and immunogenic in thousands of subjects compared to the common human adenovirus vector (Ad-5) (Ewer at al. supra), Ad-5 was associated with a major safety signal and was associated with enhanced HIV infection in the major "STEP" trial of merck HIV vaccine (Cohen (2007) Science 318: 28-29).

In a second aspect, the present invention provides a composition comprising an adenovirus genome wherein the E1 gene is replaced by an expression cassette comprising a DNA sequence encoding a fluorescent marker protein flanked by a first pair of unique restriction sites that are not present elsewhere in the adenovirus genome for use in the method of the first aspect.

The composition of the second aspect may advantageously be used in the method of the first aspect to allow for the clear identification of recombinant adenoviruses comprising nucleotide sequences encoding heterologous genes of interest. All the obvious advantages of using the method of the first aspect can also be found in the composition of the second aspect.

Drawings

The invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 shows a timing diagram for rapid production of recombinant adenovirus for use as a vaccine using existing methods. QC-quality control; amp-amplification; GMP-production quality management standard; MVSS-master virus seed stock.

FIG. 2 shows a timing diagram of the construction of recombinant adenoviruses for use in the methods of the invention for producing vaccines.

FIG. 3 shows a schematic representation of a parental viral composition for use in the methods of the invention. In this case, the parent virus is the ChAdOx1-Bi-GMP genome, which contains 3 unique restriction sites (PsiI, AsiSI and RsrII) for insertion of the antigen or expression cassette. LPTOS-Long tetracycline regulated CMV promoter.

FIG. 4 shows the analysis of TPC-Ad gDNA disrupted by 3M guanidine hydrochloride. 200 μ l of adenovirus particles (1.4e12VP/ml) were disrupted with 3M guanidine hydrochloride on ice for 45 min. Mu.l of the sample were incubated at 65 ℃ for 40 minutes with (+) or without (-) 2. mu.g proteinase K and then separated by 0.7% agarose. M1 kbp Generuler ladder (Thermofoisher).

FIG. 5 shows TPC-Ad gDNA isolation, purification, desalting and filtration after 3M guanidine hydrochloride disruption, after centrifugation at 68000rpm for 18 hours in a 2.8M CsCl gradient. The isolated DNA was desalted into 10mM Tris pH7.8 and filtered through a 0.2. mu.M syringe filter. An aliquot of the DNA was isolated by 0.7% agarose. M ═ 1kbp Generuler (thermolasher).

FIG. 6 shows the protein analysis of TPC-Ad gDNA isolated after centrifugation in a 2.8M CsCl gradient. Samples containing 50ng of TPC-Ad gDNA were separated by SDS reduction in 4-12% Bis-Tris NuPAGE midgel and stained with silver stain.

Fig. 7A and 7B show the binding positions of qPCR primers and probes in the ChAdOx1, ChAdOx2, and ChAd63 adenovirus genomes.

FIG. 8 shows an in vitro recombination reaction scheme of the claimed method to produce recombinant ChAdOx1 using ChAdOx1-Bi-GFP as parental adenovirus genomic DNA.

Figure 9 shows the percentage of cells expressing mCherry and GFP at 30 hours after transfection with a recombination reaction containing various amounts of TPC-Ad gDNA and mCherry ORF PCR products. 60, 40 and 20ng PsiI digested TPC-Ad gDNA was recombined with 40, 20 and 10ng mCherry ORF PCR products using NEBuilder. The recombination reaction was incubated at 50 ℃ for 40 minutes and then at 20 ℃ for 2 minutes prior to transfection. Using lipofectamine 2000 at 1: 5 ratio T-Rex-293 cells seeded in 96wp were transfected with the recombinant reaction. Tetracycline-containing medium was added 5 hours after transfection. The number of cells expressing GFP and mCherry was determined by FACS analysis 30 hours after infection.

FIG. 10 shows an overview of a method for rapidly producing recombinant adenovirus for use as a vaccine.

Detailed Description

In a first aspect, the present invention provides a method for producing a recombinant adenovirus for use as a vaccine, the recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest, the method comprising the steps of: (i) inserting a heterologous gene of interest into the adenovirus genome by recombining terminal protein complex adenovirus genomic DNA (TPC-Ad gDNA) with a synthetic DNA comprising a nucleotide sequence encoding the gene of interest and having at least 15bp of bases at its 5 'end and at least 15bp of bases at its 3' end, which is homologous to the insertion site sequence of the adenovirus genomic DNA in an in vitro recombination reaction; (ii) (ii) transfecting cells grown in individual containers with a dilution of the in vitro recombination reaction mixture from (i) such that a plurality of such individual containers comprise individual cells infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest; (iii) identifying those containers in which the individual cells have been infected with a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest.

The prior art provides a number of methods for producing recombinant adenoviruses, for example Hillgenberg et al (2006), Choi et al (2012), and micik et al (2018), all of which provide elegant approaches. However, none of the methods provided address the need for a long cloning process to isolate recombinant cloned adenoviruses for use as vaccines. The method of the present invention advantageously provides a method for eliminating the long cloning process when generating recombinant adenoviruses. This in turn eliminates a large delay in the production of recombinant adenoviral vectors, which makes such vectors more readily suitable for use as vaccines, particularly as personalized vaccines for rapid response vaccines against cancer treatments or fulminant pathogens.

In a preferred embodiment of the invention, the TPC-Ad gDNA comprises serotype-matched terminal proteins and an adenovirus genome. The use of serotype-matched adenovirus genomes and terminal proteins can rescue the virus with high efficiency after recombination and transfection of cells.

In particular embodiments of the invention, the gene of interest encodes a single epitope, an epitope string, an antigen segment, or a complete antigenic protein. Providing various genes of interest allows for the development of improved vaccines to protect or treat patients at risk of developing or suffering from a variety of diseases, including cancer or fulminant disease.

In alternative embodiments, the polynucleotide is a synthetic DNA molecule, a purified DNA restriction fragment, or a Polymerase Chain Reaction (PCR) product. This method allows for the flexible selection of the source of DNA for the production of recombinant adenovirus, which will result in a more rapid completion of the method, which is critical for the production of recombinant adenovirus for use as a vaccine for the prevention or treatment of a number of diseases, including cancer or fulminant disease.

In further alternative embodiments, the polynucleotide has between 5 and 50bp of bases at its 5 'end and between 5 and 50bp of bases at its 3' end which are homologous to the insertion site sequence of the adenoviral genomic DNA, or the polynucleotide has between 10 and 20bp of bases at its 5 'end and between 10 and 20bp of bases at its 3' end which are homologous to the insertion site sequence of the adenoviral genomic DNA, or the polynucleotide has 15bp of bases at its 5 'end and 15bp of bases at its 3' end which are homologous to the insertion site sequence of the adenoviral genomic DNA. Providing a polynucleotide with suitable homologous ends allows for efficient recombination with the adenoviral genomic DNA, thereby allowing for reliable production of recombinant adenovirus in an in vitro recombination reaction using the method.

In a preferred embodiment, the insertion site sequence of the adenoviral genomic DNA is located within the E1 locus. Deletion of the E1 gene allows insertion of heterologous expression cassettes and reliable and high level expression of the antigen of interest in recombinant virus infected cells. This provides advantageous properties for recombinant adenoviruses for use as vaccines.

In certain embodiments, the TPC-Ad gDNA is digested at a unique restriction site within the E1 locus of the adenoviral genomic DNA flanked on its 5 'end by a tetracycline regulated CMV promoter which drives expression of the gene of interest, and which is flanked on its 3' end by a bovine growth hormone polyadenylation sequence. Digestion of adenoviral genomic DNA at unique restriction sites in the E1 locus provides suitable end sequences for recombination with DNA sequences encoding the antigen of interest, while also removing the intact parental adenoviral DNA from any recombination reactions. Advantageously, this allows for more efficient recombination with DNA sequences encoding the antigen of interest, and also reduces the number of parental adenoviruses regenerated using this method.

In a specific example, the in vitro recombination reaction comprises 40ng of digested TPC-Ad gDNA and 44fmol of the 3 'and 5' ends of the synthetic DNA encoding the gene of interest. Providing such a number of reactants allows for optimized recombination and the production of recombinant adenoviruses comprising nucleotide sequences encoding heterologous genes of interest. Advantageously, this allows for transfection of suitable cells with an amount of recombinant adenoviral genomic DNA that increases the production of individual clones using the methods of the invention.

In a further embodiment, the cells to be transfected the day before transfection are at 3.75X 10⁵The cells are seeded in a single container at a density of cells/ml. Seeding cells at this density can increase the efficiency of recombinant adenovirus rescue in the cells.

In a further embodiment, cells are transfected while growing at about 80% confluence in a single vessel. This increases expression of adenovirus early genes and increases the efficiency of recombinant adenovirus rescue in cells.

In a further embodiment, the cell stably expresses the tetracycline repressor. Using such cells, e.g., T-Rex-293 cells, expression of the gene of interest can be inhibited during viral rescue following transfection. After transfection, the cells are fragile, inhibition of heterologous gene expression minimizes cell death, and effectively rescues the virus at this step of the method.

In particular embodiments, the transfected cells stably express the tetracycline repressor. Expression of the tetracycline repressor in cells used to rescue recombinant viruses prevents expression of genes of interest that may be toxic to the cells, thus increasing viral rescue.

In another specific example, the in vitro recombination reaction mixture is diluted and aliquoted in transfection medium in order to transfect cells grown in 60 separate containers. Advantageously, each recombinant reaction is divided into 60 aliquots and transfected, which delivers a single recombinant adenovirus to a subset of but not all 60 separate containers. This allows the user to identify a number of wells containing a single recombinant adenovirus, while including negative control wells that do not contain recombinant adenovirus.

In a preferred embodiment, transfected cells are frozen and thawed to release cell-associated virus, and the presence of recombinant adenovirus is identified by quantitative pcr (qpcr) using cell lysate from each well of transfected cells, a set of primers, and a probe designed to bind to the left end of the genome downstream of the adenovirus Inverted Terminal Repeat (ITR) and the genome upstream of the insertion site of the gene of interest in the non-coding region. This simplified and accelerated sample extraction and screening process allows for easy and rapid identification of the recombinant adenovirus of interest and exclusion of the parental adenovirus present in the sample.

In a further preferred embodiment, the adenovirus genome is derived from a human adenovirus or a simian adenovirus, preferably the human adenovirus is not a human adenovirus serotype 5. In the most preferred embodiments, the simian adenovirus is a chimpanzee adenovirus, such as ChAdOx1(Antrobus et al (2014) Mol ther.22(3): 668) and 674), ChAdOx2(Morris et al (2016) Future virol.11(9):649 and 659), ChAd3 or Chad 63. The use of recombinant adenoviruses produced by this method is permitted for use as vaccines in human subjects by using human or simian adenoviruses. The use of simian adenoviruses, particularly the use of either ChAdOx1 or ChAdOx2, provides improved vaccines that have a low probability of developing pre-existing anti-adenoviral immunity when administered to a human subject.

In a particular embodiment, each container is an individual well in a multi-well plate. The use of such a small volume container allows for rapid, economical and efficient transfection of cells and screening of the resulting recombinant adenovirus. The use of a multi-walled plate also allows for automation of the method and all related processes.

In a preferred embodiment, the TPC-Ad gDNA is provided from a stock library of TPC-Ad gDNA that has been subjected to and passed through the necessary Quality Control (QC) assays to allow for manufacturing quality management practice (GMP) bio-manufacturing. The use of TPC-Ad gDNA from Quality Control (QC) stock libraries can improve the rapidity of the process for producing recombinant adenoviruses for use as vaccines. QC testing is performed prior to the start of the process, allowing the viral components to be tested in advance and additionally reducing the testing and assay burden during the production and manufacture of adenoviruses.

In certain embodiments, the recombinant adenovirus produced by the method of the first aspect of the invention may be used as a vaccine for the prevention and/or treatment of a disease in a human or animal. In particular, the recombinant adenoviruses produced by this method are very useful in the generation of personalized vaccines for the treatment of cancer. The present invention overcomes the major impediments to providing such treatment using viral vectors: i.e., the process of generating and developing viral vector vaccines is slow. The rapid generation of recombinant adenovirus by the novel instant methods disclosed herein can provide sufficient time for clinical evaluation of patients, identification of the patient's own cancer-specific antigens, and generation of an appropriate recombinant adenovirus vaccine to treat the individual patient. This was not possible until the claimed method of the first aspect of the invention was developed.

In a second aspect, the present invention provides a composition comprising an adenovirus genome wherein the E1 gene is replaced by an expression cassette comprising a DNA sequence encoding a fluorescent marker protein flanked by a first pair of unique restriction sites that are not present elsewhere in the adenovirus genome for use in the method of the first aspect.

As mentioned above, the prior art provides a number of methods for producing recombinant adenoviruses. However, each of these methods starts by using an unmodified adenovirus genome as starting material, and therefore, each method must perform complicated steps to generate digested adenovirus genomic DNA suitable for use in vitro recombination reactions. The compositions provided by this aspect of the invention overcome this obstacle and allow a single step restriction digestion reaction to prepare adenoviral DNA for recombination with a suitable heterologous nucleic acid molecule. In addition to simplifying the preparation of adenoviral genomic DNA, the use of such compositions allows for large scale preparation of digested genomic DNA from adenoviral stock which has been tested to confirm sterility, mycoplasma deficiency, identity and genetic stability, and which can be stored in advance to simplify the production of recombinant adenoviruses which fully meet GMP requirements and ready for clinical use as required.

In a particular embodiment, the adenovirus genome is derived from a human adenovirus or a simian adenovirus, preferably the human adenovirus is not a human adenovirus serotype 5. In a more preferred embodiment, the adenoviral genome is derived from a simian adenovirus, most preferably the simian adenovirus is a chimpanzee adenovirus, such as ChAdOx1(Antrobus et al. supra), ChAdOx2(Morris et al. supra), ChAd3 or Chad 63. Use of a human or simian adenovirus, allowing the recombinant adenovirus produced by the method to be used as a vaccine in a human subject. The use of simian adenoviruses, particularly the use of either ChAdOx1 or ChAdOx2, provides improved vaccines that have a lower probability of developing pre-existing anti-adenoviral immunity when administered to a human subject.

In a preferred embodiment, the fluorescent marker protein is Green Fluorescent Protein (GFP). The presence of a fluorescent marker protein allows rapid detection of cells infected with non-recombinant, intact adenoviruses that express the gene of interest in this respect without recombination, GFP being a particularly convenient marker protein that can be detected directly by fluorescence microscopy or easily indirectly, for example using anti-GFP antibodies.

In preferred embodiments, the first unique pair of restriction sites is selected from the group consisting of PsiI, AsiSi, and RsrII sites.

In certain embodiments, the expression cassette further comprises a long tetracycline-regulated CMV promoter 5 'of the DNA sequence encoding the fluorescent marker protein and a bovine growth hormone polyadenylation sequence 3' of the DNA sequence encoding the fluorescent marker protein, wherein the first pair of restriction sites are located between the long tetracycline-regulated CMV promoter and the DNA sequence encoding the fluorescent marker protein and between the DNA sequence encoding the fluorescent marker protein and the bovine growth hormone polyadenylation sequence. Advantageously, in the method of the first aspect of the invention, the inclusion of a GFP coding sequence in the parental adenovirus genomic DNA can be used as an effective negative control to identify any cell in which the parental adenovirus is being regenerated. When seeking recombinant adenoviruses comprising nucleotide sequences encoding heterologous genes of interest for use as vaccines, simple screening procedures can eliminate those viruses expressing GFP. In addition, the presence of GFP can be used as a useful marker when generating a parental adenovirus genomic DNA library for use in the method of the first aspect.

In preferred embodiments, the expression cassette further comprises a second pair of unique restriction sites, different from the first pair of unique restriction sites, located 5 'of the long tetracycline regulated CMV promoter and 3' of the bovine growth hormone polyadenylation sequence. Advantageously, the addition of a second pair of unique restriction sites allows the removal of the entire GFP expression cassette and this allows the generation of a recombinant adenovirus comprising a nucleotide sequence encoding a heterologous gene of interest, which is expressed using a different promoter system. In this case, the desired promoter and polyadenylation sequence may be designed into a synthetic DNA comprising the nucleotide sequence encoding the gene of interest.

In a further preferred embodiment, the second pair of unique restriction sites is selected from the group consisting of the PsiI, AsiSI or RsrII sites.

In additional embodiments, the adenovirus genome is further engineered to comprise additional unique restriction sites at the S15/E4 locus. This allows recombination of a second synthetic DNA comprising a nucleotide sequence encoding the gene of interest into the adenoviral genomic DNA.

In still further embodiments, the additional unique restriction site is selected from the group consisting of a PsiI, AsiSi, or RsrII site.

In particular embodiments, the adenoviral genome is complexed with heterologous or non-serotype matched terminal proteins, but in preferred embodiments, the adenoviral genome is complexed with autologous or serotype matched terminal proteins.

In a further embodiment, the adenovirus genome lacks Gateway recombination sequences.

In a preferred embodiment, the above composition has been subjected to and passed through the necessary Quality Control (QC) assays to allow for manufacturing quality control practices (GMP) bio-manufacturing. The use of starting materials from a stock library for Quality Control (QC) assays may improve the rapidity of the method for producing recombinant adenoviruses for use as vaccines. QC testing is performed prior to the start of the process, allowing the viral components to be tested in advance and additionally reducing the testing and assay burden during the production and manufacture of adenoviruses.

In a final aspect, the invention provides a recombinant adenoviral vector immunogen comprising any of the compositions of the second aspect of the invention and expressing a pathogen or tumor epitope or antigen against an immune response generated in a mammal.

Throughout the specification and the appended claims, the word "comprise" and variations such as "comprises", "comprising", "includes" and "including" will be included. That is, these words are intended to convey that other, non-explicitly recited elements or integers may be included, where the context permits.

Examples

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

A55 kDa Terminal Protein (TP) is covalently linked to the 5' end of each strand of the adenoviral genomic DNA to generate a 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 extracellular exonuclease, acts as a primer to initiate DNA replication, and forms heterodimers with DNA polymerase. TP achieves binding of other replication factors by increasing the template activity by more than 20-fold to enhance replication through subtle changes in the origin of replication compared to the protein-free template. TPC-Ad gDNA was isolated from the disrupted purified virus particles using guanidine hydrochloride and purified by cesium chloride density gradient ultracentrifugation.

Will contain 1X 10¹¹And 1X 10¹²The purified virus solution of virus particles (500. mu.l-1 ml) was aliquoted into 1.5ml or 2ml tubes and an equal volume of filtered sterile 6M HCl in nuclease-free water was addedGuanidine (gnhcl), final gnhcl concentration 3M. After gentle mixing, the diluted virus solution was incubated on ice for 45-60 minutes.

A2.8M cesium chloride (CsCl) solution was prepared by adding 9.4281g of CsCl to 20ml of filter sterilized 3M guanidine hydrochloride (GndHCl), and the solution was made up with nuclease-free water. 2ml of 2.8M CsCl solution was added to an appropriately sized ultracentrifuge tube in a MSCII hood, and then the virus/GndHCl solution was gently spread on top of the 2.8M CsCl. The virus sample preparation was then centrifuged through the CsCl solution for 18 hours at 20 ℃ in a Beckman TLA100.3 rotor using a bench-top Optima TLX ultracentrifuge at 68,000 rpm.

After centrifugation, TPC-Ad gDNA was aliquoted into 100. mu.l aliquots which were then transferred to microcentrifuge tubes. As the amount of viral material present in the starting material increased, a pellet was seen at the end of the CsCl centrifugation step, which was then resuspended in 100. mu.l 10mM Tris HCl, pH7.8, prepared in nuclease-free water. The presence of purified DNA in aliquots removed from CsCl centrifuge tubes was visually confirmed by the following procedure: mu.l aliquots were placed on the sealing film, 1. mu.l of working stock solution (1: 10,000) SYBR safe was added, and visually observed with an orange filter under blue light.

TPC-Ad gDNA purified from the CsCl centrifugation step was then desalted using a Zeba column (equilibrated with 10mM Tris HCl pH 7.8) prepared in nuclease free water. The DNA concentration was determined spectrophotometrically and DNA purity was assessed by gel electrophoresis (fig. 4 and 5). Protein levels in TPD-gDNA preparations were qualitatively determined by SDS-PAGE reducing 4-12% gradient gels (FIG. 6). TPC-Ad gDNA was then stored at-80 ℃ until required.

Example 2 preparation of TPC-Ad gDNA for recombination by unique restriction enzyme digestion

The parental adenovirus genome, such as ChAdOx1-Bi-GFP, comprises a GFP coding sequence at E1, as shown in FIG. 3, flanked by a long tetracycline regulated CMV promoter (LPTOS) and a 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, resulting in 3 fragments: the left arm of the adenovirus genome, the GFP ORF and the right arm of the adenovirus genome. The parental virus may 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.

120ng of TPC-Ad gDNA was incubated overnight in an incubator at 37 ℃ with 10U of PsiI in the recommended reaction buffer diluted with nuclease-free water to a final reaction volume of 30. mu.l. Restriction enzymes were inactivated by incubation at 65 ℃ for 20 minutes, digestion was confirmed once no virus production was confirmed by gel electrophoresis or transfection into cells, and the digested TPC-Ad gDNA was used directly for recombination reactions.

Example 3-rapid generation of adenovirus: recombination and transfection

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

NEBuilder (NEB) and In-fusion (Takara) are commercially available systems that can seamlessly assemble multiple DNA fragments regardless of fragment length or end compatibility. These products can be used to insert antigen/expression cassettes into the TPC-Ad gDNA suitably prepared by example 2. The recombination reaction mixture includes an exonuclease and a polymerase, in the case of NEBuilder, a DNA ligase which co-acts to produce double stranded DNA molecules. Exonuclease produces single-stranded 3' overhangs that facilitate annealing of fragments whose ends share complementarity (overlapping regions), and polymerase fills the gap of each annealed fragment. In the NEBuilder reaction, DNA ligase seals gaps in the assembled DNA, forming a completely sealed DNA molecule, rather than relying on host cell DNA repair mechanisms to fill in the gapped DNA as in the Infusion reaction (Infusion reaction).

40ng of PsiI digested TPC-Ad gDNA (10. mu.l of restriction reaction from example 2) was mixed in a thin wall PCR tube with 44fmol of the 5 '/3' end of the desired antigen sequence synthesized with a minimum of 15bp of sequence complementary to the 5 'and 3' ends of the TPC-Ad gDNA insertion site. The contents of the tube were collected at the bottom of the tube by brief spinning in a microcentrifuge. The final volume of the recombination reaction was then brought to 30. mu.l by adding the recommended volumes of NEBuilder reaction mixture or In-fusion reaction mixture and nuclease-free water. The reaction mixture was incubated at 50 ℃ for 40 minutes and then at 20 ℃ for 2 minutes. The recombination reaction can be immediately transfected into complementing cells and the recombinant adenovirus can be rescued.

To rescue adenovirus, dividing cells were required to express the E1 protein and support viral replication, so the goal was to achieve a level of approximately 80% of the day's fusibility on transfection. Thus, 24 hours prior to transfection, T-Rex-293 cells stably expressing the tetracycline repressor were plated at 3.75X 10⁴The density of individual cells/well was seeded in 96-well plates in DMEM containing 10% Fetal Calf Serum (FCS) and blasticidin (5. mu.g/ml).

Preheated Optimem was added to the recombination reaction to bring the final volume in the reaction tube to 100. mu.l. In the recombination reaction, 0.5. mu.l Lipofectamine 2000 per 100ng DNA was added to 100ml Optimem in another tube. Both tubes were incubated at room temperature for 5 minutes. The diluted recombination reaction was then added to the diluted Lipofectamine 2000. The tubes were gently mixed and then incubated at room temperature for an additional 20 minutes before the mixture was diluted with Optimem to a final volume of 3 ml.

The medium was separated from T-Rex-293 cells grown in 96-well plates and 50. mu.l of diluted lipofection was added directly to each of the 60 wells. The cells were then incubated at 37 ℃ and 8% CO₂The following incubations were carried out for 4 to 6 hours. The transfection medium in each well was then replaced with 100ml DMEM containing 10% Fetal Calf Serum (FCS) and blasticidin (5. mu.g/ml) and the cells were incubated at 37 ℃ with 8% CO₂The following incubation was performed.

After 5 days, the plates were subjected to 3 freeze/thaw cycles to disrupt the cells and release the relevant virus. 10ml of cell lysate was taken from each individual well and DNA was isolated therefrom by Quantitative Polymerase Chain Reaction (QPCR) using commercially available DNARELEASY reagent analysis. The remaining cell lysate from each well was addedTo the corresponding well of a 96-well plate inoculated 24 hours ago, the well was inoculated at 2.1X 10⁴Concentration of cells/well T-Rex-293 cells were seeded in DMEM containing 10% Fetal Calf Serum (FCS). When the complete cytopathic effect was evident in the wells (between 4 and 6 days post infection), recombinant adenovirus was harvested from each well.

Example 4 quantification of adenovirus genome copy number from cell lysates or purified viruses by QPCR

The amount of ChAdOx1, ChAdOx2 or ChAd63 viral genome in HEK293 or T-Rex-293 cell lysates was measured by QPCR. The number of viral genomes (possibly associated with 1: 1 viral particles) was determined by quantitative pcr (qpcr) from cell lysates treated with dnareliasy. A set of primers and probes have been designed that bind to the left end of the genome downstream of the Inverted Terminal Repeat (ITR) and upstream of the antigen insertion region in the non-coding region (see FIGS. 7A and 7B). These primer and probe sequences were:

primer and method for producing the same

ChAd fwd 5’GTGGGAAAAGTGACGTCAAACGAG3’(SEQ ID NO:1)

ChAd rev 5’TGCATCCGCCTAGAAACACCTCA3’(SEQ ID NO:2)

Probe needle

ChAd Universal Probe 5 'GAGAGCGCGGGAAAATTGAGTATT 3' (SEQ ID NO:3)

Only one mismatch in the reverse primer of ChAdOx 2; the sequence of the relevant region in AdCh63 is identical to that in ChAdOx2, and therefore this approach may also be successful for AdCh 63. The relevant sequences were not present in the AdHu5 vector.

Cells and media were harvested from flasks that showed complete cytopathic effect (CPE). For small amounts of cells and medium, direct harvest is possible, but for large amounts of cells, they can be collected by centrifugation at 1500g for 5 minutes and resuspended in 1/10 medium volume of adenovirus lysis buffer (50mM Tris,2mM MgCl)₂pH 9.0). The cells to be harvested were then frozen and thawed three times. Mu.l of lysate was added to 15. mu.l of DNARELEASY reagent and the samples were processed in a thermal cycler with the following cycles: 65 ℃ for 15 minutes, 96 DEG CFor 2 minutes, 65 ℃ for 4 minutes, 96 ℃ for 1 minute, 65 ℃ for 1 minute, 96 ℃ for 30 seconds, held at 20 ℃. The samples were diluted to a total volume of 1ml, using 5. mu.l per QPCR reaction.

The QPCR reaction was carried out by: initial hot start activation was performed at 95 ℃ for 10 minutes, followed by denaturation at 95 ℃ for 15 seconds, 45 cycles, followed by denaturation and annealing at 60 ℃ for 1 minute.

A standard curve was established using the pTOPO-ChAdOx1 LF1 plasmid, which is 4,118 base pairs in length and gives a defined number of genomic copies per ng of DNA, as shown in Table 1.

Example 5 Generation of recombinant ChAdOx1 expressing mCherry

The mCherry gene was used as a model antigen for insertion of ChAdOx 1. The mCheerry gene was amplified using primers containing 15bp homology to the PsiI insertion site of the TPC-AdgDNA. TPC-gDNA was digested with PsiI and the enzyme was then heat inactivated prior to recombination. A series of TPC-gDNA and mcerry ORF concentrations were tested for recombination efficiency using NEBuilder and In-fusion. The reaction was incubated at 50 ℃ for 40 minutes, then at 20 ℃ for 2 minutes, then immediately using lipofectamine 2000 at a 1: 5 into T-Rex-293 cells seeded in 96-well plates. 30h after transfection, the number of GFP (from undigested TPC-Ad gDNA) and mCherry cells (from the recombination reaction) was determined by FACS (FIG. 9).

Table 1: adenovirus genome copy number criteria

Amount of DNA std	Number of copies
		5ng	1×109
0.5ng	1×108
		50pg	1×107
5pg	1×106
		0.5pg	1×105
0.05pg	1×104