Bacterial artificial chromosomes

Abstract

The present invention relates to the use of a bacterial artificial chromosome (BAC) for the preparation of a vaccine wherein the BAC comprises:-an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and-a viral expression cassette comprising cDNA of an attenuated RNA viral genome, and comprising a cis-regulatory element for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious viral RNA.

Description

Field of the Invention

[0001] The present invention relates to a plasmid vector system suitable for operating, maintaining and propagating infectious cDNA of an RNA virus genome, and to the use of said vector system. Background Art

[0002] Previously, copy DNAs (cDNAs) of several flaviviruses and other RNA viruses have been cloned into different low-copy bacterial vectors to overcome their inherent toxicity (attributed to the large size and cryptic expression of viral sequences) (Bredenbeek et al. (2003) J. Gen. Virol. 84, 1261-1268; Durbin, et al. (2006) Hum Vaccin. 2, 255-260; Fan and Bird (2008) J. Virol. Methods. 149, 309-315; Li et al. (2011) PLoS One 6, e18197; Pu et al. (2011) J. Virol. 85, 2927-2941; Rice et al. (1989) New Biol. 1, 285-296) Almazán et al. (2008) Methods Mol Biol. 454, 275-91). The cloned cDNAs have been used as templates to generate infectious recombinant viruses, either by in vitro synthesis or transfection of the RNA genome (Bredenbeek et al., 2003, cited above), or by incorporating the viral cDNA into an expression cassette that contains a promoter, such as the CMV-IE (cytomegalovirus immediate early) promoter that allows transcription of viral RNA from transfected plasmid DNA (Enjuanes et al. (2001) J. Biotechnol. 88, 183-204; Hall et al. (2003) Proc. Natl. Acad. Sci. USA. 100, 10460-10464). The viral expression cassettes that direct the expression of attenuated hand, foot and mouth disease (Ward et al. (1997) J. Virol. 71, 7442-7447) and Kunjin virus (Hall et al. (2003) Proc Natl Acad Sci USA. 100, 10460-10464) have been used as experimental DNA vaccines. Although low-copy number vector systems containing viral expression cassettes can be maintained in bacterial host cells in a stable manner, they have important drawbacks: they only allow purification of infectious viral cDNA in amounts that are just sufficient for small-scale experimental applications. Thus, their use as a conventional source of infectious viral cDNA (such as in the production of live cDNA vaccines) is not possible.

[0003] The production of viral DNA vaccines requires the large-scale amplification of cloning vectors to obtain sufficient DNA, but these amplification methods are severely restricted. To avoid mutations, vectors containing viral DNA are propagated under conditions that prevent mutagenic events (recombination, mutation, increased mismatch repair, etc.). The stability of bacterial artificial chromosomes (BACs) is known, and they can contain inserts of up to 500 kb or more.

[0004] However, the size of such vectors with foreign DNA is a heavy burden on bacteria, and their replication requires a large amount of metabolic effort. In addition, nucleotide depletion can lead to an increase in mutations. Eventually, there will be unwanted expression of foreign DNA (so-called cryptic expression), which can result in toxic recombinant proteins. The production of toxic proteins from cryptic transcripts is inherent to flavivirus DNA and can only be solved by reducing the plasmid copy number. In fact, the higher the copy number of the vector, the higher the concentration of toxic proteins. As a result, the bacterial host may counterselect mutations in which these proteins are not expressed.

[0005] Pu et al. (2011) J. Virol. 85, 2927–2941, described in detail various attempts to solve the inherent toxicity of flavivirus cDNA in bacteria. These include in vivo ligation of plasmids containing parts of the viral genome, specific hosts, mutants that avoid cryptic expression, and low-copy-number plasmids.

[0006] Therefore, the application of BACs that occur as single copies in bacteria provides a solution to these problems.

[0007] The low copy number is not a drawback for these applications, where the BAC DNA is subsequently subcloned or amplified to increase the concentration, and where some mutations introduced by these techniques are not crucial for the envisioned experiments. However, the aforementioned amplification methods cannot be applied to the manufacture of DNA vaccines, which makes BACs not the preferred vector for large-scale plasmid preparation of DNA vaccines. Ultra-large-scale culture is required to obtain large amounts of BAC.

[0008] The application of inducible BAC vectors is known from Wild et al. (2002) Genome Res. 12, 1434-1444, whereby the copy number of BACs is increased from 1 copy per cell to up to 100 copies per cell or even more. Although this system provides a method for increasing the yield of BAC DNA, there are legitimate concerns that a strong increase in the activity of the replication system after induction will increase the mutation frequency. Therefore, the manufacture of DNA vaccines requires such a system that can obtain a high copy number of vectors, but where the vectors replicate without introducing intolerable mutations. Summary of the Invention

[0009] The present invention solves the problem of amplification of viral cDNA in the art with respect to vaccine preparation in a vector system by providing a vector that can be stably maintained in a host cell at a low copy number, but can be amplified in large amounts by modulating the culture conditions of its host without unwanted mutagenic events. Another object of the present invention is to provide such a vector that is capable of shuttling between yeast and bacterial hosts, thereby providing a very versatile system that can be used to manipulate vectors in yeast and bacterial genetic systems in a controlled manner.

[0010] The present invention unexpectedly demonstrates that an inducible increase in the copy number of a BAC vector provides DNA with a surprisingly low mutation rate. Even more surprisingly, the few mutations that occur are mostly frameshift mutations or stop codons, resulting in truncated forms after expression. Point mutations (which have no effect or result in modified amino acids) are underrepresented.

[0011] This unexpected effect results in an advantageous situation where high amounts of vector can be obtained and a limited number of errors result in non-functional viral genomes rather than mutant viral genomes with increased virulence compared to the original cloned construct.

[0012] The present invention provides a bacterial artificial chromosome that contains an inducible bacterial ori sequence that allows induction of the amplification of the bacterial artificial chromosome to high copy numbers, for example, by modulating the culture conditions of the bacterial host. The bacterial artificial chromosome used herein also contains a viral expression cassette that contains the cDNA of an RNA virus genome flanked by cis-regulatory elements that promote transcription of the viral cDNA after introduction of the bacterial artificial chromosome into mammalian cells and allow processing of the transcribed RNA into infectious viral RNA. The viral cDNA contained in the viral expression cassette can correspond to those of a wild-type RNA virus genome or be a chimeric viral cDNA construct in which heterologous DNA sequences have been inserted and / or native viral sequences have been deleted, truncated or mutated. Generally, the heterologous DNA sequences encode one or more peptides / proteins that are heterologously expressed by the recombinant virus after the bacterial artificial chromosome of the present invention is introduced into mammalian cells, and the bacterial artificial chromosome of the present invention contains a viral expression cassette containing the chimeric viral cDNA. The bacterial artificial chromosome may also contain a yeast autonomous replication sequence for shuttling into yeast or maintaining the bacterial artificial chromosome in yeast. The possibility of shuttling into yeast cells or maintaining the bacterial artificial chromosome in yeast cells provides the advantage of genetic manipulation in yeast and bacterial genetic systems in a controlled manner. Similarly, the present invention provides a single vector system that is suitable for the manipulation, maintenance and propagation of infectious cDNA of an RNA virus genome.

[0013] In the absence of the stimulus of the inducible ori, the bacterial artificial chromosome of the present invention can be used to achieve stable cloning of infectious virus cDNA in a bacterial host, and when the stimulus is present, the cDNA can be easily amplified, subsequently isolated and used. The bacterial artificial chromosome of the present invention is particularly useful in the development, stability maintenance and production of virus cDNA to be used as a live vaccine against RNA virus pathogens. Alternatively, the bacterial artificial chromosome is used to maintain and propagate natural or recombinant viruses from cDNA (e.g., for research purposes).

[0014] In the present invention, a BAC with an inducible bacterial ori is used to prepare a vaccine of a viral expression cassette, and the viral expression cassette includes cis-regulatory elements for transcribing virus cDNA in mammalian cells and for processing the transcribed RNA into infectious virus RNA, and the cDNA of an RNA virus.

[0015] Surprisingly, the production of multiple copies of the BAC containing the viral DNA does not result in the defects known to occur in high-copy number systems.

[0016] Generally, toxic proteins are produced in a bacterial system, which is attributed to the cryptic expression of viral sequences. In fact, the production of flavivirus infectious clones has traditionally been hampered by the toxicity of its full-length cDNA in bacteria. A variety of methods have been employed to overcome this problem, including the use of very low-copy number plasmids and bacterial artificial chromosomes (discussed in Edmonds (2013) J. Virol. 87, 2367-2372). This is a phenomenon involving insertions that are cloned into the BAC, thus hindering bacterial growth and metabolism. Bacteria with mutations in which cryptic expression does not occur have a growth advantage and will overgrow the original population. In the prior art, this is reflected by the size of bacterial colonies. Non-mutant constructs produce toxic proteins and generally yield small colonies. Mutant constructs produce less or no toxic proteins, which results in the appearance of larger colonies.

[0017] Based on this prior art knowledge, it was thus expected that induction of plasmid replication would lead to an increase in toxic transcripts and an accompanying increase in mutants in which cryptic expression does not occur.

[0018] Surprisingly, the inducible replication system seems to be insensitive to the toxicity of cryptic proteins. In fact, the bacterial colonies are somewhat larger compared to the high-copy number systems of the prior art, indicating that the bacterial host is less sensitive to the final toxic proteins. More importantly, very large colonies represent the absence of mutant plasmids.

[0019] The discovery that the inducible system is insensitive to toxic proteins was unexpected. The prior art did not teach that the system would be insensitive to toxic proteins (or that the toxic proteins were not produced).

[0020] Another disadvantage of the inducible system is the inherent production of multiple copies of the BAC. In fact, the authors of the inducible system explained that the most important feature of BAC clones is their stability due to their very low copy number. As cited above, Wild et al. (2002) showed that the copy number can even be further reduced by adding glucose. This single-copy state improves the retention stability of the BAC library by reducing the chance of intracellular recombination between clones. This demonstrates that the inducible system published by Wild et al. does not reduce the variation of unwanted recombination events that will occur soon as long as multiple copies of the BAC are present in the host cell. Those skilled in the art should understand that induction and subsequent high copy numbers will reintroduce recombination events. Therefore, those skilled in the art will avoid using the said system to prepare DNA for vaccination purposes.

[0021] In the present invention, BACs have been amplified in the inducible ori system, and the recombination events of the amplified BACs have been tested. Unexpectedly, the recombination events are very rare.

[0022] In addition, in addition to testing recombination events, other mutations in the amplified BACs have also been tested. The frequency of mutation is very low. In addition, the missense portion is surprisingly lower than the theoretical expectation. If mutations occur, they are mainly nonsense mutations or frameshift mutations that result in non-functional viral RNA.

[0023] The present invention allows for a significant expansion in the scale of DNA vaccine production. For example, the current major producers of live-attenuated yellow fever vaccines are unable to meet the existing demand. Using the technology of the present invention, it will be possible to produce DNA vaccines at a significantly reduced cost and higher quality compared to current live-attenuated vaccines, thereby meeting the long-standing demand.

[0024] Similarly, for other viral diseases, such as JEV, WNV, measles, rubella, and HIV vaccines, a vaccine preparation platform that can provide sufficient amounts of DNA is needed.

[0025] The first aspect of the present invention relates to the use of a bacterial artificial chromosome (BAC) for the preparation of a vaccine, wherein the BAC comprises:

[0026] - An inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and - A viral expression cassette comprising a cDNA of an attenuated RNA virus genome and comprising cis - regulatory elements for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into an infectious RNA virus.

[0027] An embodiment of the cDNA of the attenuated RNA virus genome is a chimeric viral cDNA construct of the RNA virus genome, wherein a heterologous DNA sequence has been inserted, or wherein a native viral sequence has been deleted, truncated or mutated.

[0028] Embodiments of the viral expression cassette include:

[0029] - A cDNA of a positive - strand RNA virus genome,

[0030] - An RNA polymerase - driven promoter located before the 5' end of the cDNA for initiating transcription of the cDNA, and

[0031] - An element for RNA self - cleavage located after the 3' end of the cDNA for cleaving the RNA transcript of the viral cDNA at a set position.

[0032] Embodiments of positive - strand RNA viruses are: flavivirus, hepatitis C virus, pestivirus, alphavirus, picornavirus, coronavirus, hepatitis E virus, and calicivirus.

[0033] In a typical embodiment, the viral expression cassette comprises the cDNA of yellow fever virus, such as the cDNA of the live - attenuated YFV - 17D yellow fever virus vaccine.

[0034] In other embodiments, the viral expression cassette comprises the cDNA of a virus belonging to the group: negative - strand RNA virus, double - strand RNA virus or ambisense RNA virus.

[0035] In a particular embodiment, the bacterial artificial chromosome further comprises a yeast autonomous replication sequence for shuttling into yeast and retaining the bacterial artificial chromosome in yeast.

[0036] An example of a yeast ori sequence is the 2μ plasmid origin or ARS1 (autonomously replicating sequence 1) or a functional homologous derivative thereof.

[0037] In certain embodiments, the RNA polymerase - driven promoter is an RNA polymerase II promoter, such as the cytomegalovirus immediate - early (CMV - IE) promoter, the simian virus 40 promoter or a functional homologous derivative thereof.

[0038] In other embodiments, the RNA polymerase-driven promoter is an RNA polymerase I or III promoter.

[0039] Examples of elements for RNA self-cleavage are: the genomic ribozyme of hepatitis delta virus or the cDNA of a functionally homologous RNA element.

[0040] In a specific embodiment, the viral expression cassette comprises the cDNA of the attenuated live YFV-17D vaccine, wherein one or more cDNA sequences encoding the viral particle surface protein are deleted, truncated or mutated, such that the functional viral particle surface protein of YFV-17D is not expressed, and a cDNA sequence encoding a heterologous protein is inserted into the YFV-17D cDNA. An example of the heterologous protein is the viral particle surface protein of a flavivirus.

[0041] Embodiments of the viral expression cassette comprise the cDNA of the attenuated live YFV-17D vaccine, wherein one or more unrelated cDNA sequences are inserted, which are expressed as one or more heterologous proteins in the viral polyprotein.

[0042] In other embodiments, the viral expression cassette comprises viral cDNA, wherein an exogenous cDNA sequence is inserted, which is heterologously expressed by the recombinant virus.

[0043] Another aspect relates to a method for preparing a vaccine against an RNA virus, the method comprising the steps of: a) providing a bacterial host transfected with a BAC as described in the first aspect and its various embodiments

[0044] b) amplifying the BAC by adding a compound capable of activating the inducible ori

[0045] c) isolating the amplified BAC,

[0046] d) formulating the BAC into a vaccine.

[0047] Another aspect relates to: the BAC as described in the first aspect and its various embodiments, which is used as a vaccine.

[0048] Another aspect of the present invention relates to: the BAC as described in the first aspect and its various embodiments, which is used for preventing RNA virus infection.

[0049] Another aspect relates to: the use of the BAC as described in the first aspect and its various embodiments as a live DNA vaccine.

[0050] Another aspect relates to: the use of the BAC as described in the first aspect and its various embodiments for proliferating a native or recombinant virus from the cDNA.

[0051] Another aspect relates to the use of the bacterial artificial chromosome (BAC) as described in the first aspect and its various embodiments as a BAC for the preparation of vaccines. DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 . Generation of the pShuttleBAC series of RNA virus expression plasmids. Panel (A) shows the construction of pShuttleBAC / Pme as the starting vector construct. Panel (B) shows the construction of a flavivirus expression vector derived from pShuttleBAC / Pme by insertion of viral cDNA, which is carried out by homologous recombination between the SV40 promoter (SV40p) and the HDV ribozyme (HDrz).

[0054] Figure 2 . Enhancing plasmid stability of the pShuttleBAC construct in Escherichia coli. General plasmid maps show the design principles of a prior art flavivirus cDNA plasmid (A) and the novel pShuttleBAC vector series (B, DNA-YFVax). Escherichia coli transformed with a single colony of each of (A) and (B) was grown overnight at 37°C and plated on selective medium. Overall, the colonies grown from the construct of type A were much smaller in size than those of type B (shown in Figure 2 C and D, respectively). In addition, the construct of type A produced progeny with a wide range of colony sizes ( Figure 2 the right panel of C shows the normalized colony diameter), indicating the selection and isolation of mutant plasmid clones that sometimes render these cDNAs less toxic to Escherichia coli. Cloning analysis identified a variety of possible potential mutations, including transposon insertions in the viral E / NS1 region. In contrast, plasmid clones of the construct of type B of the pShuttleBAC series did not segregate and showed a more uniform colony size, even after repeated passage in Escherichia coli, indicating its high genetic stability.

[0055] Figure 3A. After transfection of Vero-B cells with pShuttle / YF17D (wild type, WT) and its replication-deficient derivative pShuttle / YF17DΔGDD (ΔGDD), replication intermediates of YFV-17D RNA replication were detected by Northern blotting. Antisense-oriented antigenomic (-)-RNA (A, upper panel, 11 kb) and sense-oriented viral genomic (+)-RNA (A, lower panel, 11 kb) were detected only in WT-transfected cells 5 days post-transfection. Ongoing replication in the presence of actinomycin D (ACD), an inhibitor of DNA-directed RNA synthesis, confirmed that after initial initiation of YFV-17D genome transcription from pShuttle / YF17D, viral replication continued autonomously in a plasmid-independent manner.

[0056] Figure 3B . Correct YFV-17D RNA transcript processing was detected by 5'- and 3'-RACE (rapid amplification of cDNA ends). pShuttle / YF17D initiated transcription of nascent YFV-17D RNA ( Figure 3B shown in bold) which, as confirmed by rapid amplification of cDNA ends (RACE), began and ended with the correct 5' and 3' ends (shown in the upper and lower panels, respectively).

[0057] Figure 4 A-C. Similar CPE induced by YFV-17D from different origins. YFV-17D virus derived from in vitro transcribed and capped RNA using pACNR-FLYF17DII as a template (A), or YFV-17D virus harvested after transfection with pShuttle / YF17D plasmid DNA (B) induced the same virus-induced cytopathic effect (CPE) in BHK-21 cells 5 days post-infection (p.i.); C, untransfected cells for comparison.

[0058] Figure 4 D-E. Similar plaque phenotypes of YFV-17D from different origins. YFV-17D virus derived from in vitro transcribed and capped RNA using pACNR-FLYF17DII as a template (D), or YFV-17D virus harvested after transfection with pShuttle / YF17D plasmid DNA (E) produced a comparable number (3×10 5 plaque-forming units (pfu) mL -1 compared to 2×10 5 pfu mL -1 ) and plaques of the same morphology (diameter 6.4±0.7 mM compared to 6.1±1.1 mM; t-test n = 8, p-value = 0.6).

[0059] Figure 5 Detection of infectious recombinant DENV2 by immunofluorescence assay (IFA). Recombinant DENV2 NGCs produced in BHK-21 cells transfected with pShuttle / DV2 showed dose-dependent infection of Vero-B cells, shown as: viral foci immunofluorescently stained for viral E protein 5 days after transfection (A, undiluted supernatant, B, 100-fold diluted supernatant, C, uninfected cell control).

[0060] Figure 6 .Survival of AG129 mice infected with Stamaril (open squares) or transfected with pShuttle / YF17D (crosses). 10-12 days after ip challenge, interferon type I and II receptor-deficient (AG129) mice begin to lose weight and develop a uniform onset of symptoms, i.e., ruffled fur, tremors, and flaccid hindlimb paralysis. Control animals transfected with a replication-deficient NS5ΔGDD plasmid variant (ΔGDD, open circles) show no disease. However, they remain susceptible to a second transfection 20 days after the initial transfection. The challenge (solid triangles) then resulted in death within a comparable time frame and displaying similar symptoms. Plasmid DNA was transfected ip using calcium carbonate microflowers in 33% propylene glycol as vehicle.

[0061] Figure 7 . Detection of YFV-17D RNA (B) and morbidity (A) in infected AG129 mice. (A) Infected or transfected with pShuttle / YF17D (pYF17D), AG129 mice lost about 20% of their body weight, and then they had to be euthanized after an average of 12-13 days (MDD, mean day to death). In contrast, mice transfected with pShuttle / YF17DΔGDD (ΔGDD) gained weight and were then (ΔGDD+2°Stamaril) challenged them, which died within about two weeks from YFV-17D infection. (B) Comparable amounts of YFV-17D RNA could be detected by qRT-PCR in brain samples of AG129 mice collected at death from (A).

[0062] Figure 8Map of.pShuttle / YF17D (synthetic construct #1). Notes: SV40p: Simian virus 40 promoter / origin, YFV-17D: Yellow fever virus vaccine strain 17D cDNA, HDVrz: Hepatitis delta virus ribozyme cDNA; 2μ: 2-micron origin of Saccharomyces cerevisiae; TRP1: TRP1 gene providing prototrophic growth towards tryptophan; parABC: partitioning gene of F-plasmid; repE: repE gene of F-plasmid; oriS: origin of F-plasmid; oriV: origin of plasmid RK2; CmR: Chloramphenicol resistance gene.

[0063] Figure 9 . Growth of Escherichia coli colonies after transfection with different YFV-17D cDNA vectors. (A) Escherichia coli EPI-300T colonies after transformation with pACNR-FLYF17DII and grown at 37 °C for 16 h. Two subpopulations of colony sizes with large size differences can be observed, small colonies (diameter less than 0.2 mm) and large colonies (diameter about 0.4 mm). Most are small colonies. (B + C) Escherichia coli EPI-300T colonies after transformation with pShuttle / YFV17D. Plated on plates without inducer (B) or containing 0.01% L-arabinose (C) (for inducing high-copy replication mediated by the inducible high-copy origin). The large black circle is a 2.5-mm-diameter Zirkonia bead embedded in agar as a calibrator. The inset is a schematic line graph showing the outlines of the colonies observed in each group.

[0064] Figure 10 . Size distribution of Escherichia coli colonies after transformation with different YFV-17D cDNA vectors. (A) Escherichia coli EPI-300T colonies after transformation with pACNR-FLYF17DII. (B + C) Escherichia coli EPI-300T colonies after transformation with pShuttle / YFV17D. Plated on plates without inducer (B) or containing 0.01% L-arabinose (C) (for inducing high-copy replication mediated by the inducible high-copy origin).

[0065] Figure 11a. Map of pShuttle / ChimeriVax-JE. pShuttle / ChimeriVax-JE consists of the SV40 promoter / origin (nucleotides 1 - 481) and YFV-17D (nucleotides 2452 - 10862), into which the neuroattenuated JEV vaccine strain JE SA14-14-2 from 477 - 2477 is inserted. The penultimate of the last two amino acids of the JEV E-ORF is a mutation from a histidine to a glycine codon to create a KasI site, and the NS2A and NS4B-ORFs contain two matching G4055a and G7349a mutations seen in that change methionine in YFV-17D NS2A to valine and lysine in the NS4B ORF to glutamine, respectively. Other silent mutations create restriction markers at position 406 (XhoI), position 4009 (BstEII), and position 7315 (NheI).

[0066] Figure 11 b. Map of pShuttle / ChimeriVax-WN. pShuttle / ChimeriVax-WN consists of the SV40 promoter / origin (nucleotides 1 - 481) and YFV-17D (nucleotides 2452 - 10862), into which a neuroattenuated derivative of the WNV strain NY-99 from 477 - 2477 is inserted. The penultimate of the last two amino acids of the WN E-ORF is a mutation from a histidine to a glycine codon to create a KasI site, and the NS2A and NS4B-ORFs contain two matching G4055a and G7349a mutations seen in that change methionine in YFV-17D NS2A to valine and lysine in the NS4B ORF to glutamine, respectively. Other silent mutations create restriction markers at position 406 (XhoI), position 4009 (BstEII), and position 7315 (NheI).

[0067] Figure 12 . Map of pShuttle / EV71. pShuttle / EV71 contains the cDNA of the EV71 strain BrCr-TR (Genbank AB204852.1) inserted between the 5'-end of the SV40 promoter / origin and the 3'-end of a 30-nucleotide-long polyA repeat plus the hepatitis delta virus ribozyme.

[0068] Figure 13 Describes the sequences having SEQ ID NO 1 - 7.

[0069] Define

[0070] The term "bacterial artificial chromosome (BAC)" refers to a plasmid DNA construct used for cloning DNA sequences in bacterial cells (e.g., Escherichia coli). Generally, DNA sequences in the range of 30,000 - approximately 300,000 base pairs can be inserted into a BAC. A BAC with an inserted DNA can be taken up by a bacterial cell. As the bacterial cell grows and divides, the BAC DNA is stably maintained in the bacterial cell at a very low copy number per bacterial cell, preferably not exceeding 3 copies per cell, e.g., 1 copy per cell. Replication of the BAC initiates at the origin of replication (ori) sequence, typically the oriS sequence. This replication is tightly regulated by gene products encoded by the BAC, generally repE and / or repF. The BAC also encodes proteins (e.g., parA, B, and C) that direct the partitioning of BAC copies to daughter cells during cell division. Typically, the BAC vector also contains selectable markers, such as antibiotic resistance or reporter enzyme markers, such as lacZ, which allows for blue - white screening. An example of a commonly used BAC is pBeloBac11 (Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8794–8797). The sequence of this vector is reported under GenBank accession number U51113. pBeloBac11 is a circular plasmid that contains oriS, the repE gene that produces the proteins that initiate and regulate replication at oriS, and the partitioning genes par A, B, and C. Optionally, pBeloBac11 contains a chloramphenicol - resistance - encoding gene. The vector also contains the lacZα gene, which can be disrupted or removed from the vector when an insert is cloned into the BAC.

[0071] The term "inducible bacterial ori sequence" refers to a plasmid ori sequence that functions in a bacterial host cell and responds to an amplification - mediated protein. Preferably, in the absence of the amplification - mediated protein, the replication function of the inducible ori is tightly inhibited or absent. Also preferably: in the presence of the amplification - mediated protein, the inducible ori amplifies the plasmid to a high copy number, preferably to more than 20 copies per cell, more preferably to more than 100, e.g., more than 500 or 1000 copies per cell. For the applications of the present invention, it is also preferred (but not necessary) that the inducible ori responds to a single amplification - mediated protein.

[0072] In the present invention, oriV is particularly useful as an inducible bacterial ori due to its broad host range, its known ability to replicate DNA fragments of 100-kb or greater, its high copy number, and its requirement for only one inducer protein. Examples of bacterial artificial chromosomes containing inducible oriV are: pBeloBAC / oriV (Wild et al. (2002) Genome Res. 12, 1434-1444) and pBAC-LacZ (Addgene plasmid 13422: pBAC-lacZ, Addgene, Cambridge, Massachusetts, USA). The pBAC-lacZ plasmid is a mini-F', which can replicate in standard E. coli strains, but because it is maintained as a single-copy episome, its DNA yield is low. pBAC-lacZ also contains a second higher-copy-number origin of replication (oriV), which is only active in the presence of the trans-acting factor encoded by the trfA gene. Transcription from the self-inducible promoter in E. coli cells expressing trfA allows an increase in the copy number of the pBAC-lacZ plasmid by inducing the expression of trfA. TransforMax TM EPI300 TM E. coli cells (Epicentre, Madison, Wisconsin, USA) contain an inducible mutant trfA gene, the gene product of which is required to initiate replication from the oriV origin of replication. Expression of trfA can be induced by adding L-arabinose to the medium, resulting in the activation of oriV.

[0073] "Yeast origin of replication" refers to a sequence in a plasmid, such as a bacterial artificial chromosome, which allows the plasmid to replicate and be maintained in yeast cells. The origin of replication present in the 2μ plasmid has been shown (Huberman et al. (1987) Cell 51, 473-481; Brewer and Fangman (1987) Cell 51, 463-471; Hartley and Donelson (1980) Nature 286, 860-865.) to be suitable as an ori for shuttling the bacterial artificial chromosomes of the present invention into yeast. Other suitable yeast ORIs have been described (Liachko et al. (2013) Genome Res. 23, 698-704, such as ARS1 (Autonomously Replicating Sequence 1) and its functional homologous derivatives, as used in yeast centromere plasmids (YCp), or the synthetic CEN6 / ARSH4 origin (Frazer and O'Keefe (2007) Yeast. 24, 777-789).

[0074] As used herein, the term "viral expression cassette" refers to a cDNA of an RNA viral genome flanked by cis-regulatory elements that, upon introduction of the bacterial artificial chromosome into mammalian cells, promotes transcription of the viral cDNA and allows processing of the transcribed RNA into infectious viral RNA, as detailed below.

[0075] "Infectious viral RNA" refers to viral RNA that, upon introduction into its mammalian host, is sufficient to provide all viral functions required for viral replication and production of infectious viral progeny. This includes: (i) serving as a transcription template for viral RNA synthesis and genome amplification, and (ii) serving as a translation template for synthesis of viral proteins required for viral replication. Other additional functions, such as engaging host cell factors involved in the innate antiviral response (Moon et al. (2012) RNA. 18, 2029-40), may also be required to be provided by the infectious viral RNA.

[0076] As used herein, "attenuation" relates to a change in the virulence of a pathogen, by which the harmful properties of a pathogenic organism are weakened (or reduced); an attenuated pathogen can be used as a live vaccine. Attenuated vaccines can be derived in different ways from live organisms that have been weakened, typically from culturing under suboptimal conditions (also referred to as attenuation), or from genetic modifications that have the effect of decreasing their ability to cause disease.

[0077] In a first aspect of the present invention, there is provided a bacterial artificial chromosome, said bacterial artificial chromosome comprising an inducible bacterial ori sequence which, in the presence of a stimulus, induces amplification of the bacterial artificial chromosome to a high copy number in bacterial cells, preferably to more than 10 copies per cell, more preferably to more than 100, such as more than 500 or 1000 copies per cell. Generally, the amplification of the bacterial artificial chromosome caused by the stimulus does not exceed more than 10,000 copies per cell, such as no more than 5000 copies. The bacterial artificial chromosome of the present invention further comprises a viral expression cassette, said viral expression cassette comprising a cDNA of an RNA viral genome flanked by cis-regulatory elements, which promotes transcription of the viral cDNA after introduction of the bacterial artificial chromosome into mammalian cells and allows the transcribed RNA to be processed into infectious viral RNA. The viral cDNA comprised in the viral expression cassette of the bacterial artificial chromosome of the present invention may be derived from a virus belonging to the group consisting of positive-strand viruses, negative-strand viruses, double-stranded RNA viruses or viruses that utilize the ambisense RNA strategy for replication. The viral cDNA comprised in the viral expression cassette can correspond to a wild-type RNA viral genome or be a chimeric viral cDNA construct in which heterologous DNA sequences have been inserted and / or native viral sequences have been deleted. Preferably, the heterologous DNA sequences encode one or more proteins which are heterologously expressed by the recombinant virus after introduction of the bacterial artificial chromosome of the present invention comprising the viral expression cassette containing the chimeric viral cDNA into mammalian cells. Optionally, the bacterial artificial chromosome of the present invention further comprises a yeast autonomous replication sequence for shuttling into yeast or maintaining the bacterial artificial chromosome in yeast. The possibility of shuttling into yeast cells or maintaining the bacterial artificial chromosome in yeast cells provides the advantage of allowing genetic manipulation to be carried out under control in yeast and bacterial genetic systems.

[0078] In the absence of a stimulus for the inducible ori, the bacterial artificial chromosome of the present invention can be used for the retention and stable cloning of infectious viral cDNA in a bacterial host, while in the presence of the stimulus, the cDNA can be amplified and subsequently isolated for use. The bacterial artificial chromosome of the present invention is particularly useful in the development, stable maintenance and production of viral cDNA to be used as a live vaccine against RNA viral pathogens. Alternatively, the bacterial artificial chromosome is used for the maintenance and propagation of natural or recombinant viruses from cDNA, for example, for research purposes. The bacterial artificial chromosomes of the present invention, particularly those comprising yeast origins of replication, also have other advantages: they provide a versatile system for genetic engineering of viral cDNA. This versatility is particularly important in research and development applications of the bacterial artificial chromosome (for example, in the design of cDNA live vaccines against RNA viral pathogens or in research aimed at elucidating the roles and functions of certain viral gene products using reverse genetics methods).

[0079] If the bacterial artificial chromosome of the present invention contains a viral expression cassette containing cDNA of a positive-strand RNA virus genome, preferably there is a polymerase-driven promoter before the 5' end of the viral cDNA, which initiates transcription of the viral cDNA after introduction into mammalian cells, and preferably there is an element for RNA self-cleavage after its 3' end for cleaving the RNA transcript of the viral cDNA at a set position. When the bacterial artificial chromosome is introduced into mammalian cells, these cis-regulatory elements together allow the transcription and process the viral cDNA into infectious viral RNA. Preferably, the polymerase-driven promoter is a promoter operated by RNA polymerase II, such as the cytomegalovirus immediate early (CMV-IE) promoter (Thomsen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 659-663), the simian virus 40 promoter (Deboist and Chambon (1981) Nature 290, 304-310) or a functionally homologous derivative thereof, such as the CMV-IE chicken β-actin chimeric (CAG) promoter (Niwa et al. (1991) Gene 108, 193-199) or an inducible form of the RNA polymerase II-operated promoter, such as the tetracycline-operator minimal CMV-IE promoter (Gossen et al. (1995) Science. 268, 1766-1769; Baron and Bujard (2000) Methods Enzymol. 327, 401-421). Alternatively, the polymerase-driven promoter is an RNA polymerase I (Russel and Zomerdijk (2006) Biochem. Soc. Symp. 73, 203-216) or an RNA polymerase III promoter, such as the U6 or H1 promoter. Also preferably, the element for RNA self-cleavage is the cDNA of the genomic ribozyme of hepatitis delta virus (Chadalavada et al., (2007) RNA 13, 2189-2201) or the cDNA of a functionally homologous hepatitis delta virus-like self-cleaving ribozyme RNA element, such as that described in Webb and Luptak (2011) RNA Biol. 8, 719-727).Preferably, the viral cDNA of the positive-strand RNA virus contained in the viral expression cassette is derived from a virus belonging to one of the following viral families: Flaviviridae, including yellow fever virus and other flaviviruses; Hepacivirus, including hepatitis C virus; Pestivirus, including bovine viral diarrhea virus, and classical swine fever virus; Togaviridae, including alphaviruses, chikungunya virus and rubivirus genus, rubella virus; Picornaviridae, including enteroviruses, such as poliovirus and rhinovirus, and foot-and-mouth disease virus, Coronaviridae, including HCoV-229E, SARS-CoV, MERS-CoV (originally described as novel coronavirus 2012 / London1 / novel CoV 2012) and feline coronavirus, and Hepevirus, including hepatitis E virus, and Caliciviridae, including Norwalk virus and norovirus. The viral cDNA contained in the viral expression cassette may correspond to those of the wild-type RNA virus genome or be a chimeric viral cDNA construct, wherein heterologous DNA sequences have been inserted, and / or, native viral sequences have been deleted, truncated or mutated. Preferably, the heterologous DNA sequences encode one or more peptides / proteins that are heterologously expressed by the recombinant virus after introducing the bacterial artificial chromosome of the present invention containing the chimeric viral cDNA into a mammal.

[0080] In a specific embodiment of the present invention, the bacterial artificial chromosome of the present invention comprises a viral expression cassette containing the cDNA of the live attenuated yellow fever virus (YFV)-17D vaccine ( Figure 6 ). The bacterial artificial chromosome according to this specific embodiment can be used for the stable cloning and propagation of YFV-17D cDNA. In addition, the bacterial artificial chromosome can be used as a DNA vaccine of live YFV-17D, as an alternative to the currently used live attenuated YFV-17D virus vaccine ( and similar preparations, such as etc.). Regarding the existing YFV-17D virus vaccine, the YFV-17D DNA vaccine of the present invention has advantages: it can be produced at a lower cost without eukaryotic cell cultures or chicken embryo eggs. In addition, its distribution does not require a cold-chain and it can be administered needle-free.

[0081] In a more specific embodiment, the bacterial artificial chromosome of the present invention comprises a viral expression cassette containing the cDNA of the attenuated live YFV-17D vaccine, wherein a heterologous DNA sequence has been inserted and / or a native viral sequence has been deleted. For example, referring to US6962708, the nucleotide sequence encoding the prM-E protein in the cDNA of YFV-17D can be deleted, truncated or mutated such that the functional prM-E protein of YFV-17D is not expressed, and a nucleotide sequence encoding the viral envelope protein of a second different virus such that the viral envelope protein of the second virus is expressed by the altered genome of the YFV-17D vaccine. Preferably, the second virus is also a flavivirus, such as Japanese encephalitis (JE, e.g., JE SA14-14-2), dengue (DEN, e.g., any type of dengue 1-4; e.g., dengue-2 strain PUO-218) (Gruenberg et al. (1988) J. Gen. Virol. 67, 1391-1398.), Murray Valley encephalitis (MVE), Saint Louis encephalitis (SLE), West Nile virus (WN), tick-borne encephalitis (TBE) (i.e., Central European encephalitis (CEE) and Russian spring-summer encephalitis (RSSE) viruses), and hepatitis C (HCV) virus. Other flaviviruses used as the second flavivirus include: Kunjin virus, Powassan virus, Quang Binh forest disease virus, Zika virus, Usutu virus, and Omsk hemorrhagic fever virus. Introduction of the bacterial artificial chromosome containing the chimeric YFV-17D cDNA into mammalian cells results in the generation of a chimeric virus composed of the genes and gene products responsible for intracellular replication belonging to YFV-17D and the genes and gene products of the envelope of the second virus. Since the viral envelope contains antigenic determinants responsible for inducing neutralizing antibodies, as a result of infection with this chimeric virus, antibodies against the second virus are produced. Alternatively, the nucleotide sequence encoding the prM-E and / or NS1 protein in the cDNA of YFV-17D can be deleted, truncated or mutated such that the functional prM-E and / or NS1 protein of YFV-17D is likewise not expressed, and a nucleotide sequence encoding a peptide / protein containing a specific epitope / antigen such that the protein is expressed by the altered genome of the YFV-17D vaccine.Alternatively, the cDNA encoding the heterologous protein can be inserted into other positions in the YFV-17D cDNA of the bacterial artificial chromosome of the present invention, for example, inserted between the E and NS1 genes (Bonaldo et al. (2007) Virol. J. 4, 115.), inserted into the C gene (Jones et al. (2005) Virology 331, 247-259; Schoggings et al. (2012) Proc. Natl. Acad. Sci. USA 109, 14610-14615) or inserted into the untranslated region of the YFV-17D cDNA (Jones et al. (2005), cited above). Preferably, the protein containing the epitope / antigen is a tumor antigen or a viral antigen or a bacterial antigen of a parasitic pathogen. Introduction of the bacterial artificial chromosome containing the chimeric YFV-17D cDNA into mammalian cells results in the generation of a chimeric virus, which consists of the genes and gene products responsible for intracellular replication belonging to YFV-17D and the genes and gene products of the protein containing the epitope / antigen. Since the viral envelope contains antigenic determinants responsible for inducing neutralizing antibodies, as a result of infection with this chimeric virus, antibodies against the protein containing the epitope / antigen are produced.

[0082] If the bacterial artificial chromosome of the present invention contains a viral expression cassette containing cDNA of a negative-strand RNA virus genome, the construct as described above for positive-strand RNA viruses must be modified in the following manner: the viral genome cDNA, with respect to the cis-elements driving its expression, is present in its sense (antigenomic) orientation, (Radecke et al. (1995) EMBO J. 14, 5773-5784), and it also contains in the sense orientation cDNA encoding viral gene products in the sense orientation, which together with the viral RNA form part of the viral replicase complex required for rescue of viral RNA replication. These cDNAs are flanked by regulatory cis-elements that are required for expression of these viral gene products from the plasmid to form the viral replicase complex. In the case of non-segmented negative-strand RNA viruses, (Conzelmann (1998) Annu. Rev. Genet. 32, 123-162) these viral gene products are the N (NP), P, and L proteins. Expression of the antigenomic cDNA of the negative-strand RNA genome can be driven by an RNA polymerase I or II promoter (Martin et al., (2006) J Virol. 80, 5708-5715); while the expression cassette for the viral proteins that form the viral replicase complex can be polycistronic (as occurs naturally in the virus itself) or monocistronic (using different RNA polymerase promoters (Morita et al. (2012) Biotechniques 0, 1-5)) for balanced expression of each replicase component. Rescue of negative-strand RNA viruses with a segmented RNA genome from the artificial bacterial chromosome of the present invention requires modification of the system (as described for negative-strand RNA viruses with an unsegmented RNA genome); more specifically, it requires incorporation of expression cassettes for each genome segment (Neumann and Kawaoka (2004) Curr. Top. Microbiol. Immunol. 283, 43-60) driven by appropriate RNA polymerase I and II promoters (Fodor et al. (1999) J. Virol. 73, 9679-9682). Because of the limited vector capacity of plasmid vector systems in the prior art for this purpose, in the prior art, all the functions required to rescue the virus must be provided by co-transfection of several plasmids, for example, up to 8 or 12 plasmids are used to rescue influenza virus, as described in the previous literature: Hoffmann et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11411-11416 and Fodor et al. 1999 (cited above).The bacterial artificial chromosome of the present invention has a high vector capacity to contain large inserts, which allows for the expression of negative-strand RNA virus genomes or genomic segments, plus the expression of other viral protein-coding sequences from a single bacterial artificial chromosome in a manner similar to that of positive-strand RNA virus genomes.

[0083] Preferably, the viral cDNA of the negative-strand RNA virus contained in the viral expression cassette and other expression cassettes required for rescuing viral replication are derived from a virus belonging to one of the following viral families: Orthomyxoviridae, including influenza A, B, and C viruses, and Paramyxoviruses, including measles virus, mumps virus, and respiratory syncytial virus. The viral cDNA contained in the viral expression cassette can correspond to those of the wild-type RNA virus genome or be a chimeric viral cDNA construct, in which heterologous DNA sequences have been inserted, and / or native viral sequences have been deleted, truncated, or mutated. Preferably, the heterologous DNA sequences encode one or more peptides / proteins that are heterologously expressed by the recombinant virus after introducing the bacterial artificial chromosome of the present invention containing the viral expression cassette with the chimeric viral cDNA into a mammal.

[0084] If the bacterial artificial chromosome of the present invention contains a viral expression cassette with a cDNA containing a double-stranded RNA virus genome, the construct must be modified such that all viral RNA genome segments required for rescuing viral RNA replication, as described by Boyce et al. (2008) J. Virol. 82, 8339-8348, i.e., 10 RNAs in the case of bluetongue virus, can be expressed by an RNA polymerase II promoter, which allows for proper processing (mainly capping) of the nascent transcripts. Alternatively, RNA polymerase I and III promoters can also be used for this purpose. Preferably, the viral cDNA of the double-stranded RNA virus contained in the viral expression cassette is derived from a virus belonging to one of the following viral families: Reoviridae, including reovirus, rotavirus, and bluetongue virus. The viral cDNA contained in the viral expression cassette can correspond to those of the wild-type RNA virus genome or be a chimeric viral cDNA construct, in which heterologous DNA sequences have been inserted, and / or native viral sequences have been deleted, truncated, or mutated. Preferably, the heterologous DNA sequences encode one or more peptides / proteins that are heterologously expressed by the recombinant virus after introducing the bacterial artificial chromosome of the present invention containing the viral expression cassette with the chimeric viral cDNA into a mammal.

[0085] If the bacterial artificial chromosome of the invention contains a viral expression cassette containing the cDNA of a virus that uses the bidirectional RNA strategy for replication, the construct must be modified in such a way that, as described by Lowen et al. (2004) Virology 330, 493-500, all viral RNA genomic segments required for rescue of viral RNA replication are expressed by RNA polymerase I or II promoters. Preferably, the viral cDNA of the virus that uses the bidirectional RNA replication strategy contained in the viral expression cassette is derived from a virus belonging to one of the following viral families: Bunyaviridae, including Rift Valley fever virus, hantavirus, and Schmallenberg virus, and Arenaviridae, including Lassa virus. The viral cDNA contained in the viral expression cassette may correspond to those of the wild-type RNA virus genome or be a chimeric viral cDNA construct, in which heterologous DNA sequences have been inserted, and / or native viral sequences have been deleted, truncated, or mutated. Preferably, the heterologous DNA sequences encode one or more peptides / proteins that are heterologously expressed by the recombinant virus after introduction of the bacterial artificial chromosome of the invention containing the viral expression cassette containing the chimeric viral cDNA into a mammal.

[0086] As described above and as illustrated in the examples, the invention allows the production of high amounts of viral cDNA of sufficiently high quality for use in DNA vaccines.

[0087] The formulation of DNA into vaccine preparations is known in the art and is described in detail, for example, in Chapters 6-10 of Methods in Molecular Medicine, "DNA Vaccines", Volume 127, (2006) Springer Saltzman, Shen, and Brandsma (eds.). Humana Press. Totowa, NJ. and in Chapter 61 of Vaccines (6th Edition), Alternative vaccine delivery methods, pages 1200-1231, (2013) (Plotkin et al. eds.). A detailed description of acceptable carriers, diluents, excipients, and adjuvants suitable for use in DNA vaccine preparations can be found in WO2005042014, as follows.

[0088] "Acceptable carrier, diluent, or excipient" refers to other substances acceptable in the application of human and / or veterinary drugs, especially drugs for immunotherapy.

[0089] For example, acceptable carriers, diluents or excipients can be solid or liquid fillers, diluents or encapsulating substances that can be safely used for systemic or local administration. Depending on the specific route of administration, a variety of carriers well known in the art can be employed. These carriers can be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsions, isotonic saline and salts such as inorganic acid salts including hydrochlorides, bromides and sulfates, organic acid salts such as acetates, propionates and malonates, and pyrogen-free water.

[0090] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is: Remington's Pharmaceutical Sciences (Mack Publishing Co., New Jersey, USA, 1991), which is incorporated herein by reference.

[0091] Any safe route of administration can be used to provide the DNA vaccine to a patient. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, transdermal, etc. can be used. Intramuscular and subcutaneous injections may be suitable, for example, for administering immunotherapeutic compositions, protein vaccines and nucleic acid vaccines. It is also contemplated that particle bombardment or electroporation can also be specifically used to deliver nucleic acid vaccines.

[0092] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, pills, allantoin, suppositories, aerosols, transdermal patches, etc. These dosage forms can also include injectable or implantable controlled release devices that are specifically designed for this purpose, or other forms of implants that are modified to function in this form. The controlled release of these therapeutic agents can be achieved by coating them, for example, with hydrophobic polymers including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. In addition, the controlled release can be achieved by using other polymer matrices, liposomes and / or microspheres.

[0093] DNA vaccines suitable for oral or parenteral administration can exist in the following forms: discrete units such as capsules, sachets or tablets each containing a predetermined amount of plasmid DNA, powders or granules, or solutions or suspensions in the form of aqueous liquids, non-aqueous liquids, water-in-oil emulsions or oil-in-water liquid emulsions. The compositions can be prepared by any pharmaceutical method, but all methods include the step of combining one or more of the reagents as described above with a carrier consisting of one or more essential components. Generally, the compositions are prepared by mixing the DNA plasmid and a liquid carrier or a finely ground solid carrier or both, and then, if necessary, shaping the product into the desired form.

[0094] The above compositions can be administered in a manner compatible with the dosage formulation and in an effective amount. The dose administered to the patient should be sufficient to effectively produce a beneficial response in the patient over a suitable period of time. The amount of the reagent to be administered may depend on the subject to be treated, including its age, sex, weight and general health, and the factors will be determined by the judgment of the attending physician.

[0095] The DNA vaccines are generally used for prophylactic or therapeutic immunization of humans, but for certain viruses, they can also be administered to vertebrates (usually mammals, birds and fish), including domestic animals such as livestock and companion animals. It is contemplated that the vaccination is carried out on animals (zoonoses) that are reservoirs of live viruses, such as monkeys, mice, rats, birds and bats.

[0096] In certain embodiments, the vaccine may comprise an adjuvant, i.e., one or more substances that enhance the immunogenicity and / or efficacy of the vaccine composition. However, live vaccines may ultimately be disrupted by adjuvants that can stimulate an innate immune response independent of viral replication. Non-limiting examples of suitable adjuvants include: squalane and squalene (or other animal oils); block copolymers; detergents such as Tween 80; Freund's adjuvant A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium diphtheriae-derived adjuvants, such as Corynebacterium parvum; Propionibacterium-derived adjuvants, such as Propionibacterium acne; Mycobacterium bovis (Bacillus Calmette-Guérin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumor necrosis factor; interferons, such as interferon γ; combinations, such as saponin-aluminum hydroxide or Freund's adjuvant A-aluminum hydroxide; liposomes; ISCOM (t) and ISCOMATRIX (B) adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides, such as muramyl dipeptide, or other derivatives; avridine; lipid A derivatives; dextran sulfate; DEAE-dextran or with aluminum phosphate; hydroxypolymethylene, such as Carbopol' EMA; acrylic copolymer emulsions, such as Neocryl A640; vaccinia or animal poxvirus proteins; subviral particle adjuvants, such as cholera toxin or mixtures thereof.

[0097] The present invention is also illustrated by the following examples: Examples

[0098] Example 1: Animals, viruses, cells, bacteria, and yeasts used in the experimental work of Examples 2 and 3

[0099] Animals.

[0100] 129 / Sv mice with knockout of interferon type I and II receptors (AG129 mice; B&K Universal Ltd. / UK) were housed indoors.

[0101] Viruses and cells.

[0102] Vero-B cells (African green monkey kidney cells; American Type Culture Collection (ATCC) CCL-81) and BHK-21 cells (baby hamster kidney cells; ATCC CCL-10) were obtained from ATCC. All cells were cultured essentially as described in the following reference (De Burghgraeve et al. (2012) PLoS ONE 7, e37244). Yellow fever virus vaccine strain 17D Purchased from Sanofi Pasteur MSD, Brussels, Belgium.

[0103] Bacteria and yeast.

[0104] The bacterial strains used for routine cloning and propagation for pShuttle - BAC amplification were Escherichia coli Top10 (Invitrogen) and Epi300 - T (Epicenter), respectively. Bacteria transformed with full - length flavivirus cDNA plasmids pACNR - FL17DII, pACNR - DENV2, and p4 (see below) were grown at 28 °C, and plasmid DNA production was increased by chloramphenicol amplification. Epi - 300T cells containing the shuttle plasmid were grown at 37 °C and amplified as described below. The yeast strain Saccharomyces cerevisiae YPH500 (genotype: MATαura3 - 52 lys2 - 801_amber ade2 - 101_ochre trp1 - Δ63his3 - Δ200leu2 - Δ1) was grown on a selective medium from Difco - BD Biosciences and Sigma - Aldrich. Transformation of competent yeast cells was carried out using the lithium acetate method, and the yeast was grown at 28 °C.

[0105] The primers used in the experimental section are shown below:

[0106] Table 1: Primer list

[0107]

[0108]

[0109]

[0110]

[0111] Example 2: Preparation of bacterial artificial chromosomes of the present invention containing viral cDNA of dengue virus type 2 (DENV2), dengue virus type 4 (Denv4), and YFV - 17D, respectively

[0112] Bacterial artificial chromosomes containing viral cDNA of dengue virus type 2 (DENV2), dengue virus type 4 (Denv4), and YFV - 17D, respectively, were prepared as described below.

[0113] Materials and methods

[0114] Plasmid constructs (bacterial artificial chromosomes).

[0115] All plasmid constructs were generated by standard techniques and confirmed by Sanger sequencing. The inducible shuttle vector pShuttleBAC / Pme was generated in multiple steps as follows ( Figure 1)。First, the lacZ gene in pBAC / LacZ (Addgene plasmid #13422), a derivative of arabinose-inducible pBeloBAC / oriV (Wild et al. (2002) Genome Res. 12, 1434-1444), was replaced with a synthetic DNA cassette that contains: (i) the simian virus 40 (SV40) promoter / origin (SEQ ID 1) driving the hph gene to provide hygromycin B resistance, (ii) the synthetic cDNA of the genomic ribozyme of hepatitis delta virus (HDrz) (Chadalavada et al. (2007) RNA 13, 2189-2201) (SEQ ID NO 2), and (iii) the Saccharomyces cerevisiae episomal 2μ plasmid origin, and (iv) the TRP1 gene to provide prototrophic growth for tryptophan. Structural module (i) was PCR amplified from pBABE-hygro using primers #334 and #425 (Morgenstern et al. (1990) Nucleic Acids Res. 18, 3587-3996; Addgene plasmid #1765). Structural modules (ii-iv) were PCR amplified from pJet(-) / Trp1_2 micron-YF3'_HDrz_BstE using primers #426 and #231. Plasmid pJet(-) / Trp1_2 micron-YF3'_HDrz_BstE is a derivative of pJet1.2 / blunt (CloneJET PCR Cloning Kit, Fermentas) that contains a fusion of the 3' end of YFV-17D cDNA (containing viral nucleotides 9466 to 10862) to (ii) HDrz (assembled from DNA nucleotides #109, 110, and 111), and (iii+iv) the 2μ-TRP1 sequence that was originally derived from pRE637 (Esteban and Fujimura, (2003) Proc. Natl. Acad. Sci. USA 100, 2568-2573) (using PCR primers #194 and #195). Structural module (i) was fused to (ii-iv), and the fusion was performed by overlap extension PCR and then cloned into the SalI site of pBAC / LacZ to generate the pShuttleBAC / SV40_Hygro_HDrz intermediate construct. Subsequently, the hph gene filler element in pShuttleBAC / SV40_Hygro_HDrz was replaced with a multiple cloning site (SfiI-PmeI-BstEII) in the final pShuttleBAC / Pme shuttle vector ( Figure 1A), the replacement is carried out by inverse PCR across the entire plasmid (using primers #474 and #475), followed by religation using T4 DNA ligase, and then transformation and cloning in E. coli.

[0116] The viral expression constructs pShuttle / YF17D, pShuttle / DV2, and pShuttle / DV4 were generated by homologous recombination in Saccharomyces cerevisiae to introduce the cDNAs of YFV-17D (SEQ ID NO 3), DENV2 strain New Guinea-C (NGC), and DENV4 strain Dominican, respectively, into pShuttleBAC / Pme( Figure 1B). To this end, the viral cDNA was amplified by three rounds of PCR with terminal extension, which contained a -76 bp SV40 promoter / initiation at its 5' end (Ghosh et al. (1981) Proc. Natl. Acad. Sci. USA 78, 100-104) and an 86-nucleotide HDrz sequence at its 3' end (Chadalavada et al., (2007) RNA 13, 2189-2201). The first PCR (10 cycles) used the viral-specific primer combinations #454 plus #457, #455 plus #458, and #856 plus #857, followed by 10 cycles each with SV40- and HDrz-specific #453 plus #456, and the final #453 plus #111 primers. The corresponding viral cDNA templates were pACNR-FLYF1DII (Bredenbeek et al., (2003), cited above), pACNR-DENV2, and p4 (Durbin et al. (2001) Am J Trop Med Hyg. 65, 405-413). Plasmid pACNR-DENV2 is a derivative of pACNR-FLYF17D in which the YFV-17D sequence was replaced with pDVWS601 derived from DENV2 NGC cDNA (Gualano et al. (1998) Gen. Virol. 79, 437-446), and also contains additional translationally silent AgeI and BstEII sites at nucleotide positions 7537 and 10232 of the viral genome, respectively. The linearized vector portion required for recombination was prepared by inverse PCR on pShuttleBAC / Pme, which was pre-linearized with PmeI using primers #552 plus #553. The vector amplicons were treated with DpnI, then gel-purified and transformed into yeast YPH500 to reduce the background formed by the possible remaining uncleaved plasmid template. Yeast clones were grown in the absence of tryptophan to select for recombinant shuttle plasmids. The plasmid DNA recovered from the yeast minipreps was transformed into Escherichia coli Epi300-T cells (Epicentre), and amplified as described in the literature (Wild et al. (2000), cited above) by adding 0.1% (w / v) L-arabinose to an overnight culture diluted 6-fold in fresh LB medium (supplemented with 20 mM magnesium chloride) and growing at 37 °C with vigorous shaking for 6 hours.

[0117] A YFV-17D variant containing a non-transforming lethal mutation (RdRpΔGDD) in its NS5 ORF was generated as follows: a 3.6 kb long BglII-PstI restriction fragment derived from the yeast episomal plasmid (YEp)p404Gal1 / HA-NS5ΔGDD_ura3, which contains the appropriate mutated YFV-17D cDNA downstream of nucleotide 9294, was homologously recombined into pShuttle / YF17D (which was linearized by ClaI (downstream of YFV-17D nucleotide 9656) and KasI (upstream of HDrz nucleotide position +27)) to generate pShuttle / YF17DΔGDD.

[0118] Results

[0119] A series of synthetic DNA constructs (pShuttleBAC series) have been assembled from several DNA building blocks as RNA virus expression plasmids ( Figure 1 ). The commonly used vector construct pShuttleBAC / Pme is a bacterial artificial chromosome that contains a second origin of replication (oriV) for conditional amplification in Escherichia coli, and a yeast 2μ origin of replication and a TRP1 auxotrophic marker for episomal replication in Saccharomyces cerevisiae ( Figure 1 A). The cDNA of the YFV-17D vaccine has been inserted to generate pShuttle / YF17D through homology between the HDV ribozyme and the SV40 promoter present in pShuttleBAC / Pme ( Figure 1 B). The map of pShuttle / YF17D is shown in Figure 8 (SEQ ID NO 4). The cDNA of other flaviviruses has also been inserted. Different from the prior art flavivirus cDNA clones (such as pACNR-FLYF17DII), pShuttle / YF17D shows superior genetic stability in Escherichia coli ( Figure 2 ), and can also be induced to obtain a high plasmid DNA yield.

[0120] Example 3: In vitro and in vivo characterization of the bacterial artificial chromosome of the present invention containing the cDNA of the YFV-17D vaccine

[0121] It was found that the characteristics of YFV-17D expressed by the bacterial artificial chromosome of the present invention are consistent with those of the original vaccine virus (such as replication efficacy, virus yield, and plaque phenotype). In addition, when the naked YFV-71D plasmid DNA was injected i.p. into AG129 mice, it caused the same pathology, morbidity, and mortality as the parental virus. This convenient, robust, and reproducible system provides a DNA vaccine against YFV at low cost without the need for eukaryotic cell cultures or chicken embryo eggs. Cold chain is no longer required, and it can be administered by needle-free means.

[0122] Materials and methods

[0123] In vitro transcription, capping and electroporation.

[0124] The plasmid pACNR-FLYF17DII containing full-length YFV-17D cDNA (Bredenbeek et al. (2003) J. Gen. Virol. 84, 1261-1268) was linearized with AflII and purified by proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. Alternatively, a full-length cDNA template for in vitro transcription (IVT) was prepared by PCR of pACNR-FLYF17DII using primers #173 and #55 and KAPA HiFi DNA polymerase to overcome limited plasmid yields. Run-off RNA transcripts were generated in vitro using Sp6 RNA polymerase (Ribomax Large Scale RNA Production Kit, Promega). The transcripts were capped using purified vaccinia virus 7-methylguanosine transferase (Scriptcap 7mG Capping System, Epigentek) and used for electroporation of BHK-21 cells (using excess total RNA extracted from BHK-21 cells as carrier RNA). The cell culture medium was harvested when cytopathic effects were nearly complete in transfected cells. The medium was cleared of cell debris by centrifugation and then used to prepare a virus stock on BHK-21 cells.

[0125] Plasmid transfection

[0126] Vero-B cells were seeded in 6-well plates to 70% confluence in medium containing 10% fetal bovine serum and transfected the next day with 2.5 μg of plasmid DNA using Transit-LT1 reagent (Mirus) at a DNA-carrier ratio of 1:3. Plasmid pmRFP1 was co-transfected at a ratio of 1:10 as a visual control to ensure the same transfection efficiency. For long-term maintenance and production of virus stocks, the medium was changed after overnight incubation to contain only 2% serum.

[0127] Mapping of the virus RNA termini.

[0128] Analysis of the proper processing of viral RNA derived from the pShuttleBAC vector was performed by rapid amplification of cDNA ends (RACE). 5′ RACE, performed after a new reverse extension / amplification protocol, has been described in detail in Dallmeier and Neyts (2013) Anal. Biochem. 434, 1-3. 3′ RACE of non-polyadenylated YFV-17D genomic RNA was performed essentially as previously described. Briefly, using the activity of T4 RNA ligase 2 (New England Biolabs) in the presence of 15% polyethylene glycol (PEG)-8000, in a total reaction volume of 10 μL, overnight at 16 °C, 5 μL of DNase I-treated total RNA from pShuttle / YF17D transfected Vero cells (approx. 1 μg of RNA) was ligated to a 5′-adenylated and 3′-dideoxycytidine (ddC)-modified adapter #393 (miRNA cloning adapter 1, IDT DNA Technologies). The ligation product was amplified by one-step RT-PCR (Qiagen), using YFV-17D- and adapter-specific primers #391 and #394, respectively. After blunting the 3′ adenine overhangs generated by Taq DNA polymerase, the 265 bp amplicon was gel-purified and cloned into pJet1.2 / blunt (CloneJet PCR cloning kit, Fermentas) and analyzed by Sanger sequencing.

[0129] Detection of intracellular viral replication-competent RNA forms.

[0130] After denaturing agarose gel electrophoresis, Northern blotting and detection of viral replication intermediates were performed essentially as described in Dallmeier et al. (2008) PLoS Pathog. 4, e1000230, with slight modifications to the gel system and probe design used. Briefly, 3 μg of total RNA was heat-denatured and run on a gel containing 1% formamide and 0.01 μg ethidium bromide mL -11% agarose gel (pH 7.0) in 2 mM ethylenediaminetetraacetic acid (EDTA), 5 mM sodium acetate, and 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), followed by capillary transfer onto a positively charged nylon membrane (Roche Diagnostics) in 20x SSC (3 M sodium chloride, 300 mM sodium citrate, pH 7.0) for separation. A strand-specific DIG-labeled single-stranded DNA probe complementary to the 5' end of the NS5 region of YFV-17D (nucleotides 7637 - 8136) was generated using primers #947 and #948 according to Knuchel et al. (2000) J. Histochem. Cytochem. 48, 285 - 294. Hybridization and immunodetection were performed according to the section on membrane hybridization in the DIG Application Manual (Roche Diagnostics).

[0131] Viral quantification was performed by plaque assay and RT-qPCR.

[0132] Infectious virus released from transfected cells was quantified by virus plaque assay on a confluent monolayer of BHK-21 cells 7 days post-transfection using 1% microcrystalline cellulose (Avicell) overlaid in 0.5x maintenance medium as previously described (Kaptein et al. (2010) Antimicrob Agents Chemother. 54, 5269 - 8520). To quantify viral RNA load in infected mouse tissues, snap-frozen autopsy specimens were disrupted in RLT buffer (RNeasy, Qiagen) in a Precellys bead mill and then total RNA was extracted according to the manufacturer's instructions. Quantitative reverse transcriptase PCR (RT-qPCR) for YFV-17D RNA was performed exactly as described in (Kaptein et al. (2010), cited above) using primers and a probe targeting an approximately 150 nucleotide stretch of the NS3 gene and serial dilutions of the same cloned YFV-17D cDNA fragment as standards.

[0133] Immunofluorescence assay.

[0134] The cleared supernatant of pShuttle / DV2-transfected BHK-21 cells 7 days post-transfection (p.t.) was used to incubate sub-confluent Vero-B cultures, which were fixed in 4% paraformaldehyde for 5 days and immunostained essentially as described previously (De Burghgraeve et al. (2012) PLoS ONE 7, e37244). Intracellular E protein expression was detected by DENV2 serotype-specific monoclonal antibody (mAb) 3H5.1 (Millipore) and Alexa Fluor-488-conjugated secondary antibody (Millipore). After DAPI staining, cells were observed using a FLoid cell imaging station (Life Technologies).

[0135] In vivo transfection in mice.

[0136] 10 - 20 μg of plasmid DNA was mixed with 20 μg of calcium carbonate microflowers in 33% propylene glycol as a vehicle (Fumoto et al. (2012) Mol. Pharm. 9, 1962 - 1970) and injected intraperitoneally into adult (ca. 20 g) AG129 mice. Alternatively, a half-dose of live attenuated YFV-17D vaccine ( Sanofi Pasteur MSD, Brussels) was injected intraperitoneally. Body weight and behavior were monitored daily.

[0137] Results

[0138] If transfected into mammalian cells, transcription of correctly processed YFV-17D RNA is initiated by pShuttle / YF17D ( Figure 3B ), which initiates intracellular self-sustaining viral replication ( Figure 3A ). Cells transfected with pShuttle / YF17D eventually secrete infectious virus particles, which are phenotypically indistinguishable from the parental YFV-17D virus with respect to their ability to induce cytopathic effects in tissue culture ( Figure 4 A - C), virus yield, and plaque phenotype ( Figure 4 D + E). Thus, the recombinant virus produced can be considered biologically identical (quantitatively and qualitatively). Similarly, cells transfected with pShutte / DV2 produce recombinant infectious DENV2 New Guinea strain C ( Figure 5 ).

[0139] Intraperitoneal transfection of pShuttle / YF17D in AG129 mice (an established lethal in vivo mouse model of YFV-17D infection) (Meier et al. (2009) PLoS Pathog. 5, e1000614; Thibodeaux et al. (2012) Vaccine 30, 3180 - 3187) resulted in a response indistinguishable from that of a true YFV vaccine Similar virus-induced mortality ( Figure 6 ) and morbidity ( Figure 7 ). Thus, this convenient, robust, and reproducible system enables the development of a DNA vaccine against YFV at low cost without the need for eukaryotic cell cultures or chicken embryo eggs. Cold chain is no longer required, and it can be administered by a needle-free method.

[0140] Example 4: Morbidity and mortality induced by pShuttle / YF17D (DNA-YFVax) in AG129 mice infected by subcutaneous route and by needle-free jet injection of naked plasmid DNA.

[0141] To evaluate the feasibility of other routes for administering pShuttle / YF17D (DNA-YFVax) in addition to the intraperitoneal route, arrays (n = 3) of 9-week-old male AG129 mice (male, weighing 22 - 25 g) were injected with 25 μg of DNA-YFVax in 100 μL of phosphate-buffered saline (PBS) by (i) subcutaneous (s.c.) injection using a syringe and a G27 needle or (ii) transdermal (t.d.) administration by a needle-free jet injector (Injex-30, Injex Pharma GmbH, Berlin, Germany) approved for human clinical use for transdermal application (e.g., insulin or anesthetic). AG129 mice were injected i.p. with 25 μg of DNA-YFVax formulated with calcium carbonate microcrystals in 200 μL of 33% propylene glycol (as previously used as a control group). Morbidity and mortality were scored as before, and the experiment was terminated after 30 days (see Table 2).

[0142] Table 2: Mortality of male AG129 mice injected with DNA-YFVax by different routes.

[0143]

[0144]

[0145] MDD – Mean days to death; n.a. – Not applicable

[0146] At least some of the AG129 mice injected with DNA-YFVax via the s.c. and t.d. routes developed signs of YFV-17D-induced disease (weight loss, ruffled fur, hunchback, flaccid hindlimb paralysis) and had to be euthanized. Most importantly, all mice injected by needle-free jet injection died uniformly within 17 ± 2 days from YFV-17D-induced encephalitis, while all control mice injected i.p. survived during the course of the experiment (30 days). During the observation period, the lack of mortality in the control group (compared to the 13 ± 2 MDD observed in Example 3) could be easily explained by the heavier body weight of the male animals used.

[0147] Note that the use of calcium carbonate as a carrier for DNA-YFVax is not necessarily the most ideal for vaccine delivery; i.e., sometimes some inhibitory effects were even observed in hamsters (see Example 5), depending on the injection route and the variability between different batches of calcium carbonate microflowers. In summary, DNA-YFVax can be successfully delivered in different formulations via different injection routes (including by needle-free jet injection).

[0148] Example 5: Seroconversion in Syrian Golden Hamsters after Immunization with pShuttle / YF17D (DNA-YFVax)

[0149] To evaluate the induction of protective immunity against YFV by DNA-YFVax, a preclinical Syrian golden hamster (Mesocricetus auratus) model (Tesh et al. (2001) J Infect Dis. 183, 1431-1436) was used. For this purpose, groups of male hamsters (8-10 weeks old, 90-100 g) were immunized by intraperitoneal (i.p.) injection of 1 / 5 dose of (100 μL), or, as described previously, 20 μg pShuttle / YF17D (DNA-YFVax) formulated in 200 μL of calcium carbonate microcrystals in 33% propylene glycol. In a repeated format, 10 μg DNA-YFVax was administered alternatively. Blood was collected by cardiac puncture under full surgical anesthesia weekly, serum was harvested by centrifugation, and frozen at -80 °C. Two untreated hamsters served as donors of normal serum. To score immunity relevant to protection, the sera were analyzed (i) by indirect immunofluorescence assay (IIFA), and (ii) by plaque-reduction neutralization test (PRNT).

[0150] Indirect immunofluorescence assay (IIFA). YFV-17D ([ and DNA-YFVax)-specific IgG antibodies in immunized hamster sera were determined by a commercially available YFV IIFA kit (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany, catalogue numbers FI-2665-1005G and FI-2665-1010G) approved for human clinical use (Niedrig et al. (2008) Clin Vaccine Immunol. 15, 177-81), with minor modifications according to the manufacturer's instructions. Hamster sera were diluted 20-, 66-, 200-, 660-, and 2000-fold in sample buffer, and 30 μL of the serum dilutions were applied to YFV-IIFA slides. The slides were incubated at room temperature for 30 min and then washed in PBS containing 0.2% Tween-20 for 5 min. To detect antibodies induced in golden hamsters, FITC-labeled anti-hamster IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc., catalogue number 307-095-003) was diluted 1:50 in PBS containing 2% BSA and used in place of the anti-human secondary antibody provided in the kit. The slides were counterstained with DAPI, and endpoint titers were determined by fluorescence microscopy.

[0151] Plaque reduction neutralization test (PRNT). Neutralizing antibody titers in yellow fever-immunized hamster sera were determined by PRNT. Briefly, 0.5 x 10 6 BHK cells / well were plated in 12-well plates and grown overnight in growth medium (MEM medium supplemented with 10% FCS, 1% sodium bicarbonate, and 1% glutamine). All sera were assayed in triplicate at serial dilutions of 1:20, 1:66, 1:200, 1:660, 1:2000, and 1:6600. 30 μL of the serum dilutions were mixed with 30 μL of test medium (identical to growth medium but containing only 2% FCS) containing 40 plaques formed by YFV-17D virus ( lot G5400P1, passaged once on Vero-B cells). After preadsorbing for 1 h at 37 °C, 440 μL of test medium was added, and 500 μL of each mixture was added to the BHK cells. After incubating at room temperature for 1 h, the cells were washed and overlaid with test medium (supplemented with LMP agarose (Invitrogen) to a final concentration of 0.5%). Subsequently, the cells were incubated at 37 °C for 5 days, fixed with 8% formaldehyde for 2 h, and stained with Giemsa stain. Plaques were counted, and 50% neutralization titers were calculated as described by Reed and Munch (Reed and Muench (1938) Am. J. Hyg. 27, 493-497).

[0152] Overall, the detection of cross-reactive antibodies by IIFA (Table 3) was consistent with the detection of neutralizing antibodies by PRNT (Table 4). Regarding vaccine efficacy, DNA-YFVax was shown to be non-inferior to In both experimental groups, 2 out of 3 individuals (1 / 5 compared to 20 μg DNA-YFVax), and 4 out of 5 individuals compared to 2 out of 4 individuals (1 / 5 compared to 10 μg DNA-YFVax) seroconverted to high titers of YFV cross-reactive and neutralizing antibodies (see Tables 3 and 4). These values are considered to represent fully protective immunity (immunity associated with protection), where a PRNT titer > 40 is protective in the lethal YFV challenge hamster model (Julander et al. (2011) Vaccine 29, 6008-6016). Most importantly, the WHO has stated that a log neutralization index (LNI) > 0.7 is associated with post-vaccination immune protection in primates, and this is also the case for humans (WHO position paper (2013) Wkly. Epidemiol. Rec. 88, 269-283.). This benchmark of a PRNT Log10 titer > 0.7 was exceeded by DNA-YFVax in hamsters by several orders of magnitude, more than 150-fold (see Table 4).

[0153] In addition, compared to the immune response was more homologous in individuals vaccinated with DNA-YFVax, with seroconversion to high PRNT titers consistently detected within 3 weeks, rather than only after 4 weeks, i.e., 3 weeks later than other seroconverters from the group.

[0154] Table 3: YFV cross-reactive antibody responses in Syrian golden hamsters immunized with DNA-YFVax

[0155]

[0156] ND = not detected; *(x / x) = number of seroconverted individuals / number of tested individuals

[0157] Table 4. Neutralizing antibody responses in Syrian golden hamsters immunized with DNA-YFVax

[0158]

[0159]

[0160] ND = not detected

[0161] Example 6: Assessment of the genetic instability of the cloned YFV-17D cDNA.

[0162] (a) Starting materials: Large-scale plasmid preparations of the above-mentioned pACNR-FLYF17DII (Bredenbeek et al. (2003)) and pShuttle / YFV-17D were performed using standard techniques. For this purpose, pACNR-FLYF17DII was transformed into a standard Escherichia coli K12-derived strain, plated on LB-agar containing 100 μg / mL ampicillin, and grown overnight at 28 °C (instead of 37 °C) to facilitate plasmid stability. The small colonies were scaled up and grown in two consecutive overnight cultures at 28 °C with vigorous shaking in LB containing 100 μg / mL to ultimately reach a 1 L batch culture. This batch was grown overnight at 28 °C and finally amplified by adding chloramphenicol to a final concentration of 20 μg / mL and continuing at 28 °C for 8 hours. Similarly, pShuttle / YFV-17D was transformed into the Escherichia coli strain EPI300-T (Epicentre), plated on LB-agar containing 20 μg / mL chloramphenicol, and grown overnight at 37 °C. The latter plasmid was scaled up accordingly, and all growth was carried out at 37 °C in the presence of 20 μg / mL chloramphenicol. The final overnight batch culture was diluted 1:6 into fresh LB medium containing 20 μg / mL chloramphenicol and 0.01% L-arabinose and grown for no more than 6 hours. The plasmids were purified using standard column affinity purification (Qiagen), dissolved in TE (10 mM Tris-HCl, 1 mM EDTA) to a final concentration of 1 μg / mL, and stored at -20 °C.

[0163] (b) Colony growth and size. Both plasmids were transformed into Escherichia coli EPI300-T and streaked on MacConkey agar (2% peptone, 0.5% NaCl, 1% lactose, 0.15% bile salts, 0.003% neutral red, 0.0001% crystal violet, 1.35% agar) containing the appropriate antibiotic as the selective medium. Sterile Zirkonia beads (2.5 mm in diameter) were embedded in the agar to serve as a calibrator for absolute size measurements. For one aliquot of bacteria transfected with pShuttle / YFV-17D, the agar additionally contained 0.01% L-arabinose. After incubation at 37 °C for 16 hours, pictures were taken using a conventional digital camera (Canon Powershot SX10IS) and saved as JPEG files (see Figure 9 ). The pictures were imported into OpenCFU version 3.8.11 for image analysis regarding colony counting and size Geissman (2013) PLoS One. 8, e54072.

[0164] E. coli clones carrying pShuttle / YFV-17D ( Figure 9 B) grew to much larger sizes than clones containing pACNR-FLYF17DII ( Figure 9 A), and their colonies were also more homogeneous than those containing pACNR-FLYF17DII ( Figure 9 A). Unexpectedly, this seemingly lower toxicity of pShuttle / YFV-17D was even maintained in the arabinose-induced state ( Figure 9 C). The larger colonies present in the pACNR-FLYF17DII transformant colonies ( Figure 9 A) were more likely to contain plasmids with mutations that eliminated the cryptic expression of the toxic viral protein (see Example 6).

[0165] Image analysis showed that clones containing pShuttle / YFV-17D had significantly higher homogeneity compared to clones containing pACNR-FLYF17DII (Table 5). In fact, the pACNR-FLYF17D transformants could be clearly divided into at least two subpopulations of different sizes, indicating (i) a large standard deviation from the mean (Table 5), a non-Gaussian size distribution ( Figure 10 A), and (iii) a large difference between, for example, the calculated arithmetic mean and median colony sizes (Table 5). In contrast, the transformants carrying pShuttle / YFV-17D showed more homogeneous colony sizes (Table 5) and a bell-shaped Gaussian size distribution ( Figure 10 B). Unexpectedly, the latter was also fully suitable for the arabinose-induced state of pShuttle / YFV-17D (Table 5, Figure 10 C).

[0166] Table 5. Descriptive statistical scores for transformant colony sizes (in mm)

[0167]

[0168]

[0169] Example 7 Mutation patterns and frequencies during the propagation of cloned YFV-17D cDNA in E. coli.

[0170] To implement the clonal genetic stability of plasmids containing YFV-17D cDNA, the two plasmids described in Example 6 were transformed into Escherichia coli EPI300-T and streaked on MacConkey agar containing the appropriate antibiotic as the selective medium as described above (see Example 6b). The pACNR-FLYF17DII clone was incubated at 28 °C for 24 hours, while the pShuttle / YF17D clone was incubated at 37 °C for 16 hours.

[0171] Two series of 24 - 48 colonies each were picked from each plasmid for growth of the plasmid in 200 μL of liquid medium (LB supplemented with 20 mM MgCl2 and the appropriate antibiotic). One pACNR-FLYF17DII series was selected to start from small colonies (pAS series), and one was selected to start from large colonies (pAL series). For pShuttle / Y17D where no major size difference was observed (see Example 6b), colonies with a reasonable size difference were selected as the two culture series, starting from smaller (pSS series) and larger colonies (pSL series), respectively.

[0172] Bacteria containing the pACNR plasmid were incubated at 28 °C for 24 hours, while the pShuttle colonies were incubated overnight at 37 °C and then incubated for 6 hours in 600 μL of LB containing chloramphenicol and 0.01% arabinose. PCR amplification (GoTaq Green master mix, Promega) was directly performed on portions of these cultures [regarded as plasmids of the 0th generation (P0)] using primers #208 and #94 (corresponding to YFV-17D nucleotides 1 - 940), and #953 and #954 (corresponding to YFV-17D nucleotides 2500 - 3600), respectively.

[0173] The amplicons were affinity purified (Qiagen) and directly sequenced using primers #208 and #953, respectively (Bigdye, Applied Biosystems). Upon analysis, the cDNA regions were expected to contain the cDNAs of previously known virulence determinants, namely the cryptic promoter for abnormal transcription and translation in the viral 5' untranslated region in Escherichia coli (Li et al. (2011), cited above; Pu et al. (2011), cited above), and especially the hydrophobic protein extension in the viral E-NS1 region (Yamshchikov et al. (2001) Virology 281, 272 - 280).

[0174] Another aliquot of each cultured clone was diluted 1 / 100 in fresh medium and grown as described above to obtain the next passage (P1). The latter was repeated up to 10 passages (P10). Plasmids from P1, P3, and P10 were analyzed by PCR and sequencing as described above. For P10, the plasmids were grown in a larger volume of 5 mL medium, and the plasmids were isolated by standard alkaline miniprep. These plasmid minipreps were subjected to (i) PCR analysis (targeting nucleotide regions 1–940 and 2500–3600), followed by agarose gel electrophoresis, (ii) direct sequencing (if the PCR amplicons could be detected), and (iii) restriction digestion analysis with PstI. The sequencing results for P0, P1, P3, and P10 are summarized in Tables 6 and 7. The results of the PCR and restriction analysis for P10 are summarized in Table 8.

[0175] Mutations found in early passages.

[0176] Direct sequencing of plasmids from early passages (low passage numbers P0–P3) of the pAS and pAL series (Table 6) showed a rather high mutation frequency, up to 13% in the initial clone plasmid preparations (see Example 6a). Almost all of the mutations found were nonsense or frameshift (FS) mutations, which were attributed to premature stop codons (PMSt) and single nucleotide deletions / insertions, respectively. These mutations apparently completely abolished the expression of the full-length YFV-17D open reading frame (ORF). In the pSL series, similar mutations were found at P1, although at a lower frequency. Missense mutations that did not abolish the expression of the viral ORF were found in the pSS series at P0.

[0177] In summary, the large-scale preparation of the initial clone plasmids carrying the cDNA of YFV-17D (see Example 6a) contained an easily detectable amount of mutant plasmid variants. In the case of pACNR-FLYF17DII, most of the mutations apparently abolished the expression of the full-length viral polyprotein, resulting in the production of viral RNA from the corresponding non-replicative mutant cDNA. This was the case for more than 10% of all plasmid clones. In the case of pShuttle / YFV-17D, the mutation frequency was lower (less than 10%), and most importantly, a smaller fraction of the mutants would thus constitute a priori non-replicative viruses.

[0178] Mutations found in P10 passages.

[0179] When analyzing the plasmids from the pAL and pAS series at passage P10, most plasmids contained large structural rearrangements, where 10 out of 48 (21%) and 44 out of 48 (92%) plasmid clones were completely unable to amplify the nucleotide region 2500 - 3600, respectively (Table 8). This was accompanied by abnormal restriction maps of the latter mutant plasmids, where DNA up to several thousand bases was lost from the plasmids. It should be noted that all the mutant clones tested still contained the nucleotide region 1 - 940 (Table 6), and thus were more likely to be descendants of the original pACNR - FLYF17DII (Table 8) rather than unrelated contaminants. Deletion of the said toxic cDNA fragment was expected and has been reported in the prior art (Yamshchikov et al. (2001), cited above).

[0180] In contrast, no similar rearrangements and deletions were ever found in the pShuttle - YFV17D series. Here, only 2 out of 48 (4%) showed missense mutations that significantly altered the likelihood of the in - frame ATG start codon (nucleotides 2957 - 2959) in the E - NS1 coding region. This did not abrogate the expression of the viral ORF.

[0181] In summary, repeated passaging of the initial clone pACNR - FLYF17DII (see Example 6a) led to extensive deletions in the viral cDNA and thus to a loss of up to 90% of the functional cDNA, and more importantly, all plasmid clones most significantly disabled the viral RNA produced from the corresponding mutant cDNA. In the case of pShuttle / YFV - 17D, the mutation frequency was much lower (less than 5%), and most importantly, no mutations were observed that would constitute a priori replication - disabled viruses (which would render it unusable, for example, as an attenuated live DNA vaccine).

[0182] Table 6: Mutations that occurred in the cloned YFV - 17D cDNA during E. coli passaging (early passage).

[0183]

[0184] n.a. – Not applicable; PMSt – Produce premature STOP codons (nonsense mutations); FS – Frameshift due to single - nucleotide insertions or deletions; MS – Missense mutations that alter codons; S – Silent synonymous codon exchanges

[0185] *One FS observed in the non - coding region immediately upstream of the start codon

[0186] **A mixed population of all three variant codons, but not the synonymous wild - type codons

[0187] Table 7: Mutations that occurred in cloned YFV-17D cDNA during E. coli passage (late P10 generation)

[0188]

[0189] na – not applicable; DEL – large (kilobase range) deletion, see Table 8 for more details; MSi – possible missense mutation in the in-frame ATG start codon

[0190] *pSS and pSL clones grew to the same colony size when plating overnight cultures derived from P0 and were therefore no longer considered separately at P10.

[0191] Table 8: Major structural changes in plasmids carrying cloned YFV-17D cDNA during passage in E. coli (P10 generation)

[0192]

[0193] nd – not determined

[0194] *pSS and pSL clones grew to the same colony size when plating overnight cultures derived from P0 and were therefore no longer considered separately at P10.

[0195] Example 8: Construction of pShuttle / YFV-JE, pShuttle / YFV-WN and pShuttle / YFV-USU as expression vectors for use as chimeric flavivirus vaccines

[0196] Recombinant chimeric derivatives of YFV-17D have been developed and used as vaccines, wherein YFV-17D serves as a carrier of heterologous antigens (Guy et al. (2010) Vaccine 28, 632-49; U.S. Patent Application 20100278773), for example, carriers of surface glycoproteins of other pathogenic flavivirus antigens, such as the prM and E proteins of Japanese encephalitis virus (JEV) and West Nile virus (WNV), developed into ChimeriVax-JE ( The pShuttle / YFV-17D (DNA-YFVax) of the present invention can be modified so that it can directly start these aforementioned ChimeriVax vaccine viruses from transfected plasmid DNA and can therefore completely replace the attenuated live ChimeriVax-JE containing live viruses. and ChimeriVax-WN20 vaccine.

[0197] Expression of ChimeriVax-JE, pShuttle / ChimeriVax-JE (Figure 11 The BAC of a) was generated by replacing nucleotides 477 - 2477 of the neuroattenuated JEV vaccine strain JE SA14 - 14 - 2 with nucleotides 482 - 2451 of YFV - 17D in pShuttle / YFV17D (Chambers (1999) J. Virol. 73(4), 3095 - 3101; Arroyo et al. (2001) J. Virol. 75, 934 - 942.) plus two adaptative mutations in the NS2A and NS4B genes on the YFV - 17D backbone (Pugachev et al. (2004) J. Virol. 78, 1032 - 1038). This was achieved by homologous recombination and incorporation of three plasmid fragments; two PCR amplicons of pShuttle / YFV17D (nucleotides 7228–481, and nucleotides 3966–7342) and the chimeric YFV - JEV cDNA fragment (IDT Integrated DNA Technologies, Inc., Ghent, Belgium) were made by conventional DNA synthesis methods. The latter contained nucleotides 359–4105 of ChimeriVax - JE( Figure 11 a). The final construct had the sequence shown in SEQ ID NO:5.

[0198] ChimeriVax - WN02 expressing BAC, pShuttle / ChimeriVax - WN02( Figure 11 b) was generated by replacing the prM - E gene region of pShuttle / ChimeriVax - JE with the corresponding region of WNV (NY99 strain), which contains three neuroattenuating mutations in the E protein as described by Monath et al. (2006) (Proc Natl Acad Sci USA. 103, 6694 - 6699), namely L107F, A316V, and K440R. For this purpose, a chimeric YFV - WNV cDNA fragment was prepared by conventional DNA synthesis (IDT Integrated DNA Technologies, Inc., Ghent, Belgium) and recombined into the XhoI (nucleotide 406) and KasI (nucleotide 2477) sites of pShuttle / ChimeriVax - JE. The final construct had the sequence shown in SEQ ID NO:6.

[0199] Example 9: Construction of pShuttle / EV71 as an expression vector for different picornaviruses

[0200] Enteroviruses are (+)-RNA viruses that belong to the picornavirus family of small non-enveloped viruses with RNA genomes of positive sense orientation. Typically, picornavirus genomes are uncapped and instead carry a VPg protein covalently linked to the 5' end. Lacking a cap structure, the internal ribosome entry site (IRES) recruits the cellular translation machinery to the viral RNA for viral protein expression. In principle, picornavirus replication and the production of progeny infectious virus can be initiated intracellularly following heterologous transcription of the viral genome. In the prior art, the viral genome is transcribed from cDNA under the control of a phage promoter and is only transcribed upon co-transfection with the homologous phage RNA polymerase and expressed in producer cells (the two-plasmid system). Similarly, the phage polymerase can be expressed intracellularly following transduction of the corresponding cDNA with a helper virus (e.g., recombinant baculovirus) (Yap et al. (1997) Virology. 231, 192-200).

[0201] As an alternative to the more complex methods in the prior art, the picornavirus cDNA can be expressed intracellularly from a derivative of pShuttle-BAC (the single-plasmid system) for direct initiation of viral replication, as exemplified below for human enterovirus 71 (EV71). To this end, the EV71 genome is cloned as an expression cassette into pShuttle-BAC, which carries a 5' SV40 promoter and a 3' terminal polyA tail, followed by a hepatitis delta virus ribozyme. This is accomplished by PCR amplification of the EV71 cDNA using primers #991 and #992 and re-amplification using primers #453 and #990 to generate the corresponding expression cassette. Different nucleic acid sources can be used as templates for this PCR to appropriately amplify the approximately 7.4 kb long EV71 cDNA; (i) the cloned EV71 cDNA, e.g., Chua et al. (2008) J. Gen. Virol. 89, 1622-1632) and Zhang et al. (2013) Virus Genes. 47, 235-243) or (ii) the reverse transcription product of the full-length genomic RNA of EV71 from any tissue culture or human or animal tissue; or, (iii) the EV71 cDNA can be prepared by conventional gene synthesis methods. Irrespective of the source of the cDNA, the resulting expression cassette will be inserted into pShuttle / BAC-Pme, which has been linearized by digestion with the restriction endonuclease PmeI (preferably by recombination in yeast). The construct has the sequence shown in SEQ ID NO:7, with respect to the EV71 strain BrCr-TR (Arita et al. (2005) J. Gen. Virol. 86, 1391-401).

[0202] Similar strategies can be followed for the cloning of other enteroviruses such as human rhinovirus 14 (hRV14), with only the first set of primers used for the initial amplification of viral cDNA being changed. Suitable primers for hRV14 are #988 and #989.

[0203] Infectious EV71 and hRV14 viruses will be generated by transfection of pShuttle / EV71 and pShuttle / hRV14 plasmids in cultured mammalian cells (e.g., human cervical cancer (HeLa) cells) or by in vivo transfection, respectively. Their attenuated variants can be generated in a similar manner and used as live attenuated vaccines.

Claims

1. Use of a bacterial artificial chromosome (BAC) for the preparation of a DNA vaccine comprising the BAC, wherein the BAC comprises: - an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and - a viral expression cassette comprising a cDNA of an attenuated RNA virus genome and comprising cis-regulatory elements for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious RNA virus in the mammalian cells.

2. The application according to claim 1, characterized in that, For the preparation of a DNA vaccine for preventing RNA virus infection.

3. The use according to claim 1, characterized in that the DNA vaccine is for immunization of humans and / or vertebrates, and the BAC comprises cis-regulatory elements for transcribing the viral cDNA in a human or vertebrate and for processing the transcribed RNA into infectious RNA virus in the human or vertebrate.

4. The application according to claim 1, characterized in that The cDNA of the attenuated RNA virus genome is a chimeric viral cDNA construct of an RNA virus genome, in which a heterologous DNA sequence has been inserted or in which a native viral sequence has been deleted, truncated or mutated.

5. The application according to claim 1, characterized in that, The viral expression cassette comprises - a cDNA of a positive-strand RNA virus genome, - an RNA polymerase-driven promoter located before the 5' end of the cDNA for initiating transcription of the cDNA, and - an element for RNA self-cleavage located after the 3' end of the cDNA for cleaving the RNA transcript of the viral cDNA at a set position.

6. The application according to claim 5, wherein, The RNA polymerase-driven promoter is a eukaryotic RNA polymerase-driven promoter selected from RNA polymerase I, RNA polymerase II and RNA polymerase III promoters.

7. The application according to claim 5, wherein The positive-strand RNA virus is selected from the group consisting of flavivirus, hepatitis C virus, pestivirus, enveloped virus, picornavirus, coronavirus, hepatitis E virus and calicivirus.

8. The application according to claim 1, characterized in that, The viral expression cassette comprises a cDNA of yellow fever virus.

9. The application according to claim 1, characterized in that, The viral expression cassette comprises a cDNA of the live attenuated YFV-17D yellow fever virus vaccine.

10. The application according to claim 1, wherein The viral expression cassette comprises a cDNA of a virus belonging to the group consisting of negative-strand RNA virus, double-stranded RNA virus or ambisense RNA virus.

11. The application according to claim 1, characterized in that The bacterial artificial chromosome further comprises a yeast autonomous replication sequence for shuttling into yeast and retaining the bacterial artificial chromosome in yeast.

12. The application according to claim 11, wherein The yeast ori sequence is a 2μ plasmid origin or ARS1 (autonomous replication sequence 1) or a functional homologous derivative thereof.

13. The application according to claim 6, wherein The RNA polymerase II promoter is a cytomegalovirus immediate early (CMV-IE) promoter, a simian virus 40 promoter or a functional homologous derivative thereof.

14. The application according to claim 5, characterized in that The element for RNA self-cleavage is a cDNA of the genomic ribozyme of hepatitis delta virus or a functional homologous RNA element.

15. The application according to claim 9, characterized in that, The viral expression cassette contains the cDNA of the attenuated live YFV-17D vaccine, wherein one or more cDNA sequences encoding the viral particle surface protein are deleted, truncated or mutated, so that the functional viral particle surface protein of YFV-17D is not expressed, and a cDNA sequence encoding a heterologous protein is inserted into the YFV-17D cDNA.

16. The application according to claim 15, wherein The heterologous protein is the viral particle surface protein of a flavivirus.

17. The application according to claim 15 or 16, characterized in that The viral expression cassette contains the cDNA of the attenuated live YFV-17D vaccine, wherein one or more irrelevant cDNA sequences are inserted, and their expression is one or more heterologous proteins in the viral polyprotein.

18. The application according to claim 1, wherein The viral expression cassette contains viral cDNA, wherein an exogenous cDNA sequence heterologously expressed by the recombinant virus is inserted.

19. A method for preparing a DNA vaccine containing a BAC against an RNA virus, the method comprising the following steps: a) Providing a bacterial host transfected with a BAC, which contains: - an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and - a viral expression cassette containing the cDNA of the attenuated RNA virus genome and containing cis-regulatory elements for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious viral RNA in the mammalian cells, b) Amplifying the BAC by adding a compound that activates the inducible ori c) Isolating the amplified BAC, d) Formulating the BAC into a vaccine.

20. Use of a BAC in the preparation of a vaccine, the BAC containing: - an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and - a viral expression cassette containing the cDNA of the attenuated RNA virus genome and containing cis-regulatory elements for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious viral RNA in the mammalian cells.

21. The use according to claim 20, for the preparation of a vaccine for preventing RNA virus infection.

22. Use of the bacterial artificial chromosome according to claim 1 for maintaining the cDNA of a natural or recombinant RNA virus genome and for propagating a natural or recombinant virus from the cDNA.

23. A DNA vaccine containing a bacterial artificial chromosome (BAC), wherein the BAC contains: - an inducible bacterial ori sequence for amplifying the BAC to more than 10 copies per bacterial cell, and - a viral expression cassette containing the cDNA of the attenuated RNA virus genome and containing cis-regulatory elements for transcribing the viral cDNA in mammalian cells and for processing the transcribed RNA into infectious RNA virus in the mammalian cells.

24. The DNA vaccine according to claim 23, wherein the DNA vaccine is capable of preventing RNA virus infection in a human or vertebrate, and wherein the BAC contains cis-regulatory elements for transcribing the viral cDNA in the human or vertebrate and for processing the transcribed RNA into an infectious RNA virus in the human or vertebrate.

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