(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`AMCAMAA AOAAAA
`
`Z
`
`(43) International Publication Date
`30 October 2014 (30.10.2014)
`
`WIPOIPCT
`
`GD)
`
`International Patent Classification:
`CI2N 15/69 (2006.01)
`
`(21)
`
`International Application Number:
`
`(74)
`
`(81)
`
`PCT/EP2014/058459
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(7)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`25 April 2014 (25.04.2014)
`
`English
`
`English
`
`Priority Data:
`1307528.8
`
`26 April 2013 (26.04.2013)
`
`GB
`
`Applicant: KATHOLIEKE UNIVERSITEIT LEUVEN
`[BL/BE}; K.U. Leuven R&D, Waaistraat 6 - box 5105, B-
`3000 Leuven (BE).
`
`(84)
`
`Inventors: DALLMEIER, Kai; c/o Labo Virology and
`Chemotherapy, Kapucijnenvoer 33 blok i
`- bus 1030, B-
`3000 Leuven (BE). NEYTS, Johan; c/o Dept. Microbio-
`logy and Immunology, Minderbroedersstraat 10 blok x -
`bus 1030, B-3000 Leuven (BE).
`
`(54) Title: BACTERIAL ARTIFICIAL CHROMOSOMES
`
`(10) International Publication Number
`WO 2014/174078 Al
`
`IPLODGE BVBA; Technologielaan 9, B-3001
`Agent:
`Heverlee (BE).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT,
`HN,HR, HU,ID,IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI NO, NZ,
`OM,PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SL SK, SM,
`TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW,
`KM,ML, MR, NE, SN, TD, TG).
`
`[Continued on next page]
`
`>
`
`Size distribution of pACNR series
`
`(57) Abstract: The inventionrelates to the use of a bacterialartificial chro-
`mosome (BAC)for the preparation of a vaccine, wherem the BAC com-
`prises: - an inducible bacterial ori sequence for amplification of the BAC to
`more than 10 copies per bacterial cell, and - a viral expression cassette com-
`prising a cDNA of an allenuated RNA virus genome and comprising cis-
`regulatory elements for transcription of said viral cDNAin mammalian cells
`and for processing of the transcribed RNA into infectious viral RNA.
`
`2
`
`4
`
`5
`
`&
`
`
`
`Feecuency(Numibayofchenest
`
` Radifardtary
`
` alll.
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`46
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`OF
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`40 th az 43 14 45
`B
`8
`7
`fadisjarbleery sstits}
`
`'C
`
`Size distribution of the pShuttle dones + arabinose
`
`Figure 10
`
`
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`wo2014/174078A1|IIMIMNIINUINAYRITIANMITTATANTTCATUA
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`

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`WO 2014/174078 A1IIfMUIMOTIITIT ATTAINTTRTA
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`Declarations under Rule 4.17: —_before the expiration of the time limit for amending the
`
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`—__of inventorship (Rule 4.17(iv)) claims and to be republished in the event of receipt of
`.
`amendments (Rule 48.2(h))
`Published:
`
`
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`— ith ‘tofdescriptionlisti (Rule 5.2(
`— with international search report (Art. 21(3))
`with
`sequence listingpart of description (Rule
`5.2(a))
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`WO 2014/174078
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`PCT/EP2014/0358459
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`BACTERIAL ARTIFICIAL CHROMOSOMES
`
`FIELD OF THE INVENTION
`
`The present invention relates to a plasmid vector system suitable for manipulating,
`
`maintaining and propagating infectious cDNA of RNA virus genomes as well as to
`
`the use of such vector systems.
`
`BACKGROUND OF THE INVENTION
`
`Previously, copy DNA (cDNA) of several flaviviruses and other RNA viruses has been
`
`cloned in different low copy bacterial vectors to overcome their intrinsic toxicity
`
`(due to the large size and cryptic expression of the viral sequences) (Bredenbeek ef
`
`al. (2003) J. Gen. Virol. 84, 1261-1268; Durbin, et a/. (2006) Hum Vaccin. 2, 255-
`
`260; Fan and Bird (2008) J. Virol. Methods. 149, 309-315; Li et a/, (2011) PLoS
`
`One 6,
`
`€18197;
`
`Pu ef a/. (2011) J. Virol. 85, 2927-2941; Rice et al. (1989) New
`
`Biol. 1, 285-296) Almazan et al.
`
`(2008) Methods Mo/ Biol. 454, 275-91). The
`
`cloned cDNAs have been used as templates for production of infectious recombinant
`
`viruses, either by /n vitro synthesis and transfection of
`
`the RNA genomes
`
`(Bredenbeek et a/., 2003, cited above), or by incorporating the viral cDNA in an
`
`expression
`
`cassette, which
`
`comprise
`
`a
`
`promoter
`
`such
`
`as
`
`the CMV-IE
`
`(Cytomegalovirus Immediate Early) promoter allowing the transcription of the viral
`
`RNA from transfected plasmid DNAs (Enjuanes et a/. (2001) J. Biotechnol. 88, 183-
`
`204; Hall ef a/. (2003) Proc. Natl. Acad. Sci. USA. 100, 10460-10464). Such viral
`
`expression cassettes directing the expression of attenuated foot-and-mouth disease
`
`(Ward ef a/. (1997) J. Virol. 71, 7442-7447) and Kunjin viruses (Hall et a/. (2003)
`
`Proc Natl Acad Sci USA. 100, 10460-10464) have been used as experimental DNA
`
`vaccines. Although the low copy number vector systems comprising a viral
`
`expression cassette can be maintained in the bacterial host cell in a stable manner,
`
`they have the important disadvantage that
`
`they only allow the purification of
`
`infective viral
`
`cDNA in amounts that are merely sufficient
`
`for
`
`small
`
`scale
`
`experimental use. Therefore, their use as a routine source of infective viral cDNA is
`
`impossible, for instance in the production of a life cDNA vaccine.
`
`The production of viral DNA vaccines requires a substantial amplification of cloning
`
`vectors to obtain sufficient DNA, but these amplification methods are subject to
`
`severe constraints.
`
`In order to avoid mutations, vectors comprising viral DNA are
`
`propagated under conditions which prevent mutagenic events (recombination,
`
`mutations,
`
`improving mismatch
`
`repair,
`
`and
`
`the
`
`like). Bacterial Artificial
`
`Chromosomes(BAC) are known for their stability and can contain inserts up to 500
`
`kb or more.
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`However the size of such a vector with foreign DNAis a serious burden for bacteria,
`
`and its replication requires a substantial metabolic effort. Furthermore, exhaustion
`
`of nucleotides can lead to increased mutations. Finally, unwanted expression of
`
`foreign DNA (so-called cryptic expression) may occur, which can lead to toxic
`
`recombinant proteins. The production of toxic proteins by cryptic transcripts is
`
`inherent to flavivirus DNA and can only be solved by lowering the copy number of
`
`plasmids.
`
`Indeed,
`
`the higher
`
`the copy number of a vector,
`
`the higher
`
`the
`
`concentration
`
`of
`
`toxic
`
`proteins. As
`
`a
`
`consequence,
`
`bacterial
`
`hosts may
`
`counterselect for mutants wherein these proteins are not expressed.
`
`10
`
`Pu et a/. (2011) J. Virol. 85, 2927-2941, describes in detail various attempts to
`
`solve the intrinsic toxicity of flavivirus cDNA in bacteria. These include the in vivo
`
`ligation of plasmids comprising parts of the viral genome, specific hosts, mutants to
`
`avoid cryptic expression and also low copy number plasmids.
`
`The use of BACs which occur as a single copy in a bacterium provides thus a
`
`15
`
`solution for these problems.
`
`20
`
`25
`
`The low copy number is not a drawback for those applications wherein BAC DNA is
`
`subsequently subcloned or amplified to increase the concentration and wherein the
`
`introduction of some mutations by these techniques is not critical for the envisaged
`
`experiments. However,
`
`such amplification methods cannot be applied in the
`
`manufacture of DNA vaccines, making BACs a non-preferred vehicle for large scale
`
`plasmid preparations for DNA vaccines. Very large scale cultures are required to
`
`obtain substantial amounts of BAC.
`
`The use of inducible BAC vectors is known from Wild et a/. (2002) Genome Res. 12,
`
`1434-1444 whereby the copy number of the BAC increases from 1 copy per cell to
`
`up to 100 copies per cell, or even more. Although this system provides a method to
`
`increase the yield of BAC DNA,
`
`there is a legitimate concern that the strongly
`
`increased activity of
`
`the replication system upon induction will
`
`increase the
`
`mutation frequency. The manufacture of DNA vaccines thus requires a system
`
`wherein a high copy number of a vector is obtained, but wherein replication of the
`
`30
`
`vectors occurs without intolerable introduction of mutations.
`
`SUMMARY OF THE INVENTION
`
`35
`
`The present invention resolves the problem of the amplification of viral cDNA for
`
`the preparation of a vaccine in the vector systems of the prior art by providing a
`
`vector that can be stably maintained in the host cell at low copy number, but can
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`WO 2014/174078
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`PCT/EP2014/058439
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`be significantly amplified by modifying the culture conditions of its host, without
`
`unwanted mutagenesis events.
`
`It is a further object of the present invention to
`
`provide such vector that can be shuttled from and to both a yeast and bacterial
`
`host, thus providing a very versatile system amenable to manipulate the vector in
`
`both a yeast and bacterial genetic system.
`
`The present invention demonstrates, against what was expected, that the inducible
`
`increase in copy number of a BAC vector provides DNA which has a surprisingly low
`
`mutation rate. Even more surprisingly, the few mutations that occur are mostly
`
`frameshift mutations or
`
`stop codons,
`
`leading to truncated versions upon
`
`expression. Point mutations, which are either without effect or which lead to
`
`modified amino acids, are underrepresented.
`
`This unexpected effect
`
`leads to the advantageous effect that high amounts of
`
`vector are obtained and that the limited amount of errors that does occur leads to a
`
`non-functional viral genome, rather than to a mutated viral genome of which the
`
`virulence can be increased comparedto originally cloned construct.
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`10
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`15
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`The present
`
`invention provides a bacterial artificial chromosome comprising an
`
`inducible bacterial ori sequence, which allows to induce the amplification of said
`
`bacterial artificial chromosome to a high copy number, for instance by modifying
`
`20
`
`the culture conditions of the bacterial host. Bacterial artificial chromosomes as used
`
`herein further comprise a viral expression cassette comprising a cDNA of a RNA
`
`virus genome flanked by cis-regulatory elements, which upon introduction of said
`
`bacterial artificial chromosome in a mammalian cell promote the transcription of
`
`said viral cDNA and allow for the processing of the transcribed RNA into infectious
`
`viral RNA. Viral
`
`cDNA contained in the viral expression cassette can either
`
`correspond to that of a 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. Typically heterologous
`
`DNA sequences encode one or more peptides/proteins, which are heterologously
`
`expressed by the recombinant virus, following the introduction in a mammalian cell
`
`of a bacterial artificial chromosome according to the present invention that contains
`
`a viral expression cassette comprising such chimeric viral cDNA. The bacterial
`
`artificial chromosome can further comprise a yeast autonomously replicating
`
`sequence for shuttling to and maintaining said bacterial artificial chromosome in
`
`yeast. The possibility to shuttle to and maintain the bacterial artificial chromosome
`
`according to the present invention in a yeast cell provides the advantage that it is
`
`amenable for genetic manipulation in both the yeast and bacterial genetic systems.
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`4
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`As such the present
`
`invention provides a
`
`single vector system suitable for
`
`manipulating, maintaining and propagating infectious cDNA of RNA virus genomes.
`
`In absence of a stimulus of the inducible ori, the bacterial artificial chromosomes
`
`according to the present invention can be used for archiving and stable cloning of
`
`infectious viral CDNA in a bacterial host, while in presence of such stimulus said
`
`cDNAcan readily be amplified and subsequently isolated to be used. The bacterial
`
`artificial chromosomes according to the present invention are particularly useful in
`
`the development, stable maintenance and production of viral cDNA to be used as a
`
`life vaccine against RNA viral pathogens. Alternatively, said bacterial artificial
`
`chromosomes are used for
`
`the maintenance and propagation of native or
`
`recombinant viruses from cDNA,for instance for research purposes.
`
`In the present invention, BACs with an inducible bacterial ori are used for the
`
`preparation of a vaccine of a viral expression cassette comprising cDNA of an RNA
`
`virus and cis regulatory elements for the transcription of viral CDNA in mammalian
`
`cells and processing of the transcribed RNA into infectious viral RNA.
`
`Surprisingly, the generation of multiple copies of the BAC comprising the viral DNA
`
`did not lead to the disadvantages that are known to occur with high copy number
`
`systems.
`
`Generally,
`
`toxic proteins are produced in bacterial systems, due to the cryptic
`
`expression of viral sequences. Indeed, the generation of flavivirus infectious clones
`
`has been traditionally hindered by the toxicity of their full-length cDNAs in bacteria.
`
`Various approaches 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
`
`which relates to the insert which is cloned into the BAC, and hampers bacterial
`
`growth and metabolism. Bacteria with mutants wherein cryptic expression does not
`
`take place have a growth advantage and will overgrow the original population. In
`
`the prior art
`
`this is
`
`reflected by the size of bacterial colonies. Non-mutated
`
`constructs produce toxic proteins and typically small colonies are obtained. Mutated
`
`constructs produce less or no toxic proteins which results in the occurrence of
`
`larger colonies.
`
`Based upon this prior art knowledge it was expected that the induction of plasmid
`
`replication would thus result in an increase of toxic transcripts and a concomitant
`
`increase in mutants wherein cryptic expression does not take place.
`
`Surprisingly the inducible replication system appears to be insensitive to the
`
`toxicity of cryptic proteins. Indeed, compared to the prior art high copy number
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`systems,
`
`the bacterial colonies are somewhat
`
`larger, which indicates that
`
`the
`
`bacterial host is less sensitive to eventual toxic proteins. More importantly, very
`
`large colonies, representing mutated plasmids are not encountered.
`
`The finding that
`
`the inducible system is
`
`insensitive towards toxic proteins is
`
`unexpected. There were no indications in the prior art which indicated that this
`
`system would not be sensitive to toxic proteins (or perhaps that toxic proteins are
`
`not produced).
`
`A further disadvantage of the inducible system is inherent to the generation of
`
`10
`
`multiple copies of the BAC. Indeed the authors of the inducible system explain that
`
`the most important feature of BAC clones is their stability resulting from their very
`
`low copy number. Wild et a/. (2002) cited above shows that the copy number can
`
`be lowered even further by the addition of glucose. This single copy state improves
`
`stability of maintenance of BAC libraries
`
`by
`
`reducing the opportunity for
`
`15
`
`intracellular recombination between clones. This demonstrates that the inducible
`
`system as published by Wild et a/. does not
`
`lower the changes of unwanted
`
`recombination events which occur as soon as multiple copies of a BAC are present
`
`in a host cell.
`
`It
`
`is understood by the skilled person that
`
`induction and the
`
`subsequent high copy number will reintroduce recombination events. Consequently,
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`20
`
`the skilled person would refrain of using such systems for DNA preparations that
`
`are intended to be used for vaccination purposes.
`
`In the present invention BACs have been amplified in the inducible ori system and
`
`the amplified BAC has been tested for recombination events. Contrary to what
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`25
`
`would be expected, recombination events are rare.
`
`Furthermore, apart from testing for recombination events, the amplifies BACs have
`
`been tested as well for the presence of other mutations. The mutation frequency
`
`which was encountered was very low, and moreover, the fraction of missense is
`
`surprisingly lower
`
`than theoretically expected. Mutations,
`
`if occurring
`
`are
`
`predominantly nonsense or frameshift mutations leading to non-functional viral
`RNA.
`
`The present invention allows a significant upscale of DNA vaccine production. For
`
`example, the main manufacturers of the life-attenuated Yellow Fever vaccine are at
`
`present unable to meet the existing demands. With the technology of the present
`
`invention, it will be possible to produce DNA vaccinesat significant lower costs and
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`PCT/EP2014/058439
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`higher quantities than the current life-attenuated vaccine, thereby fulfilling a long
`
`felt need.
`
`Also for other viral diseases such as JEV, WNV, measles, rubella and HIV vaccines
`
`there is a need for a vaccine preparation platform which can provide sufficient
`
`amounts of DNA.
`
`A first aspect of the invention relates to uses of a bacterial artificial chromosome
`
`(BAC) for the preparation of a vaccine, wherein the BAC comprises:
`
`- an inducible bacterial ori sequence for amplification of said 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 transcription of said viral cDNA in
`
`mammalian cells and for processing of the transcribed RNA into infectious RNA
`
`virus,
`
`Embodiments of cDNAs of an attenuated RNA virus genome are a chimeric viral
`
`cDNA construct of an RNA virus genome, wherein a heterologous DNA sequence has
`
`been inserted or wherein a native viral sequence has been deleted, truncated, or
`
`10
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`15
`
`mutated.
`
`Embodiments of the viral expression cassette comprise
`
`-
`
`-
`
`20
`
`a CDNAofa positive-strand RNA virus genome,
`
`a RNA polymerase driven promoter preceding the 5’ end of said cDNA for
`
`initiating the transcription of said cDNA, and
`
`-
`
`an element for RNA self-cleaving following the 3’ end of said cDNA for
`
`cleaving the RNA transcript of said viral cDNA at a set position.
`
`Embodiments
`
`of positive-strand
`
`RNA virus
`
`are
`
`flaviviruses,
`
`hepaciviruses,
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`25
`
`pestiviruses,
`
`togaviruses,
`
`picornaviruses,
`
`coronaviruses,
`
`hepeviruses,
`
`and
`
`caliciviruses.
`
`In a typical embodiment
`
`the viral expression cassette comprises a cDNA of a
`
`yellow fever virus, for example a cDNA ofthe life-attenuated YFV-17D yellow fever
`
`30
`
`virus vaccine.
`
`In other embodiments the viral expression cassette comprises a cDNA of a virus
`
`belonging to the group of negative-strand RNA viruses, double-strand RNA viruses
`
`or ambisense RNA viruses.
`
`In specific embodiments the bacterial artificial chromosome further comprises a
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`35
`
`yeast autonomously replicating sequence for shuttling to and maintaining said
`
`bacterial artificial chromosome in yeast.
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`An example of a yeast ori sequence is
`
`the 2u plasmid origin or the ARS1
`
`(autonomously replicating sequence 1) or
`
`functionally homologous derivatives
`
`thereof.
`
`In certain embodiments the RNA polymerase driven promoter is an RNA polymerase
`
`II promoter, such as Cytomegalovirus Immediate Early (CMV-IE) promoter, the
`
`Simian virus 40 promoter or functionally homologous derivatives thereof.
`
`In other embodiments the RNA polymerase driven promoter is an RNA polymerase I
`
`or IIT promoter.
`
`Examples of an element for RNA self-cleaving is the cDNA of the genomic ribozyme
`
`of hepatitis delta virus or functionally homologous RNA elements.
`
`In a particular embodiment the viral expression cassette comprises a cDNA of the
`
`life-attenuated YFV-17D vaccine, wherein one or more of the cDNA sequences
`
`coding for the virion surface proteins are either deleted, truncated, or mutated so
`
`that such functional virion surface protein of YFV-17D is not expressed and wherein
`
`a cDNA sequences coding for a heterologous protein is inserted in the YFV-17D
`
`cDNA. An example of such heterologous protein is a virion surface protein of a
`
`flavivirus.
`
`Embodiments of a viral expression cassette comprises a cDNA ofthe life-attenuated
`
`YFV-17D vaccine, wherein one or more unrelated CDNA sequences are inserted to
`
`be expressed as one or more heterologous protein within the viral polyprotein.
`
`In other embodiments the viral expression cassette comprises a viral CDNA wherein
`
`foreign cDNA sequences are inserted to be heterologously expressed by the said
`
`recombinant viruses.
`
`A further aspect relates to methods of preparing a vaccine against RNA viruses
`
`comprising the steps of: a) providing a bacterial host transfected with a BAC as
`
`described
`
`in
`
`the
`
`first
`
`aspect
`
`and
`
`in
`
`the various
`
`embodiments
`
`thereof
`
`b) amplifying the BAC by adding a compound which activates said inducible ori
`
`c) isolating the amplified BAC,
`
`d) formulating the BAC into a vaccine.
`
`A further aspect relates to a BAC as described in the first aspect and in the various
`
`embodiments thereof for use as a vaccine.
`
`Another aspect of the present invention relates toa BAC as described in the first
`
`aspect and in the various embodiments thereof for use in the prevention of a RNA
`
`virus infection.
`
`A further aspect relates to uses of a BAC as described in the first aspect and in the
`
`various embodiments thereof as a life DNA vaccine.
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`Another aspect relates to uses of a BAC as described in the first aspect and in the
`
`various embodiments thereof for the propagation of native or recombinant viruses
`
`from said cDNA.
`
`A further aspect relates bacterial artificial chromosome (BAC) as a BAC as described
`
`in the first aspect and in the various embodiments thereof for the preparation of a
`
`vaccine.
`
`DETAILED DESCRIPITON OF THE INVENTION
`
`Legendsto the figures
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`10
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`15
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`25
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`30
`
`Figure 1. Generation of the pShuttleBAC series of RNA virus expression
`
`plasmids. (A) Sketch showing the construction of the pShuttleBAC/Pme as starting
`
`vector construct.
`
`(B) Sketch showing the construction of the pShuttleBAC/Pme
`
`derived flaviviral expression vectors by insertion of the viral cDNAs by homologous
`
`recombination between the SV40 promoter (SV40p) and HDV ribozyme (HDrz).
`
`Figure 2. Enhanced plasmid stability of pShuttleBAC constructs in E.coli.
`
`Generic plasmid maps showing the principle layout of prior art flaviviral cDNA
`
`plasmids (A) and the new pShuttleBAC series of vectors (B, DNA-YFVax). Upon
`
`transformation in E. co/i a single colony of each (A) and (B) was grown overnight at
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`37 °C and plated on selective media. Generally constructs of type A grow into much
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`smaller colony sizes as those of type B (Figure 2C and D, respectively). Moreover,
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`type A constructs give rise to progeny of a wide range of colony sizes (histogram
`
`for normalized colony diameters, right panel of figure 2C)
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`indicative for selection
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`and segregation of mutant plasmid clones that occasionally render the cDNAs less
`
`toxic to £. coli. Clonal analysis identified multiple possible underlying mutations,
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`including transposon insertion in the viral E/NS1 region. By contrast, plasmid clones
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`comprising type B constructs of the pShuttleBAC series do not segregate and show
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`a more homogenous colony size even after repeated passage in E. coli indicating
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`high genetic stability.
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`Figure
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`3A. Detection of
`
`replicative intermediates of YFV-17D RNA
`
`replication after
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`transfection of Vero-B cells with pShuttle/YF17D
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`(wildtype,
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`WT)
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`and
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`its
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`replication
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`deficient
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`derivative
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`
`
`
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`pShuttle/YF17DAGDD (AGDD) by Northern§blot. Antisense oriented
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`antigenomes, (-)-RNA (A upper panel, 11 kb) and sense orientated viral genomes,
`
`(+)-RNA (A lower panel, 11kb), could be detected 5 days after transfection only in
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`35
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`WTtransfected cells. Ongoing replication in the presence of actinomycin D (ACD),
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`an inhibitor of DNA directed RNA synthesis, confirms that after initial
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`launching of
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`YFV-17D genome transcription from pShuttle/YF17D, viral
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`replication continues
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`autonomously in a plasmid independent manner.
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`Figure 3B. Detection of proper YFV-17D RNA transcript processing by 5’-
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`and 3’-RACE (rapid amplification of cDNA ends). pShuttle/YF17D launches
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`transcription of nascent YFV-17D RNAs (bold cases in figure 3B) that start and end
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`with proper 5‘ and 3’ ends (upper and lower panel, respectively) as confirmed by
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`rapid amplification of cDNA ends (RACE).
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`Figure 4A-C. Similar CPE induced by YFV-17D of different origin. YFV-17D
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`viruses derived from /n vitro transcribed and capped RNA using pACNR-FLYF17DII
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`as a template (A) or harvested after plasmid DNA transfection of pShuttle/YF17D
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`(B) produce an identical virus induced cytopathic effect (CPE) on BHK-21 cells 5
`
`days postinfection (d p.i.); C, uninfected cells for comparison.
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`Figure 4D-E. Similar plaque phenotype of YFV-17D of different origin. YFV-
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`17D viruses derived from in vitro transcribed and capped RNA using pACNR-
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`FLYF17DII as a template (D) or harvested after plasmid DNA transfection of
`pShuttle/YF17D (E) produce a comparable number (5 days p.i. 3x 10° plaque
`forming units (pfu) mL vs 2 x 10° pfu mL") and identical morphology of plaques
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`(diameter 6,4 + 0,7 mM vs 6,1 + 1,1 mM; n = 8, p-value = 0,6 by t-test) on BHK-
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`21 cells.
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`Figure
`
`5.
`
`Detection
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`of
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`infectious
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`recombinant
`
`DENV2
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`by
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`immunofluocrescence assay (IFA). Recombinant DENV2 NGC produced by BHK-
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`21 cells transfected with pShuttle/DV2 shows dose dependent infection of Vero-B
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`cells, visualizes as viral foci by immunofluorescence staining for the viral E protein 5
`
`days p.i. (A, undiluted supernatant, B, 100-fold diluted supernatant, C, uninfected
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`10
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`15
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`20
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`25
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`cell control).
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`Figure 6. Survival of AG129 mice infected with Stamaril (open squares) or
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`transfected with pShuttle/YF17D (crosses). About 10 to 12 days after i.p.
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`challenge,
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`interferon type I and II receptor deficient (AG129) mice start losing
`
`weight and develop a uniform set of symptoms, namely ruffling of the fur, tremor
`
`and flaccid hind limb paralysis. Control animals transfected with the replication
`
`deficient NSSAGDD plasmid variant (AGDD, open circles) show no pathogenesis.
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`However, they stay susceptible to a second Stamaril ® challenge (filled triangle) 20
`
`days after initial
`
`transfection and then die within a comparable timeframe and
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`showing similar symptoms. Plasmid DNAs were transfected i.p. using calcium
`
`carbonate microflowers in 33%propylene glycol as carrier.
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`Figure 7. Morbidity (A) and detection of YFV-17D RNA (B) from infected
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`AGi129 mice.
`
`(A)
`
`If
`
`infected either with Stamaril ® or
`
`transfected with
`
`pShuttle/YF17D (pYF17D), AG129 mice loose about 20 % of body weight before
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`they have to be euthanized after an average of 12 to 13 days (MDD, mean day to
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`death). By contrast, pShuttle/YF17DAGDD (AGDD})
`
`transfected mice gain weight,
`
`before they are challenged with Stamaril ® (AGDD + 2° Stamaril) and die from
`
`YFV-17D infection within about two weeks.
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`(B) Comparable amounts of YFV-17D
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`RNA can be detected by means of qRT-PCR in brain samples of AG129 mice from
`
`(A) collected at death.
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`Figure 8. Map of pShuttle/YF17D (synthetic construct #1). Legend: SV40p:
`
`Simian virus 40 promoter/origin, YFV-17D: yellow fever virus vaccine strain 17D
`
`cDNA, HDVrz: hepatitis delta virus ribozyme cDNA; 2u: S. cerevisiae 2-micron
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`origin; TRP1: TRPi gene conferring prototrophic growth towards tryptophan;
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`parABC: partitioning genes of F-plasmid; repE: repE gene of F-plasmid; oriS: origin
`
`of F-plasmid; oriV: origin of plasmid RK2; CmR: chloramphenicol resistance gene.
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`Figure 9. E. coli colony growth after tranformation with different YFV-17D
`
`cDNA vectors. (A) —. coli EPI-300T colonies after transformation with pACNR-
`
`FLYFi7DII and growth for 16 h at 37° C. A colony size distribution with two
`
`subpopulations with major
`
`size differences
`
`can be observed, microcolonies
`
`(diameter smaller than 0.2 mm) and macrocolonies (diameter around 0.4 mm).
`
`Microcolonies
`
`represent
`
`the majority.
`
`(B+C) &.
`
`co/f EPI-300T colonies after
`
`transformation with pShuttle/YFV17D. Plating on plates without inducer (B), or with
`
`0.01% L-arabinose (C) for induction of of high copy replication mediated by the
`
`inducible high-copy origin. Large black circles are zirkonia beads of 2.5 mm
`
`diameter embedded into the agar to serve as calibrators.
`
`Inset
`
`figures are
`
`schematic line drawings representing the colony outlines observed in each setting.
`
`Figure 10. Size distribution of £. coli colonies after transformation with
`
`different YFV-17D cDNA vectors.
`
`(A)
`
`&£.
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`co/i EPI-300T colonies
`
`after
`
`transformation with pACNR-FLYF17DII.
`
`(B+C) &.
`
`co/i EPI-300T colonies after
`
`transformation with pShuttle/YFV17D. Plating on plates without inducer (B), or with
`
`0.01% L-arabinose (C)
`
`for induction of high copy replication mediated by the
`
`inducible high-copyorigin.
`
`Figure ita. Map of pShuttle/ChimeriVax-JE. pShuttle/ChimeriVax-JE contains
`
`following the SV40 promotor/origin nt 1-481 and 2452-10862 of YFV-17D,
`
`in which
`
`477-2477 of neuroattenuated JEV vaccine strain JE SA14-14-2 are inserted. The
`
`second last two last amino acids of the JEV E-ORF are mutated from a histidin to a
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`glycin codon to generate a KasI site,
`
`the NS2A and NS4B-ORFs contain two
`
`adaptive G4055a and G7349a mutations found in Imojev® changing a methionine
`
`to valine in the YFV-17D NS2A and a lysine to glutamine in the NS4B ORFs,
`
`respectively. Additional silent mutations generate restriction markers at positions
`
`406 (XhoI), 4009 (BstEII), and 7315 (NhelI).
`
`Figure 11b. Map of pShuttle/ChimeriVax-WN. pShuttle/ChimeriVax-WN contains
`
`following the SV40 promotor/origin nt 1-481 and 2452-10862 of YFV-17D,
`
`in which
`
`477-2477 of a neuroattenuated derivative of WNV strain NY-99 are inserted. The
`
`second last two last amino acids of the WN E-ORF are mutated from a histidin to a
`
`glycin codon to generate a KasI site,
`
`the NS2A and NS4B-ORFs contain two
`
`adaptive G4055a and G7349a mutations found in Imojev® changing a methionine
`
`to valine in the YFV-17D NS2A and a lysine to glutamine in the NS4B ORFs,
`
`respectively. Additional silent mutations generate restriction markers at positions
`
`406 (XhoI), 4009 (BstEII), and 7315 (NheI).
`
`Figure 12. Map of pShuttle/EV71. pShuttle/EV71 contains the cDNA of EV71 strain
`
`BrCr-TR (Genbank AB204852.1) inserted between the SV40 promotor/origin at its
`
`5’ terminus, and a 30 nt long polyA repeat plus the hepatitis delta virus ribozyme at
`
`its 3’ end.
`
`Figure 13 depicts sequences with SEQ ID NO 1-7
`
`Definitions
`
`The term "bacterial artificial chromosome (BAC)" refers to a plasmid DNA
`
`construct used to clone DNA sequencesin bacterial cells, such as F. coli. Typically
`
`DNA sequences ranging from 30,000 to about 300,000 base pairs can be inserted
`
`into a BAC. The BAC, with the inserted DNA, can be taken up by bacterial cells. As
`
`the bacterial cells grow and divide, the BAC DNA is stably maintained within the
`
`bacterial cells at a very low copy number per bacterial cell, preferably not
`
`exceeding 3 copies per cell, such as at a single copy per cell. The replication of a
`
`BAC is
`
`initiated at an origin of
`
`replication (ori) sequence,
`
`typically the oriS
`
`sequence. This replication is stringently regulated by gene products, generally the
`
`repE and/or repF, encoded by the BAC. The BAC further encodes for proteins, such
`
`as parA, B and C, directing the partitioning of the BAC copies to the daughter cells
`
`during division. Typically, BAC vectors further comprise selectable markers, such as
`
`antibiotic resistance or reporter enzyme markers, such as lacZ allowing for blue
`
`white selection. An example of a generally used BAC is the pBeloBacil (Shizuya et
`
`al. (1992) Proc. Natl. Acad. Sci. USA 89, 8794-8797.) The sequence of this vector
`
`was reported at GenBank Accession Number U51113. pBeloBaci1 is a circular
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`plasmid that includes oriS, the repE gene that produces a protein that initiates and
`
`regulates the replication at oriS, and partition genes par A, B, and C. For selection
`
`pBeloBac11 includes a chlo