# Patent

## Title
Reverse genetics of negative-strand RNA viruses in yeast

## Abstract

The present invention relates to a methodology for the generation of infectious ribonucleoparticles (RNPs) of negative-strand RNA viruses, and in particular of non-segmented negative-strand RNA viruses in yeast, especially in budding yeast. Accordingly, the patent application relates to a recombinant yeast strain suitable for the rescue of infectious non-segmented negative-strand RNA virus particles or infectious virus-like particles. The invention also relates to the use of the recombinant yeast to prepare vaccine seed and to the use of the produced RNPs or RNPs-like to prepare vaccine formulations. It also concerns the use of the recombinant yeast for the screening of libraries of DNA.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Divisional of copending application Ser. No. 12/865,567, filed on Apr. 28, 2011, which was filed as PCT International Application No. PCT/IB2009/000373 on Jan. 30, 2009, which claims the benefit under 35 USC. §119(a) to patent application Ser. No. 08/290,087,9, filed in EUROPE on Jan. 31, 2008, all of which are hereby expressly incorporated by reference into the present application.


TECHNICAL FIELD OF THE INVENTION
The present invention relates to a methodology for the generation by reverse genetics of infectious ribonucleoprotein complexes (RNPs) also designated ribonucleocapsids or ribonucleoparticles of negative-strand RNA viruses, and in particular of non-segmented negative-strand RNA viruses (Mononegavirales), in yeast, especially in budding yeast. More generally, this method provides means to implement the transcription and the replication of negative-strand virus RNA in yeast, to produce RNPs or derivatives.
Accordingly, the patent application relates to a recombinant yeast strain suitable for the expression of infectious RNPs of non-segmented negative-strand RNA viruses or RNPs encapsidating minigenomes, recombinant minigenomes or recombinant genomes, derived from the genome of non-segmented negative-strand RNA viruses (designated RNPs-like).
The invention also concerns the viral RNPs or RNPs-like obtainable from the recombinant yeast strain of the invention.
The invention also relates to the DNA constructs, especially to vectors providing expression of said viral RNPs or RNPs-like, suitable for use in the preparation of the recombinant yeast strain.
Many advantages may be seen in the preparation of RNPs or RNPs-like from yeast including the safety of the yeast as expression cells, the possibility to obtain high yield and productivity, the fact that the involved media for the yeast culture are not expensive one, the stability of the strains and the durability of the cells (yeast is able to survive over a very large if not indefinite period of time).
Vectors according to the invention encompass cloning vectors, expression vectors, especially used as complementation vectors or replacement vectors (the latter being also named genome vectors). The vectors of the invention may be designed for use as vaccine vectors or target vectors or as screening vectors.
By providing a new methodology for the preparation of infectious RNPs of non-segmented negative-strand RNA virus or RNPs-like particles, the invention further enables the preparation of new formulation of said immunogenic compositions or vaccine compositions.
The invention also provides means for the screening and the identification of antiviral compounds, or for the screening and the identification of cellular factors associated with viral replication or transcription, and for the study of virus-host interactions.
BACKGROUND INFORMATION
Negative-strand RNA viruses are associated with many diseases in humans such as influenza, rabies or measles. Other well known examples are the viral infections caused by Mumps virus, Respiratory Syncytial virus, Human Parainfluenza virus types 1-4, Ebola virus, Marburg virus, Hanta virus, Nipah virus, Vesicular Stomatitis virus, Rinderpest virus and canine Distemper virus (2). There are still many diseases associated with negative-strand RNA viruses, such as Parainfluenza virus, responsible for 30-40% of all acute respiratory infections in children and infants, for which no effective drugs or vaccines exist. Some of these viruses may re-emerge from animal species or reappear as new agents of bioterrorism. Moreover, measles virus still remains one of the leading causes of death by infectious agents worldwide. This, together with the insufficient therapy options today, has increased markedly the demand for new antiviral strategies.
Among negative-strand RNA viruses, the non-segmented negative-strand RNA viruses (Mononegavirales) are enveloped viruses that have genomes consisting of a single RNA molecule of negative sense. This order includes viruses with high medical relevance, such as the Rhabdoviridae, Paramyxoviridae, Filoviridae, and Bornaviridae families which are considered for the purpose of the invention. Although these viruses have distinct biological properties, their replicative and transcriptional system is conserved. Accordingly, the description of the invention which follows which is provided by reference to particular examples of viruses in this order, should be understood as providing disclosure of the corresponding features for other viruses of the order of Mononegavirales unless technically irrelevant for the skilled person.
The use of mammalian cells and reverse genetic tools to study negative-strand RNA virus has constituted a major advance for the comprehension of the biology of this group of pathogens and for the generation of vaccines (3). While positive-strand RNA or DNA viruses can be easily obtained in vitro after transfection of their engineered infectious cDNA or DNA in appropriate cells, the negative-strand RNA viruses cannot be rescued directly by reverse genetics from their cDNA. The genome of negative-strand RNA viruses is not able to initiate in vitro an infectious cycle because it does not code directly for proteins. Both transcription and replication require a transcriptase-polymerase enzymatic complex contained in the nucleoproteins encapsidating the viral genome (RNPs). Thus, the generation of recombinant negative-strand RNA viruses from cDNA involves reconstitution of active RNPs from individual components: RNA and proteins, to assemble nucleocapsids.
A remarkable set of work from numerous laboratories has allowed the establishment of different systems for rescuing almost all negative-strand RNA viruses from their cDNA (3). In contrast to the viruses with segmented genomes, the RNPs of non-segmented negative-strand RNA viruses (Mononegavirales) are tightly structured and contain, in addition to the nucleoprotein (N), the assembly and polymerase cofactor phosphoprotein (P) and the viral RNA polymerase large protein (L). The first infectious Mononegavirales, the rabies rhabdovirus, was recovered from cDNA in 1994 (4). The approach involved intracellular expression of rabies virus N, P, and L protein, along with a full length RNA whose correct 3′ end was generated by the hepatitis delta virus (HDV) ribozyme. A transcript corresponding to the viral antigenome (positive strand) rather than to genome (negative strand) was used to avoid a severe antisense problem raised by the presence of N, P, and L sequences in full-length RNAs. In this system, the essential helper proteins were provided by a replication-competent vaccinia vector encoding the phage T7 RNA polymerase to drive T7-specific transcription of plasmids encoding the required proteins N, P and L. Similar systems allowed recovery of infectious rabies viruses, VSV, as well as the Paramyxoviridae Sendai virus, HPIV-3 and measles virus (3).
However, in previously described methods for generating negative-strand RNA viruses by reverse genetics from infectious cDNA it is often relied on transformed mammalian cell lines that would be inappropriate for GMP (good manufacturing production) production of clinical vaccine lots, according to the certification of international safety agencies. The development of an alternative reverse genetics system for Mononegavirales in yeast would therefore be extremely advantageous. Production in yeast has especially many advantages on the industrial scale.
In order to provide an alternative to the use of mammalian cells, the inventors have considered yeast strains, especially Saccharomyces strains.
The straightforward genetics of the budding yeast Saccharomyces cerevisiae and its high conservation of basic cellular processes with higher organisms make it an excellent tool for fundamental research and drug development (5). Yeast is frequently used to produce vaccines based on recombinant proteins or virus-like-particles. For example, the efficient and safe prophylactic HPV vaccine GARADASIL® is composed of recombinant HPV VLPs antigens that are produced in yeast (6). The advantages of yeast-based vaccines are the ease of manipulation and cultivation of S. cerevisiae and the use of the fermentation process to provide large amounts of viral particles. The budding yeast is a eukaryotic organism that can be also used as a simpler system to replicate live mammalian viruses and thus, to provide substrates to produce viral live-attenuated vaccines (7). Indeed, yeast has been used successfully as a model host to replicate a wide range of viruses. These include DNA and RNA viruses that infect plants, mammals and humans (8) (9) (10) (11). However, there is not yet such technology for negative-strand RNA viruses.
Viruses that replicate in yeast comprise two families of viruses: (i) DNA viruses including dsDNA (Human papillomavirus (11), Bovine papillomavirus (12) and ssDNA (Mung bean yellow mosaic India virus) and (ii) positive strand RNA viruses family including Brome mosaic virus (8), Carnation Italian ringspot virus (13), Tomato bushy stunt virus (9), Flock House virus (10) and Nodamura virus (14). Viruses that replicate in yeast have positive-strand RNA genomes and share a common replication process: the genomic positive-strand RNA genome serves as mRNA and as template for replication. This feature facilitates the replication and the transcription of this RNA virus family in yeast. Experimentally, the strategies used to replicate positive-strand RNA virus in budding yeast have some common traits. The viral RNA-dependent RNA polymerase and viral replication essential cofactors, if required, are expressed from yeast promoters. Next, positive-strand RNA genome is introduced into yeast cells either by spheroplast transformation or by in vivo transcription from a yeast expression vector. The integrity of the 5′ and 3′ ends of the RNA is respected because they harbor important replication elements. This can be achieved by using a ribozyme to generate the exact ends. The genomic RNA contains a reporter gene, which expression is dependent on viral replication system. Stable expression of all the components of the replicative system is achieved by using yeast plasmids carrying selectable markers. The expression of the reporter gene, which depends on viral RNA replication, indicates the presence of RNA virus replication (7).
Yeast technology and the so-called <<humanized yeast>> systems have a high impact in the understanding of the host/virus-related molecular process and are potential tools to discover novel medicinal compounds (7, 15). Many studies using genome-wide screening, DNA and protein micro arrays, deletion mutants libraries, expression profiling, genome wide synthetic lethal screens and gene dosage effects have allowed the identification in yeast of multiple host factors that affect positive-strand RNA/DNA replication and are involved in unexpected novel cellular pathways (16).
DISCLOSURE OF THE INVENTION
The invention discloses such an alternative system for reverse genetics of non-segmented negative-strand RNA viruses.
The invention is directed to methods and tools which enable the preparation of virus RNPs or virus RNPs-like of non-segmented negative-strand RNA viruses, and in particular enables the preparation, by reverse genetics, of RNPs-like which express heterologous polypeptides or peptides, the term “heterologous” meaning that the polynucleotide is not one of said non-segmented negative strand RNA virus used to provide the core of the RNPs-like. The prepared virus-RNPs or virus-RNPs-like can be preserved and stored in yeast strains, thus in particular avoiding the cost of cryoconservation of virus particles or virus-like particles, which are particularly relevant in the field of vaccine industry.
The invention also provides means suitable to produce in great amount, in particular out of yeast fermentors, RNPs of RNPs-like of several viruses of a great research, industrial, medical and/or vaccine interest. These RNPs or RNPs-like can be used as seeds to reproduce the viral particles or virus-like particles of the said non-segmented negative-strand RNA virus, in cell culture in conditions that would allow their use as immunogens, preferably as vaccine components. Accordingly, an object of the present invention is a new method for generating reproducibly and with high efficiency, infectious RNPs or RNPs-like from any non-segmented negative-strand RNA virus, in particular for such viruses which replicate in the cytoplasm of cells or in the nucleus of cells, as illustrated in the present application for Schwarz strain of measles virus (MV), starting from cloned cDNA of said virus RNA or derivatives thereof. Such a method is suitable to be carried out in yeast, especially budding yeast as illustrated by Saccharomyces cerevisiae. 
The invention accordingly relates to a recombinant yeast strain, suitable for the expression of infectious non-segmented negative-strand RNA virus Ribonucleocapsids (RNPs) or infectious RNPs-like.
The recombinant yeast are obtained by transformation, i.e., by the introduction of nucleic acid in the cells. Integration of the introduced nucleic acid in the chromosomes of the yeast does not happen or is not required to carry out the invention.
In a particular embodiment of the invention, the recombinant yeast strain, suitable for the expression of infectious non-segmented negative-strand RNA virus Ribonucleocapsids (RNPs) or infectious RNPs-like, is obtained after being transformed with at least the following expression vector:

    
    
        (i) At least one genome vector, comprising, as an insert under the control of regulatory expression sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding the full-length (+) strand sequence (antigenome) of a non-segmented negative-strand RNA virus or encoding part of said antigenome, said cDNA thus comprising, in the 5′ to 3′ orientation, a sequence encoding the Trailer sequence of the genome said virus, one or more polynucleotide(s) which code(s) sequence(s) of interest cloned in sense orientation with respect to the cis-acting sequences of said virus, a sequence encoding the Leader sequence of the genome of said virus, and wherein said cDNA is flanked, in the cloned DNA molecule, by autocatalytic ribozyme sequences enabling the recovery of RNA after transcription and replication, having original 3′ and 5′ ends, of the genome of said non-segmented negative-strand virus.
    
    


In another particular embodiment of the invention, the recombinant yeast strain, suitable for the expression of infectious non-segmented negative-strand RNA virus Ribonucleocapsids (RNPs) or infectious RNPs-like, is obtained after being transformed with at least the combination of the following expression vectors:

    
    
        (i) at least one genome vector, comprising, as an insert under the control of regulatory expression sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding the full-length (+) strand sequence (antigenome) of a non-segmented negative-strand RNA virus or encoding part of said antigenome, said cDNA thus comprising, in the 5′ to 3′ orientation, a sequence encoding the Trailer sequence of the genome said virus, one or more polynucleotide(s) which code(s) sequence(s) of interest cloned in sense orientation with respect to the cis-acting sequences of said virus, a sequence encoding the Leader sequence of the genome of said virus, and wherein said cDNA is flanked, in the cloned DNA molecule, by autocatalytic ribozyme sequences enabling the recovery of RNA after transcription and replication, having original 3′ and 5′ ends, of the genome of said non-segmented negative-strand virus,
        (ii) one or more trans-complementation vectors comprising, under the control of regulatory expression sequences functional in yeast, nucleotide sequences which enable said vector(s) to collectively express the proteins necessary for the synthesis of the viral transcriptase complex of said non-segmented negative-strand RNA virus used in the genome vector, and enable assembly of the ribonucleocapsid (RNPs) or assembly of RNPs-like of said virus, which are functional in yeast for the replication and transcription, said vector(s) further comprising, under the control of regulatory expression sequences functional in yeast, a selectable marker.
    
    


Alternatively, the cDNA of the non-segmented negative-strand RNA virus encodes its (−) strand (genome), as a full-length sequence or part thereof. It is however noted that antigenomic sequence has been shown to be more efficiently used in the preparation of cDNA constructs for use in reverse genetics. Accordingly, the description which is provided herein with respect to the (+) strand of the viral RNA is transposable to the (−) strand.
In embodiments where the cDNA encodes either part of the antigenome of said non-segmented negative-strand RNA virus, or part of the genome of said non-segmented negative-strand RNA virus it necessarily comprises cis-active sequences necessary for replication and transcription, i.e., it comprises the Leader and Trailer sequences. Such a cDNA may further comprise additional regulatory region(s) of the gene transcription of the non-segmented negative-strand RNA virus such as a Promoter and/or a Terminator sequence of a gene of said virus. Such a cDNA may further or alternatively also comprise gene sequences or their coding sequences, intergenic regions or part thereof, derived from the non-segmented negative-strand RNA virus, which are contained in the full-length RNA of said virus.
In the cDNA used in accordance with the invention, the Leader sequence of said non-segmented negative-strand RNA virus comprises one viral promoter of said non-segmented negative-strand RNA virus.
The Trailer sequence also comprises a terminator sequence of the transcription.
The cDNA sequence used in accordance with the invention may further comprise, in a particular embodiment, coding sequences of the genes of said non-segmented negative-strand RNA virus and possibly also regulatory sequences of such genes (promoter, terminator, intergenic region). As stated above, in a particular embodiment, the cDNA does not contain the coding sequences of all the genes or does not contain the coding sequences of any of the viral genes.
In a particular embodiment of the invention, the cDNA comprises, as a substitution fragment of part or all of the coding sequences of the full-length antigenomic or genomic RNA or as an addition fragment in said full-length or in said part of the full-length viral RNA, a DNA insert encoding a polypeptide or a peptide of interest.
In order to insert such a DNA fragment encoding a polypeptide or a peptide of interest, it may be necessary or appropriate to insert an Additional Transcription Unit (ATU) in the cDNA. An ATU may comprise a transcription stop sequence, a poyadenylation sequence and a transcription start sequence as contained in an intergenic region of the non-segmented negative-strand RNA virus, such as for example in the N-P intergenic region, P-M intergenic region or H-L intergenic region when reference is for example made to the measles virus, or in corresponding intergenic regions of other Mononegavirales.
The DNA fragment encoding said polypeptide or said peptide of interest is advantageously inserted in an intergenic region of said the cDNA encoding the full-length antigenomic strand (or genomic strand) or part thereof if it contains such intergenic region.
The intergenic regions in the genome of the virus and especially the intergenic region between the Nucleoprotein and the Phosphoprotein is appropriate to insert said DNA fragment. Other intergenic regions may be considered also such as the intergenic region between the P and M gene, or between the H and L gene.
The DNA molecule comprising the cDNA can be introduced in the yeast by various methods such as electroporation, as insert in a vector including classical plasmids or recombinant plasmids carrying the genetic information required to replicate in eukaryotic yeast cells etc. . . . .
The genome vector(s) and the trans-complementation vector(s) of the invention is (are) especially plasmid(s).
In the recombinant yeast, the complementation vector(s) collectively provide expression of the proteins necessary for a non-segmented negative-strand RNA virus to express the transcriptase complex required to assemble ribonucleocapsids (as N protein associated with the RNA, i.e., N: RNA, and P and L proteins) necessary for transcription and replication of the virus, in the cytoplasm of cells and/or in their nucleus, especially in yeast.
Accordingly, the invention provides in a particular embodiment, one complementation vector encoding one unique protein of the transcriptase complex. In another embodiment, one vector encodes two or more of the proteins required for the preparation of the transcriptase complex.
Especially, the invention relates to a set of complementation vectors, where one vectors contains a sequence encoding the N protein of a non-segmented negative-strand RNA virus and a sequence encoding the P protein of the same non-segmented negative-strand RNA virus; and another complementation vector encoding the sequence of the L protein, in particular of the same non-segmented negative-strand RNA virus.
The transcriptase complex contains the Nucleoprotein (N) which associates to the viral genomic RNA, the Phosphoprotein (P) and the Polymerase (L), which harbour the catalytic activity. The transcriptase complex thus defined may alternatively comprise functional derivatives, especially functional fragments of said proteins.
If one trans-complementation vector comprises more than one, especially two nucleotide sequences coding for distinct proteins, each nucleotide sequence is under the control of a regulatory expression sequence. In particular, the coding sequences for said proteins are cloned in antisense orientation with respect to each other.
A regulatory expression sequence contains a promoter and if appropriate a terminator sequence for transcription.
The vector for complementation also comprises an origin replication (ori) of yeast, such a ori of the yeast 2 μm plasmid.
Suitable plasmids for the preparation of complementation vectors and/or the genome vectors of the invention are for example pESC or pYES yeast vectors.
Suitable yeast promoters for use in the complementation vectors and/or in the genome vectors are especially inducible promoters such as galactose-inducible promoters, such as GAL1 to GAL10 promoters.
The promoters controlling the transcription of the protein(s) of the transcriptase complex and the promoter controlling the expression of the selectable marker, present on a same vector are preferably different.
Suitable selectable markers for the construction of the complementation vector(s) are HIS, LEU, TRP, ADE or URA yeast genes encoding respectively histidine, leucine, tryptophane, adenine and uracile amino-acids necessary for the growth of yeasts in a medium which is devoid of said amino acids.
In the genome vector, the cDNA present in the cloned molecule—including when it is recombined with the DNA fragment which it may contain to encode a heterologous polypeptide or peptide of interest—should comply with the rules that govern the efficient replication of the non-segmented negative-strand virus from which it derives, if any.
In a particular embodiment, the invention provides the technology for the generation of a yeast strain (W303-NPLMV) which expresses the viral proteins N, P and L respectively coding for the nucleoprotein, the phosphoprotein and the polymerase of the attenuated Schwarz strain of measles virus (MV). These three components are necessary and sufficient to allow the transcription and replication of non-segmented negative-strand RNA viruses in human cells. The invention demonstrates that the yeast strain W303-NPLMV can be used as a helper-trans-complementary cell to reconstitute de novo infectious RNPs from measles virus after transformation by synthesized cloned full-length infectious viral antigenomic/genomic cDNA.
Thus, in the case of most viruses of the genus Paramyxoviridae, in particular for measles virus (MV), said cDNA (possibly including also a cloned heterologous DNA fragment) complies with the “rule of six” meaning that the total number of the nucleotides of the sequence is an integral multiple of six.
It is specified that the rule of six applies, in principle, to the rubulaviruses, respiroviruses and morbilliviruses, although in some strains, it might apply with less stringency. Especially, the rule of six does not apply for VSV and RSV.
In a particular embodiment of the invention, the genome vector also comprises a gene for a selectable marker, especially an auxotrophy marker gene, suitable for use in yeast, under the control of an expression control sequence. Genes encoding HIS, LEU, TRP, URA in yeast as stated above are appropriate. This is especially desirable when the genome vector comprises a minigenome.
In an embodiment, the genome vector comprises a reporter gene (i.e., a coding sequence for a reporter molecule such as an antibiotic, or a gene that confers resistance to an antibiotic such as KANMX4 conferring resistance to kanamycin) in the cDNA, under the control of viral expression control sequences. Many reporter genes may be inserted in the genome vector (such as reporter gene conferring resistance to an antibiotic (e.g. KANMX4 gene) and a reporter gene suitable to visualize gene expression (e.g. the eGFP gene).
In another embodiment, the reporter gene is replaced by the sequence encoding the selectable marker.
The invention relates in particular to a recombinant strain, suitable for the expression of infectious non-segmented negative-strand RNA virus Ribonucleocapsids (RNPs) or infectious RNPs-like, wherein the yeast is transformed with the following expression vectors:

    
    
        (i) a genome vector, especially a plasmid genome vector, comprising, as an insert operatively linked with expression control sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding the (+) strand full-length sequence (antigenome) of said non-segmented negative-strand RNA virus and wherein said cDNA is flanked, in the cloned DNA molecule, by autocatalytic ribozyme sequences enabling the recovery of mRNA transcripts and of antigenomic RNAs of said non-segmented negative strand or derivatives thereof and
        (ii) a genome vector, preferably a plasmid genome vector, comprising, as an insert under the control of regulatory expression sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding part of the antigenome of said non-segmented negative-strand RNA virus including in the 5′ to 3′ orientation, a viral Terminator sequence, a polynucleotide which codes a selectable marker cloned in sense orientation with respect to the cis-acting sequences of said virus and the Leader sequence of said virus,
        (iii) one or more trans-complementation vectors comprising, under the control of regulation expression sequences functional in yeast, nucleotide sequences which enable said vector(s) to collectively express the proteins necessary for the synthesis of the viral transcriptase complex of said non-segmented negative-strand RNA virus, and enable assembly of the ribonucleocapsid (RNPs) of said non-segmented negative-strand virus or assembly of RNPs-like comprising recombinant RNA derived from viral RNA of a non-segmented negative-strand RNA virus, wherein the RNPs or RNPs-like are functional for the replication and transcription, each of said vector(s) further comprises, under the control of regulatory expression sequences functional in yeast, a selectable marker and wherein
    
    


in said vectors all the selectable markers are different from each other.
In a particular embodiment of the invention, the genome vector of (ii) above does not comprise the trailer sequence of the non-segmented negative-strand RNA virus.
The DNA molecule for cloning into the genome vector (especially plasmid vector) and in particular the cDNA which it contains may be obtained by any appropriate method, including by PCR elongation or by synthesis. For the cDNA, subgenomic fragments of said virus cDNA could be obtained by PCR elongation or synthesis and cloned.
In the recombinant yeast, the complementation vector(s) collectively provide expression the proteins necessary for a non-segmented negative-strand RNA virus to express the transcriptase complex required to assemble ribonucleocapsids (as NP: RNA, P and L) necessary for transcription and replication of the virus in the cytoplasm of cells. Thus the proteins necessary to express the transcriptase complex through the transcomplementation vectors encompass or consist in the N, P and L proteins. It may also be functional derivatives thereof, i.e., modified proteins with respect to the native one, to the extent that they enable the assembly of the nucleocapsids.
A functional derivative may be derived from the known native viral protein one of the following features:

    
    
        the nucleic acid encoding the functional derivative hybridizes in high stringency conditions with a nucleic acid encoding the wild-type (reference) RNA polymerase or with the N protein and the P protein of an identified non-segmented negative-strand RNA strain or virus. High stringency conditions are defined by Sambrook et al. in Molecular Cloning: a laboratory manual (1989). These conditions of high stringency encompass: use a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation conditions of 50% formamide, 6×SSC at 42° C. and washing conditions at 68° C., 0.2×SSC and 0.1% SDS. Protocols are known to those having ordinary skill in the art. Moreover, the skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to experimental constraints;
        the nucleic acid encoding the functional variant presents at least 80%, preferably 90%, more preferably 95% or even 99% similarity with a native nucleic acid encoding the RNA polymerase, the N protein or the P protein, said similarity being calculated over the entire length of both sequences;
        the nucleic acid encoding the functional derivative differs from a native nucleic acid encoding the RNA polymerase, the N protein or the P protein by at least one nucleotide substitution, preferably 1, 2, 3, 4 or 5 substitution(s), optionally conservative substitutions (nucleotide substitution(s) not altering the amino acid sequence), by at least one nucleotide deletion or addition, preferably 1, 2, 3, 4 or 5 nucleotide(s) deletion or addition.
        The functional derivative therefore may be a fragment of the native protein, whose nucleic acid sequence complies with one of the above definitions.
    
    


A fragment is defined in the present application as a part of the full-length RNA polymerase, of the N protein or of the P protein, as long as the fragment has the same activity as the entire protein from which it is derived, at least as a ribonucleoprotein complex (RNP complex) as disclosed herein. In a particular embodiment, the fragment represents at least 70%, particularly 80%, and more particularly 90% or even 95% of the full-length protein.
Accordingly, where reference is made herewith to RNA polymerase, N or P proteins or to their coding sequences, the description similarly applies to their functional derivatives as defined herein.
In order to have a functional genome vector, it is not required that the promoter contained in the DNA molecule, outside the sequence specifying the cDNA, be in the same orientation as the cis-acting sequences, especially the promoter, of the cDNA. Rather, it is necessary that the coding sequences or the Open Reading Frames (ORF) comprised in the cDNA be in a proper orientation with respect to the cis-acting sequences, especially the promoter, of the cDNA, i.e., be operatively linked to said promoter.
In a particular embodiment of the invention, the non-segmented negative-strand RNA is one selected among the group of Rhabdoviridae, Paramyxoviridae, Filoviridae, Bornaviridae and Flaviviridae.
The invention concerns in particular the use of viruses of the genus Vesiculovirus, Lyssavirus, Morbillivirus, Respirovirus, Rubulavirus, Pneunovirus, Ebola-like virus.
Among these viruses, the Measles Virus (MV) is one of the Paramyxoviridae which is much preferred to carry out the invention and has especially been used to illustrate the invention. Strains of measles virus, which are approved strains for vaccination such as the Schwarz strain, are particularly advantageous. Other viruses of interest are Rous Sarcoma Virus (RSV) or Human Parainfluenza Virus type 2 (HPIV-2), or HPIV-3.
An approved vaccine strain of a virus suitable for carrying out the invention is especially characterized by the fact that it has proven to be attenuated and stable after numerous cell passages, over a long period of time, and accordingly does not elicit detrimental secondary effects when administered to a host, for elicitation of protection against said virus. An approved vaccine strain may be one qualified as such because it complies with the following provisions: safety, efficacy, quality, reproducibility after review in laboratory.
According to a preferred embodiment, the yeast strain of the invention is a recombinant strain suitable for the expression of infectious RNPs of non-segmented negative-strand RNA virus or infectious RNPs-like, in particular when said virus is a measles virus, and the complementation and the genome vectors according to the invention are further characterized as follows:

    
    
        (i) the complementation vectors are capable of expressing the nucleoprotein (N), the Phosphoprotein (P) and the Polymerase (L) or functional derivatives thereof which enable assembly of functional ribonucleoproteins (RNPs) comprising the transcriptase complex and,
        (ii) the genome vector comprises a cloned molecule which comprises a cDNA encoding the full-length (+) strand (antigenome) of said virus and wherein said cDNA is framed by autocatalytic ribozyme sequences.
    
    


In a particular embodiment, the genome vector thus defined further comprises a heterologous polynucleotide encoding a polypeptide or a peptide of interest, cloned in the cDNA.
In another particular embodiment of the recombinant yeast defined herein, the recombinant yeast strain is suitable for the expression of infectious RNPs or RNPs-like of non-segmented negative strand RNA virus, in particular when said virus is a measles virus and the complementation and the genome vectors of the invention are further characterized as follows:

    
    
        (i) the complementation vector(s) are capable of collectively expressing the nucleoprotein (N), the Phosphoprotein (P) and the Polymerase (L) or functional derivatives thereof which enable assembly of functional ribonucleoproteins (RNPs) or RNPs-like comprising the transcriptase complex and,
        (ii) the genome vector comprises, in an insert, a cloned DNA molecule which comprises a cDNA encoding a fragment of the (+)strand (antigenome) of said virus, including the cis-acting Leader and Trailer sequences, and furthermore one or more coding sequences, or ORF(s), heterologous to said virus, the expression of which is sought.
    
    


The particular embodiments which have been disclosed above or are described hereafter concerning the design of the various vectors, apply to these particular yeast strains.
In a particular embodiment, the nucleoprotein (N), phosphoprotein (P) and polymerase (L) are expressed by several plasmid expression vectors, wherein each vector comprises a cloned polynucleotide consisting of or containing viral coding sequences for one of the N, P or L proteins under the control of a promoter suitable for expression in yeast, especially an inducible promoter. In such a case, each of the vectors comprises a selectable marker operatively linked to regulatory expression sequences including a promoter and possibly a terminator; said promoter is different from the promoter controlling the expression of the viral coding sequence present on the same plasmid.
In another particular embodiment of the invention, the backbone of all the complementation vectors, especially of the plasmids, is identical and it is only the insert which is chosen to express either the N, P or L proteins, and the selectable marker which are different in each vector.
In a particular embodiment, the nucleoprotein (N), and the phosphoprotein (P) are expressed by a single expression vector, especially a single plasmid, and the polymerase (L) is expressed by another expression vector, especially a plasmid, said expression vectors comprising cloned polynucleotides consisting of viral coding sequences for the N and P proteins or for the L protein respectively, under the control of a promoter suitable for expression in yeast, especially an inducible promoter.
In a particular embodiment of the invention, the backbone of the vectors, e.g., of the plasmids, used to prepare the genome vector(s) and the trans-complementation vector(s) to carry out the transformation of the yeast cells are the same. In another embodiment of the invention, at least some of them are different independently from each other.
The inserts for cloning in the vectors may be prepared by any available methods, including by PCR elongation from a template, or by synthesis.
Promoters used for the design of the complementation vector, especially inducible promoters, may be identically used in the construction of the genome vector(s). The selectable markers are usually available as commercial DNA polynucleotides or cassettes and may be used either for the genome or for the complementation vectors.
In the genome vector(s), the inserted DNA molecule (insert) is cloned in the plasmid under the control of expression control sequences including a promoter and a transcription terminator sequence suitable for expression in yeast, in sense or in antisense orientation with respect to said promoter.
The N, P and L proteins expressed by the trans-complementation vectors may be of the same or of different viruses. They may be independently of each other of the same or of a different virus than the one providing the cDNA. The L protein expressed is advantageously of the same virus as the virus providing the RNA for the preparation of the cDNA.
A particular preferred yeast strain of the invention is one as defined in the present application, which is further characterized as follows:

    
    
        (i) In one or in all the genome vectors, the cDNA in the cloned DNA molecule comprises at least one of the following polynucleotides:
        
            (a) as Leader and/or Trailer sequences, the leader and/or trailer sequences of an MV virus, in particular of the Schwarz strain or any other vaccine strain;
            (b) an additional Promoter sequence derived from the MV virus, e.g., the promoter of the nucleoprotein (N), phosphorprotein (P) or polymerase (L) of an MV virus, in particular of the Schwarz strain or any other vaccine strain; and/or
            (c) an additional Terminator sequence derived from the MV virus, e.g., the terminator of the polymerase (L) or of the nucleoprotein (N), or the phosphorprotein (P) of an MV virus, in particular of the Schwarz strain or any other vaccine strain; and/or
        
        
        (ii) The cDNA of the cloned molecule is framed by different or identical autocatalytic ribozymes selected among hammerhead ribozyme and hepatitis delta virus ribozyme.
    
    


The sequences of the Schwarz strain of the MV virus, for the preparation of all the vectors of the invention, may be obtained from the particular pTM-MVSchw plasmid deposited at the CNCM (Collection Nationale des Microorganismes Paris, France) under No I-2889 on Jun. 12, 2002, which contains the cDNA encoding the full-length antigenomic strand of the virus.
Sequences of a Schwarz strain of MV virus has also been disclosed in WO 98/13505.
The sequence of the cDNA from the virus, if said virus is an MV strain, may advantageously be prepared starting from particles of a commercial batch of vaccine strains such as the vaccines available for the Schwarz strain.
Primers are especially described in the examples of the present application, in order to isolate the N gene, P gene or L gene of the MV Schwarz strain, for example starting from the sequence of the insert in the pTM-MVSchw plasmid.
The leader sequence is characterized in that, in the native genome of the non-segmented negative-strand virus, it separates the 3′ end of the virus genome (negative strand) from the beginning of the first viral gene which it contains, whereas the trailer sequence is characterized in that, it separates the 5′ end of the genome of the virus from the end of the last viral gene, which it contains. The leader and the trailer sequences both contain promoter sequences and the trailer further contains a terminator sequence.
In non-segmented negative-strand RNA viruses, leader and trailer sequences are approximately 50-nucleotide long, and have a common sequence in each genera of viruses. Leader and trailer sequences of MV are illustrated in the examples.
MV sequences illustrated in the examples and figures may be replaced by corresponding sequences of other non-segmented negative-strand RNA viruses, and the proposed GAL or the viral promoters used in the constructs may be substituted by any other promoter functional in yeast or any other viral promoter respectively. The illustrated terminator sequence may also be substituted by any other viral terminator sequence. The gene coding for resistance to an antibiotic (such as Kanamycine) or expression reporter genes may also be replaced by a marker gene or a gene encoding a polypeptide or a peptide of interest.
The autocatalytic ribozymes in the genome vector(s) of the invention enable to achieve cleavage of the cDNA encompassed within the cloned DNA molecule with exact ends, reproducing the ends of the cDNA prior to its insertion in the DNA molecule, i.e., the terminations of the leader and trailer sequences originating from the non-segmented negative-strand RNA virus genome.
The invention relates especially to a construct described in the examples (point II) which comprises the recombinant full-length antigenomic sequence of the measles virus or a construct derived therefrom, wherein the heterologous genes KANMX4 and/or eGFP are substituted by coding sequences of interest expressing polypeptides or peptides of interest or are deleted.
One particular construct prepared on the basis of the construct provided in FIG. 8 is a full-length recombinant MV antigenome, or a plasmid containing the same, wherein one or more heterologous sequence, especially an heterologous coding sequence is inserted between the L and F genes of MV or/an between the P and N genes of MV. In such a construct the non viral promoter sequence is selected for expression in yeast.
In a preferred embodiment of the invention, the recombinant yeast strain is transformed with at least one of the plasmids pCM101, pCM103, pCM104, pCM105, pCM106, pCM112, pCM113, pCM201, pCM224, pCM225, pCM226, pCM227 deposited at the CNCM (Collection Nationale de Culture de Microorganismes, Paris, France) on 31 Jan. 2008, under numbers No. I-3896, I-3897, I-3898, I-3899, I-3900, I-3901, I-3902, I-3903, I-3904, I-3905, I-3906, I-3907 respectively.
In another particular embodiment of the invention, the recombinant yeast is transformed with a plasmid selected among pCM402, pCM476, pCM503, pCM603, deposited at the CNCM on 30 Jan. 2009 under numbers CNCM I-4117, CNCM I-4118, CNCM I-4119, CNCM I-4120 respectively.
Among yeast strains suitable for carrying out the invention, Saccharomyces Cerevisiae is one of the preferred strains and strain W303 is most preferred.
Other examples of suitable yeast strains for the realisation of the invention include Pichia Pastoris, or Saccharomyces Pombe, especially Schiso Saccharomyces Pombe. 
For the purpose of the invention, the yeast strains encompass either mature cells or spheroplasts of yeasts. The yeast may be budding yeast.
The phenotype of the yeast is adapted if necessary to take into account the selectable markers present in the vectors of the invention. Accordingly, the yeast used to carry out the invention should not be capable to express the components, especially the nutrients, which are encoded by the vectors as selectable markers. In a particular embodiment, prior to recombination with the vectors of the invention, the yeasts are thus silenced for expression of markers selected among tryptophan (Trp), histidin (His), leucin (Leu), uracil (Ura) and adenine (Ade), if said vectors express these markers.
The invention thus relates to a recombinant yeast strain yCM112, yCM113 or yCM226 deposited at the CNCM on 31 Jan. 2008 under numbers NO. I-3908, I-3909, I-3910 respectively and to recombinant yeast strain yCM403 deposited at the CNCM on 30 Jan. 2009 under number I-4121.
These recombinant strains are recombinant Saccharomyces Cerevisiae. 
It has been specified that the invention especially relates to a yeast strain wherein, in at least one of the genome vector(s), the cDNA encoding a nucleic acid derived from a genome of said non-segmented negative-strand RNA virus is devoid of all the viral genes or is devoid of all the viral coding sequences. It constitutes a minigenome. In such a minigenome, heterologous coding sequences, i.e., coding sequences which are not derived from said non-segmented negative-strand RNA virus providing the minigenome may be inserted to express polypeptide or peptide of interest, and/or a selectable marker.
According to this embodiment, the cDNA is accordingly a construct which only carries the cis-acting regions of the non-segmented negative strand RNA virus sufficient for transcription and replication of the minigenome.
When the cDNA construct cloned into the DNA molecule of the genome vector(s) contains a heterologous polynucleotide encoding a particular protein, polypeptide, or peptide, including a reporter polypeptide or a selectable marker, the DNA of the heterologous polynucleotide is inserted in said cDNA by any available means. Although the obtained nucleic acid is not necessary a cDNA on its whole length since it may comprise genomic DNA thereby forming a recombinant cDNA, the expression cDNA to designate this construct is kept for convenience in the present application to designate this chimeric nucleic acid.
According to a particular embodiment, the cDNA of the cloned molecule is a recombinant cDNA which comprises a coding sequence of a cellular protein, especially of a yeast protein, especially a protein of the yeast host strain.
In a particular embodiment of the invention, the yeast strain is such that the cDNA which it contains is a recombinant cDNA which comprises a coding sequence of an antigen or an epitope, suitable for eliciting an immune response in a host in need thereof, in particular a humoral and/or a cellular immune response.
Said coding sequence of an antigen or epitope is heterologous to said non-segmented negative-strand RNA virus. It may be selected in order to provide multi-epitopic immunogenic compositions and especially compositions for eliciting an immune reaction against the non-segmented negative-strand RNA virus providing the cDNA and, furthermore, against an additional pathogenic agent or pathogenic organism.
Examples of pathogenic organisms providing heterologous polynucleotides encoding antigens or epitopes encompass viruses especially retroviruses including in particular the lentiviruses, either human or non-human, especially HIV, in particular HIV-1 or HIV-2. Particularly, such antigens are especially from envelopes of AIDS viruses including HIV-1 or HIV-2, from capsid of HIV.
According to a particular embodiment of the invention, the heterologous nucleic acid encodes a protein from an HIV retrovirus, particularly an envelope antigen of HIV and especially a peptide derived from an envelope protein or glycoprotein of HIV-1 or HIV-2. The antigens of interest in this respect are especially gp160, gp120 and gp41 of HIV-1 or gp140, GAG or TAT of HIV-1. In a particular embodiment of the invention, the heterologous amino acid sequence is derived from a recombinant gp160, gp120 of HIV-1 or gp140, GAG or TAT of HIV-1.
In another embodiment, the V1, V2 and/or V3 loops of the gp120 (or gp160) antigen are deleted or deleted in part, or substituted or substituted in part individually or in combination in such a way that conserved epitopes are exposed on the obtained recombinant gp120 antigen. The V1, V2 and V3 loops of the gp120 (or gp160) antigen of HIV-1 have been especially disclosed in Fields virology (Fields B. N. et al. Lippincott Raven publishers 1996, p. 1953-1977).
Other polynucleotides can be derived from Yellow Fever Virus, West Nile Virus, Dengue virus (DV), Japanese encephalitis virus (JEV) or SARS-associated coronavirus. Other retroviridae, or flaviviridae or coronaviridae may also provide such polynucleotides.
As examples of heterologous polynucleotides, those encoding antigens or epitopes of the respiratory viruses different from MV such as RSV, HPIV-3, MMPN, Ebola, Influenza, Parainfluenza etc. . . . are encompassed within the definition of the invention. When such a virus provides a polynucleotide for the performance of the invention, if the virus is a non-segmented negative-strand RNA virus, in a particular embodiment, it is not used for the preparation of the DNA molecule comprising the cDNA encoding the full-length or encoding part of the RNA of the non-segmented negative-strand RNA virus.
Other heterologous polynucleotides may encode antigenic polypeptides of a human papillomavirus such as HPV18, HPV16 or antigens of Hepatitis viruses including HBV or HCV.
The polynucleotide may alternatively encode an antigen expressed on tumor cells or a tumoral antigen.
The antigens or epitopes may be from the envelopes of these viruses or from other antigenic components. The immunogenic response elicited should be cellular and/or humoral. When a cellular response is desired, it is advantageously a CD4 or a CD8 T cell response.
The recombinant yeast strain according to the invention is also useful as seeds for the preparation of immunogenic compositions or vaccines. Indeed, the recombinant yeast strain according to the invention is stable, and enables the maintenance of the vectors which it contains in usual storage conditions for yeasts. Advantageously it is also stable in the sense that it enables the expression of the viral components and their transcription and replication in culture conditions or in fermentation conditions suitable for yeasts.
Thus the recombinant yeast strains also provide seeds for the preparation of immunogenic compositions or even live vaccine whose active principle is provided as RNPs or RNPs-like. In the immunogenic composition the RNPs or the RNPs-like are formulated to enable their uptake by the target cells of the host to whom they are administered.
The invention thus also relates to a novel formulated immunogenic composition, or to a novel vaccine formulation comprising the RNPs or the RNPs-like of the invention, with a transfectant agent. Examples of transfectant agents may comprise lipofectamine or calcium phosphate or liposomes such as FUGENE® of Roche company.
When produced in the recombinant yeasts of the invention, the RNPs or the RNPs-like may be purified, or partially purified in a manner that preserve the possible adjuvant effect of some of the yeast compounds, to provide active principle of an immunogenic composition.
If appropriate, the RNPs or the RNPs-like recovered from the recombinant yeast strains are further used for the transfection of mammalian cells for the expression of viral particles or viral-like particles.
Tranfection may be carried out by using any known methods including transfectant agents. Methods such as lithium acetate-polyethylene glycol method or transfection by lipofectamine or by calcium phosphate can be used to transform the mammalian cells.
The invention is also directed to a process for the recovery of RNPs or RNPs-like expressed in the recombinant yeast cell of the invention.
Such a method comprises:

    
    
        lysis of the yeast cells in an appropriate buffer solution containing protease and RNAases inhibitors;
        recovering yeast extract containing RNPs or RNPs-like and filtering to remove cellular debris;
        centrifugating through a sucrose cushion.

Particular conditions to recover the RNPs or the RNPs-like are described in the examples.

    
    


The invention concerns also a system for the preparation of RNPs or RNPs-like from a non-segmented negative-strand RNA virus by reverse genetics in yeast strains, wherein said system comprises:

    
    
        a recombinant yeast strain according to the invention,
        a culture medium for said yeast strain, which comprises an adequate culture medium for a yeast which is especially devoid of the components which are expressed by the selectable markers contained in the complementation vectors of said recombinant yeast.
    
    


In a particular embodiment of the invention, the culture medium in the system for the preparation of RNPs or RNPs-like from a non-segmented negative-strand RNA virus by reverse genetics in yeast strains comprises Raffinose in a proportion between 0.05% and 5%. Raffinose is especially added in the culture medium with the component required for induction of the promoters, if said promoters are inducible. The invention especially relates to the use of Galactose 1% and Raffinose 2% in the yeast culture media (in final volume of the culture media), when the promoters of the vectors include a Gal promoter.
For the preparation of MV RNPs or RNPs-like in Saccharomyces Cerevisiae, the system comprises a culture medium which is a drop out culture medium with selected nutrients omitted corresponding to the nutrients encoded by the selectable markers in the vectors. Where the yeast promoters are inducible by Galactose, the ratio of Raffinose which is provided in the culture medium is advantageously 2% Galactose and 1% Raffinose.
The invention also relates to the particular yeast culture media which are disclosed in the examples and to the yeast culture conditions described herein.
When one or several of the vectors comprise(s) a gene encoding resistance to an antibiotic, this antibiotic is further added in the culture medium in order to enable the selection of recombinant yeasts capable of growing in the presence of the antibiotic.
Many other applications of the invention are enabled by the use of the recombinant yeast including the use for the screening of factors including yeast factors interacting with transcription, replication or maintenance of infectious RNPs or RNPs-like of non-segmented negative-strand RNA virus.
To carry out such a screening, having recourse to genes contained in DNA libraries, including yeast or human libraries, the recombinant yeast strain of the invention is transformed with a vector expressing a minigenome as defined herein, said minigenome comprising a reporter gene under the control of the cis-acting sequences of the non-segmented negative-strand RNA virus and vectors expressing the transcriptase complex of said virus providing said minigenome, and is further transformed with a plasmid comprising regulatory expression control sequences functional in yeast operatively linked to the polynucleotide to be assayed for interaction.
If the expression product of said polynucleotide is assayed for interaction with replication of the RNA of the non-segmented negative-strand RNA virus the reporter gene in the minigenome is cloned in sense orientation with the leader and, if any, with the additional promoter sequences of the minigenome. The level of replication of the minigenome is measured in yeast transformed with the same.
If the expression product of said polynucleotide is assayed for interaction with transcription of the RNA of the non-segmented negative-strand RNA virus the reporter gene in the minigenome is cloned in sense orientation with the trailer sequence and, if any, with the additional terminator sequence and if any of the promoter sequences of the minigenome. The level of transcription of the minigenome is measured in yeast transformed with the same.
Another application of the recombinant yeast strain is for use for the screening of antiviral compounds interacting with infectious RNPs or RNPs-like of non-segmented negative-strand RNA virus produced in said yeast and especially antiviral molecules interacting with viral replication or with transcription of RNPs or RNPs-like in yeast.
In order to assay such an antiviral activity of a compound, as a result of interaction with viral replication, the yeast is transformed with the complementation vectors as defined in the present application and is further transformed with a vector genome which is a minigenome comprising a reporter gene (such as a gene coding for resistance to an antibiotic) under the control of the cis-acting sequences of the non-segmented negative-strand RNA virus, including the leader and trailer sequences and possibly additional viral promoter and terminator sequences. The reporter gene is encoded in sense orientation with the trailer and terminator sequences of the minigenome. The recombinant yeast is then cultured in a medium containing the antibiotic or the substrate for the reporter gene and is further contacted with the compound assayed for antiviral activity.
In order to assay an antiviral activity of a compound, as a result of interaction with viral transcription, the yeast is transformed with the complementation vectors as defined in the present application and is further transformed with a vector genome which is a minigenome comprising a reporter gene (such as a gene coding for resistance to an antibiotic) under the control of the cis-acting sequences of the non-segmented negative-strand RNA virus, including the leader and trailer sequences and possibly additional viral promoter and terminator sequences. The reporter gene is encoded in sense orientation with the leader and promoter sequences of the minigenome. The recombinant yeast is then cultured in a medium containing the antibiotic or the substrate for the reporter gene and is further contacted with the compound assayed for antiviral activity.
The invention also concerns a set of RNPs of a non-segmented negative-strand RNA virus or a set of virus RNPs-like of a non-segmented negative-strand RNA virus, which is expressed from a yeast according to the invention.
The invention also relates to a process for the preparation of infectious RNPs or RNPs-like of non-segmented negative-strand RNA virus which are expressed from yeast after:
(i) recombining a yeast strain with vectors according to the invention and,
(ii) growing said recombinant yeast strain, especially in a fermentor,
(iii) recovering the produced infectious virus RNPs or infectious RNPs-like.
The invention also relates to the plasmids used as vectors to carry out the invention and which are described herein. It is especially directed to the plasmids deposited at the CNCM on Jan. 31, 2008 which are described herein.
The invention also concerns the cDNA constructs of figures starting from FIG. 14. It also relates to the particular functional sequences described in these figures, such as the ribozyme sequences, the leader sequence of the MV Schwarz strain, the trailer sequence of the MV Schwarz strain, and to the insertion sites, suitable to prepare the vectors of the invention.
Other characteristics and properties of the invention in its broad definition can be derived from the examples which address the preparation of recombinant yeast strain expressing RNPs of measles virus. The figures also provide key features for the design of the vectors suitable for the preparation of the recombinant yeast strains. The features which are shown in the figures can especially be applied to corresponding features of other non-segmented negative strand RNA viruses.



LEGEND OF THE FIGURES
FIG. 1: Real time RT-PCR analysis of MV N, P and L genes expression. Real time RT-PCR was performed to quantify viral N (striped bars), P (white bars), and L (black bars) mRNA expressed in the yeast strain W303-NPLMV. (Glu: culture with glucose, Gal: culture with galactose). In all quantitative PCR calculations, the amount of RNA was standardized using yeast 18S RNA genes. All quantification data are presented as the standardized values, mean±standard deviation of triplicates.
FIG. 2A: Composition of Schwarz MV minigenomes. pYES2 plasmid expression vector containing the URA3 selectable marker, 2μ replication origin and GAL1 inducible promoter were used for the different constructions. GAL1: yeast galactose inducible promoter; Rz: autocatalytic ribozymes; Tr: MV virus Trailer sequence; TL: MV virus L terminator sequence; 3′K: KANMX4 gene 3′ non coding sequence; KAN: KANMX4 gene coding sequence; PN: MV virus N promoter sequence; Le: MV virus Leader sequence; Ter: yeast transcription terminator sequence.
FIG. 2B: The molecular events involved in the geneticin resistance mediated by minigenome α.
FIG. 2C: The molecular events involved in the geneticin resistance mediated by minigenome β.
FIG. 3: Replication of MV minigenome is possible in yeast. W303-NPLMV yeast growth in medium containing geneticin after transformation by the four different minigenomic constructs α, β, γ, ∂ (7 days culture at 30° C., pH 5.6 and 1% raffinose et 2% galactose). Only the minigenomes α or β allow yeast to grow in selective medium. C+: positive control.
FIG. 4: Replication and transcription of MV minigenomes are strictly dependent on viral N, P and L genes. Yeast W303-NPLMV coexpressing different combination of MV N, P, L genes and the α or β minigenomes (7 days culture at 30° C., pH 6.5 and 1% raffinose et 2% galactose). Only the strains coexpressing simultaneously N, P, L and a or (3 minigenomes can grow in presence of geneticin.
FIGS. 5A and 5B: Reconstitution of MV full-length genome RNP in S. cerevisiae. Yeast strain containing the N, P, L priming vector), the derivatives α/β minigenomes and the expression plasmid harboring full MV genome that will produce infectious viral RNPs particles. When yeast grows in presence of canavanin, the priming L vector is eliminated by counter selection. The complementation by L expressed from full-length MV genome is essential for yeast growth in presence of geneticin.
FIGS. 6A and 6B: Reconstitution of recombinant MV full-length genome RNA in Saccharomyces Cerevisiae. 
A. Yeast strain containing the NPL expresser plasmid pESC-LEU-N, pESC-TRPp. pESC-HIS-L and a recombinant full-length MV genome with two additional heterologous genes eGFP and KANMX4
B. Visualisation of transformed yeast expressing eGFP from MV genome.
FIG. 7: Transfection of 293 T and Vero cells with RNPs obtained from yeast as described in FIG. 6 and visualization of the expression of eGFP in yeast.
FIGS. 8A and 8B: A. Amplification of Vero cells of recombinant MV obtained from RNP transfected cells as mentioned in FIG. 7 (eGFP visualization) B. Visualization by RT PCR and sequencing of the presence of KANMX4 reporter gene in the genome of recombinant MV obtained from yeast.
FIG. 9: Genome-wide identification of host genes affecting replication and transcription of MV virus. Yeast Knock-out deletion collection (the deleted gene contain the “bar code” UPTAG and DOWNTAG unique sequences allowing the identification of the yeast strain) will be transformed by the N, P, L and the derivatives α (up)/β (down) minigenomes containing the Luciferase reporter gene and the level of transcription/replication will be measured.
FIG. 10: Identification of host genes and peptides libraries regulators of min-MV replication/transcription in yeast. Yeast W303-NPLMV coexpressing viral N, P, L genes and the derivatives α minigenome will be transformed by DNA libraries coding for yeast/mammalian genes or peptides and the level of transcription/replication will be measured.
FIG. 11A: Schematic representation of MV viral particle (A) and RNP (B). N (nucleoprotein); P (phosphoprotein); M (matrix protein); F (fusion protein); H (hemagglutinin); L (large polymerase).
FIG. 11B: Mice immunization with MV-RNP
FIG. 11C: Partial protection of mice from lethal WNV challenge after immunization with RNP from recombinant MV-sEWNV expressing the envelope protein from WNV.
FIG. 12A: Screening and identification of antiviral compounds inhibiting viral replication in yeast. Yeast W303-NPLMV coexpressing MV N, P, L genes and the derivatives β minigenome containing CAN1 gene under the control of viral Trailer sequence will be exposed to chemical compound libraries and the antiviral active compounds (i) will be identified by selecting colonies growing on canavanin.
FIG. 12B: Screening and identification of antiviral compounds inhibiting viral transcription in yeast. Yeast W303-NPLMV coexpressing MV N, P, L genes and the derivatives α minigenome containing CAN1 gene under the control of viral Leader sequence will be exposed to chemical compound libraries and the antiviral active compounds (i) will be identified by selecting colonies growing on canavanin.
FIG. 13: pYES2: 5856 nucleotides.
FIGS. 14A, 14B, 14C, and 14D: Nucleotide sequences of plasmid constructs of minigenomes, of complementation plasmids expressing N, P and L proteins, using the Schwarz strain of the measles virus The plasmids used are pYES2 plasmids for the preparation of the minigenomes and pESC for the preparation of the complementation plasmids.
FIGS. 15.1, 15.2, 15.3, 15.4, and 15.5 show the Minigenome α sequence (PCM112).
FIGS. 16.1, 16.2, 16.3, 16.4, and 16.5 show the Minigenome β sequence (pCM113).
FIGS. 17.1, 17.2, 17.3, 17.4, and 17.5 show the Minigenome γ sequence (pCM114).
FIGS. 18.1, 18.2, 18.3, 18.4, and 18.5 show the Minigenome δ sequence (pCM115).
FIGS. 19.1, 19.2, 19.3, 19.4, 19.5, and 19.6 show the Minigenome based CAN1 sequence (pCM224).
FIGS. 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, and 20.7 show the Minigenome based CAN1 sequence pCM225).
FIGS. 21.1, 21.2, 21.3, 21.4, 21.5, and 21.6 show the ADE2 plasmid containing minigenome KANMX4 based sequence (pCM226).
FIGS. 22.1, 22.2, 22.3, 22.4, 22.5, and 22.6 show the ADE2 plasmid containing minigenome KANMX4 based sequence (pCM227).
FIGS. 23.1, 23.2, 23.3, 23.4, and 23.5 show the pESC-Leu-N (pCM103) sequence.
FIGS. 24.1, 24.2, 24.3, and 24.4 show the pESC-TRP-P (pCM104) sequence.
FIGS. 25.1, 25.2, 25.3, 25.4, 25.5, and 25.6 show the pESC-LEU-NP (pCM106) sequence.
FIGS. 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, and 26.7 show the pESC-HIS-L (pCM105) sequence.
FIGS. 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, and 27.10 show the pCM105-CAN1 (pCM201) sequence.
FIGS. 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 28.10, 28.11, 28.12, 28.13, and 28.14 show the pESC-URA-MV (pCM101) sequence.
FIGS. 29.1, 29.2, and 29.3 show the Gap repair plasmid based sequence (pCM476).
FIGS. 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, and 30.9, show the Recombinant measles genome sequence: 2nd Gap repair plasmid (pCM402).
FIGS. 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 31.10, and 31.11 show the Recombinant measles genome sequence of the resulting plasmid after the Gap repair (pCM403).
FIGS. 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, and 32.10 show the Recombinant measles genome sequence (pCM503).
FIGS. 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 33.10, and 33.11 show the Recombinant measles genome sequence (pCM603).



EXAMPLES
To demonstrate the capacity of yeast strain W303-NPLMV to support the transcriptional and replicative activity of Schwarz MV RNPs, we generated various subgenomic constructions (minigenomes) derived from the Schwarz measles virus. These minigenomes contain the MV “leader-trailer” sequences necessary for viral genome transcription/replication flanking the reporter gene KANMX4 that confers to yeasts resistance to geneticin drug. Gene KANMX4 was cloned either in sense or in antisense under the control of the cis-active sequences of measles virus, cloned themselves in sense or in antisense under the control of the yeast GAL1 promoter. Autocatalytic ribozyme sequences were added at the two ends of constructions in order to prevent the expression of the minigenomes dependent on the yeast GAL1 promoter. Transformation of the yeast strain W303-NPLMV by the minigenomes allowed yeast to grow in presence of geneticin, the KANMX4 gene being expressed by functional MV RNPs.
This result demonstrates that the replication and the transcription of a minigenome derived from measles virus are possible in yeast. This system enabled us to determine the cloning parameters of the viral genome in a yeast expression vector, which are compatible with the production of functional RNPs. The minigenome can be replaced by a complete genome coding for the whole viral proteins.
Construction of Yeast Strain W303-NPLMV Expressing N, P and L Genes from Schwarz MV
In order to allow replication of measles virus RNPs in yeast, we established a strain of S. cerevisiae expressing the viral proteins N, P and L coding respectively for the nucleoprotein, the phosphoprotein and the polymerase of measles virus (Schwarz vaccine strain), which are the minimal components required for measles RNP formation. We first constructed yeast expression plasmids harboring N, P, and L. The sequences corresponding to the viral N, P and L open reading frames (ORF) were cloned in the pESC series (FIG. 14) of galactose-inducible yeast expression vectors containing the LEU, TRP and HIS selectable markers, respectively, to generate pESC-LEU-N, pESC-TRP-P, pESC-HIS-L plasmids. These plasmids were used to transform the yeast strain W303 to generate the new strain W303-NPLMV. We choose the W303 reference strain because it has been used successfully, notably to replicate papillomavirus (11). The transformed (ref. 20) yeast was cultured at 30° C. in defined drop out medium, with selected nutrients omitted (tryptophan, histidin, leucin) to provide selection for the 3 DNA plasmids together. The culture conditions were optimized to allow the expression of viral components. Optimal induction of N, P and L expression was obtained in a medium containing raffinose (see below). This nonfermentable carbon source does not interfere with galactose induction and favours cell growth.
The RNA expression of measles virus N, P and L genes under the control of the galactose inducible yeast GAL1 promoter was evaluated by real time RT-PCR in the W303-NPLMV strain grown in the presence of galactose. The viral N, P and L genes were highly induced (FIG. 1). Interestingly, the expression profile of N, P and L in yeast is close to their expression profile in MV infected-mammalian cells: N and P are expressed at a similar high level, while L is expressed at a much lower level. Indeed, to achieve optimal viral replication in mammalian cells, the L should be under expressed as compared to N and P(17). Thus, the observed expression profile of N, P and L in yeast appears favorable for measles minigenome replication.
Construction of Measles Virus Minigenomes
To demonstrate that measles virus N, P and L proteins are functional to assemble measles virus RNPs and to promote viral transcription and replication in yeast, we designed different minigenomic constructs harboring a reporter gene, which enabled us to analyze transcriptional and translational activities associated with viral RNPs in yeast. These minigenomes can be replaced by full-length genomes coding for all the viral proteins or by recombinant minigenome or full-length genomes. Minigenomes are viral subgenomic constructs from which all viral ORFs have been removed and replaced by a single reporter gene. Such constructs are able to form transcriptionally active RNPs when expressed together with the N, P and L viral proteins (3). The critical step consisted in designing precise Schwarz measles vaccine subgenomic constructs (minigenomes) harbouring the reporter gene KANMX4, in order to confer to yeast resistance to geneticin drug (G418), as a selectable marker. Minigenomes were designed such as after transformation of W303-NPLMV yeast and growth in presence of geneticin in an appropriate medium and under galactose induction, transformed yeast can grow only upon MV-dependant expression of KANMX4 gene. The minigenomes of the present invention (schematically shown in FIG. 2) contain the Schwarz MV cis-active <<leader>> (nucleotides 1-55) and <<trailer>> (nucleotides 15854-15894) sequences necessary for measles virus replication, flanking the KANMX4 reporter gene. The expression of reporter KANMX4 gene is under the control of the MV N gene promoter (contained in MV nucleotides 55-111) and MV L gene terminator (contained in MV nucleotides 15785-15854). These sequences are flanked on both sides by autocatalytic ribozyme sequences (hammerhead ribozyme in 5′ and hepatitis delta virus ribozyme in 3′). The minigenomes were constructed into the galactose-inducible yeast expression vector pYES2 (FIG. 10) containing the URA3 selectable marker (FIG. 2).
Four minigenomic constructs were generated, in which the reporter KANMX4 gene was cloned in sense (α, β) or antisense (γ), according to MV genome organization. They were placed in pYES2 plasmid either in sense (β, γ) or antisense (α, δ) according to the yeast GAL1 promoter.
1. Minigenome α
Upon transformation of W303-NPLMV yeast by the α minigenome, the inducible GAL1-dependent transcription of KANMX4 gene, which is mediated by yeast RNA polymerase, produces non functional KANMX4 mRNA (because the KANMX4 ORF is not cloned in sens with GAL1 promoter). Yeast should thus be sensible to geneticin. However, the viral N promoter-dependant transcription of KANMX4 gene, which is mediated by viral L RNA polymerase should produce functional KANMX4 mRNA and thus induce resistance to geneticin (FIG. 2B).
2. Minigenome β
In the β minigenome construct, the KANMX4 ORF is cloned in sense with GAL1 promoter and should directly confer resistance to geneticin upon galactose induction. However, the autocatalytic ribozyme sequences added at both extremities prevent the yeast GAL1 promoter-dependant expression of minigenome by cleaving RNA molecules almost simultaneously with synthesis. Resistance to geneticin will be conferred by the β minigenome only if the viral L polymerase replicates (duplicates) the positive strand RNA minigenome (generated by yeast RNA Pol II and than cannot be translated because uncapped and unpolyadenylated) to negative strand RNA minigenome (MV genome). This negative strand MV genome will be transcribed in turn by the viral L polymerase to produce functional KANMX4 mRNA allowing yeast to grow in the presence of geneticin. The geneticin resistance occurring in the case of the β minigenome need two steps; the replication step followed by the transcription step by the viral L polymerase. While the geneticin resistance occurring in the case of the α minigenome need only the transcription step and thus the yeast cells grow faster in this case compared to the 13 minigenome associated growth on geneticin (FIG. 2C).
3. Minigenomes γ and ∂.
It cannot be excluded that some transcription of the KANMX4 gene from α or β minigenomes should arise from cryptic yeast promoters within the minigenomes sequences, or from inefficient autocatalytic ribozyme activity in yeast, or from minigenomes integration in yeast chromosomes. To exclude these eventualities we constructed the γ and δ minigenomes in which the KANMX4 ORF is cloned in antisense with the viral cis-active sequences. Thus, MV-dependant replication and transcription by the viral L polymerase will produce an antisense KANMX4 mRNA non functional to confer G418 resistance. Indeed, the γ and δ minigenomes have the same architecture than α and β minigenomes respectively regarding the KANMX4 dependent transcription by GAL1 promoter.
The FIGS. 2B and 2C explain the molecular events involved in the geneticin resistance mediated by α and β minigenomes. The following molecular mechanisms shed some light on the observed phenotypes: the yeast RNA pol II transcription of the minigenomes flanked by the ribozymes produces polyadenylated and capped mRNA followed by the nuclear export to the cytoplasm. The autocatalytic activity of ribozymes liberates the minigenome. The viral proteins N, P and L encapsidate the minigenome to generate RNPs particles, which are functional to replicate and transcribe the KANMX4 gene. For survival in presence of G418 and a permanent expression of the KANMX4 resistance phenotype, yeast constrains MV minigenome to assemble and to replicate efficiently.
Transcription and Replication of MV Minigenomes in Yeast W303-NPLMV 
The α, β, γ or δ minigenomes constructs were coexpressed with N, P and L after galactose induction in the yeast strain W303-NPLMV. Interestingly, only the α and β minigenomes (KANMX4 ORF cloned in sens with the viral Leader sequence and N promoter, whatever the cloning sens regarding GAL1 promoter) allowed yeast to grow in geneticin-containing selective medium. The γ or δ minigenomes were not able to confer resistance to geneticin in the same condition (FIG. 3). We thus conclude that viral N, P, L and α/β minigenomes are functional in budding yeast Saccharomyces cerevisiae. The growth in geneticin-containing selective medium of the yeast containing the α and β minigenomes constructs demonstrate that transcription and replication of negative strand RNA minigenome virus is possible in yeast.
Transcription and Replication of MV Minigenome in Yeast is Strictly Dependent on Viral Replication Factors
In order to demonstrate that the activity of α and β minigenomes is strictly dependant on the association of functional measles virus RNPs containing the three viral components N, P and L proteins, we generated control W303 yeast strains containing either N, P, L alone or NP (FIG. 4) or NL or PL (data not shown). When transformed by α or β minigenomes, none of them was able to grow on selective medium. FIG. 4 shows that only the strains coexpressing simultaneously N, P, L and α or β minigenomes can grow in presence of G418. This excludes the possibility that yeast factors would be responsible of KANMX4 gene expression and demonstrates that negative strand RNA virus genomes can assemble in functional RNPs in yeast and display RNA dependent RNA polymerase activity.
I Production and Purification of MV-RNPs in Yeast
The minigenomic constructs demonstrate the proof-of-concept for negative strand RNA viruses replication in yeast. The system generated in the present invention that consists of the W303-NPLMV yeast strain and α- or β-type negative strand RNA virus minigenomes may be used for screening cellular factors or antiviral compounds associated with viral replication.
In another major application, the system makes it possible to generate full-length viral RNPs that could be purified from yeast and used to produce live attenuated viruses. In order to assemble functional full-length RNPs that do not contain yeast selection marker inserted inside the viral genomic sequence, yeast must harbour together with the full-length genome a minigenome expressing the resistance gene.
The yeast strain W303-NPLMV is mutated in the CAN1 gene (encoding arginine permease; null mutant of CAN1 gene confers resistance to the arginine analogue Canavanin). The viral polymerase L is expressed from a plasmid harboring the CAN1 gene (FIG. 5). The CAN1 yeast episomal plasmid vector replicates autonomously because of the presence of a yeast origin of replication (2 μm ori). Under conditions of non selective growth in the presence of Canavanin drug, CAN1 plasmid is highly unstable, being lost rapidly from yeast after each generation during cell division.
To produce infectious RNPs harboring full-length MV genome, the yeast W303-NPLMV coexpressing the N, P, L genes from MV is co-transformed by a full-length infectious cDNA corresponding to the MV antigenome and by ε minigenome (FIG. 5). The ε minigenome is an α-like minigenome lacking the MV trailer sequence, thus unable to be transcribed or replicated without complementation. The expression of viral L polymerase from CAN1 plasmid is required to prime the transcription/replication of full-length MV genome. Then, this plasmid will be lost upon yeast growth in the presence of Canavanin, thus making geneticin resistance dependent on the transcription of L polymerase from MV full-length genome (FIG. 5). The trans-complementation by L expressed from full-length MV genome after natural elimination of the L priming plasmid induced by the Canavanin drug becomes thus essential for yeast growth on geneticin. This system allows the production of viral RNPs containing a full-length genome with no selection marker inserted. The E minigenome cannot replicate in yeast or mammalian cells due to the lack of the cis-active trailer sequence (FIG. 5).
We studied the effect of Canavanin on MV minigenomes replication and observed that Canavanin does not interfere with MV minigenomes replication/transcription. We concluded that Canavanin based screening may be used. We established the optimal Canavanin concentration required for Canavanin based screening in yeast, i.e., (200 ug/ml).
We generated a plasmid expressing the viral L polymerase and the CAN1 gene. We constructed a vector containing ADE2 selectable marker from the minigenome β plasmid. The MV full-length genome was cloned into a pESC-URA3 yeast vector. After transformation, the final yeast strain will harbor five plasmids with 7 selectable markers.
Interestingly, the yeast strain W303-NPLMV containing the N, P, L/CAN1 or W303-MV-NPc plasmids and the α/β minigenome and a full-length MV genome was able to grow in medium containing Canavanin and G418, as compared to the same strain lacking the full-length MV genome plasmid that did not grow. Thus, despite the loss of the L plasmid due to the presence of Canavanin, the viral L polymerase was expressed from MV genome and the viral proteins N, P and L were likely encapsidated the minigenome to generate RNPs particles, which, in turn, produced functional KANMX4 mRNA, thus inducing resistance to geneticin.
After extraction from yeast, viral RNPs containing a full-length genome can be used to transfect mammalian cells in order to reproduce infectious virus with a high yield. This invention allows to produce in yeast fermentors a new formulation of measles vaccine or of any other similar live attenuated vaccine. We demonstrated that viral RNPs purified from mammalian cells are infectious and immunogenic (FIG. 11A, B).
II—Expression in Yeast of Recombinant MV Genome Containing Heterologous Genes KANMX4 and/or eGFP.
1. Generation of a Yeast Strain Capable of Stable and Long-Lasting Expression of Complete RNPs of Measles Virus.
In order to enable the replication of viral RNPs particles in the yeast, a strain of Saccharomyces Cerevisiae W303NPL MV-eGFP-KANMX4 has been prepared, which expresses N, P and L viral proteins of the measles virus, together with a full-length recombinant viral antigenome containing two additional genes: the eGFP reporter gene cloned between viral N and P genes, and the KANMX4 gene for resistance to geneticin between F and L viral genes (FIG. 26). This antigenome is cloned in reverse sense with respect to the GAL1 yeast promoter in order to prevent direct expression of the reporter genes by the RNA polymerase of the yeast. These reporter genes can only be expressed after replication and transcription of the antigenomic viral RNA through the action of the viral L polymerase using the viral promoters (Leader, Trailer and specific promoters of the viral genes). Only the first antigenomic RNA is produced by the RNA polymerase of the yeast.
The yeast strain W303 and a strain W303 MV-eGFP-KANMX4 expressing the recombinant genome and devoid of plasmids encoding N, P and L have been used as controls for this study.
2) Purification of Viral RNPs from the Cytoplasm of Yeasts
The strain WO303NPL MV-eGFP-KANMX4 was grown in 400 ml of SD medium for 14-18 hours until exponential phase (OD=0.6-0.8, 6-8×106 cells/ml). The yeasts have been incubated for 4-6 hours in a medium containing galactose in order to induce expression of N, P, L proteins and of recombinant antigenome. The yeasts have been yield and then transformed into spheroplasts for a part thereof and lyzed with glass microbeads for the other part. The RNPs contained in the extracts obtained by both techniques have been purified by treatment with Triton, clarification and ultracentrifugation through a 30% sucrose cushion. The material which was collected in the centrifugation pellet was taken in a Tris-EDTA buffer.
3) The RNPs Purified from the Yeast are Infectious
In order to assay its infectivity, the purified material was transfected (with lipofectamine) in Vero and 293T cells. The day after transfection for the 293T cells and 3 days after transfection for the Vero cells, fluorescent cells and fluorescent syncytia plates were apparent in the cultures (FIG. 7). These plates propagated within the following 48 hours. This experiment shows that yeast W303NPL MV-eGFP-KANMX4 has reconstituted infectious RNPs of the measles virus, which are recombinant for the eGFP reporter gene.
The Vero and 293T cells expressing the eGFP have then been grown with fresh Vero cells. FIG. 8 shows that the viruses derived from the first transfections have propagated into the culture. These viruses elicit especially the formation of typical syncytium. A Petri box was covered with syncytia (90%) in 48 hours.
After amplification of the virus on Vero cells, total RNAs of infected cells have been extracted. A PCR amplification with primers located upstream and downstream of the KANMX4 gene inserted into the measles genome has demonstrated the presence of a band corresponding to the KANMX4 insert (FIG. 8). Sequencing of the amplified band allowed to confirm its nature.
These results show that RNPs of recombinant measles obtained from yeast are infectious on cultured cells.
4) Material Et Methods
Generation of Yeast Strain W303NPL MV-eGFP-KANMX4
The yeast strain W303-1B (ATCC 201238) having genotype MATalpha leu2-112 trp1-1ura3-1 his3-11 his3-15 ade2-1 can1-100 has been transformed by pESC-LEU-N, pESC-TRP-P, pESC-HIS-L and with a recombinant genome containing the eGFP reporter gene, cloned between N and P genes, and containing the gene for selection with geneticin (KANMX4) cloned between the F and L genes.
To prepare the recombinant yeast strain, the following plasmids were prepared and used (the biological material was deposited on Jan. 30, 2009):
pCM476 (CNCM I-4117)
pCM476 is a plasmid comprising synthetic DNA fragment containing Ribozymes sequences and Leader Trailer sequences purchased from Genecust (Luxembourg). The fragment was synthesized in the pUC57 plasmid in SmaI restriction site. The EcoRI-SphI fragment containing the synthetic DNA fragment was then cloned into pYES2 vector containing Ampicillin marker.
The sequence of the synthetic DNA fragment is the following:







GCGGCCGCCAACTTTGTTTGGTCTGATGAGTCCGTGAGGACGAAACCCGG


 


AGTCCCGGGTCACCAGACAAAGCTGGGAATAGAAACTTCGTATTTTCAAA


 


GTTTTCTTTAATATATTGCAAATAATGCCTAACCACCTAGGGCAGGATTA


 


GGGTTCCGGAGTTCAACCAATTAGTCCTTAATCAGGGCACTGTATCCGAC


 


TAACTTATACCATTCTTTGGACTAGTGACGTCCGCGGTCGACACGTGAGA


 


TCTGATGGCCATCTCGGATATCCCTAATCCTGCTCTTGTCCCTGATAATA


 


GGATCTTGAATCCTAAGTGCACTAGAAGATGATCATTGATTGAACTATCC


 


TTACCCAACTTTGTTTGGTGGCCGGCATGGTCCCAGCCTCCTCGCTGGCG


 


CCGGCTGGGCAACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGG


 


GAC


pCM402 (CNCM I-4117)






pCM402 (CNCM I-4117)
pCM402 is a plasmid comprising DNA inserts for eGFP and KANMX4 markers cloned into the Measles Schwarz genome and then cloned into pYES2 vector containing Ampicillin marker.
The KANMX4 was amplified by PCR from pFA6a-kanMX4 plasmid using the primers:










MscIKAN:
CACGTACGATGGGTAAGGAAAAGACTCACG



 



KANAatII:
TCCTTGCGCGCTTAGAAAAACTCATCGAGC






The pTM-MVSchw plasmid harboring BssHII/BsiWI restriction site between Measles virus F and L genes was digested with BssHII/BsiWI and the KANMX4 fragment was cloned in the same site to obtain pCM401 plasmid.
The pTM-MVSchw plasmid harboring eGFP cloned between Measles virus N and P genes and pCM401 plasmids were digested with SalI to obtain two fragments with each plasmid. The fragments containing eGFP and KANMX4 were purified and ligated to obtain pCM402 plasmid.
pCM403
The pCM403 plasmid was obtained by gap repair in yeast: pCM476 was digested with MscI/PflMI and pCM402 was digested by NotI and then the digested plasmids were cotransformed in yeast to obtain pCM403.
pCM503 (CNCM I-4119)
The pTM-MVSchw plasmid harboring eGFP cloned between Schwarz Measles virus N and P genes was digested by NotI and the Schwarz Measles genome containing the eGFP marker was cloned into pYES2 vector containing Ampicillin marker which was digested with NotI.
pCM603 (CNCM I-4120)
pCM603 contains Schwarz Measles genome containing inserts for eGFP and KNAMX4 markers cloned in pYES2 vector containing Ampicillin marker. The pCM401 plasmid was digested with NotI and the Measles virus genome containing eGFP cloned between the N and P genes and KANMX4 cloned between Measles virus genes F and L was cloned in the plasmid pYES2 digested with NotI to obtain pCM603.
Yeast Strain yCM403 (CNCM I-4121)
The yeast strain yCM403 was obtained from Yeast S. Cerevisiae strain W303 NPL MV-eGFP-KANMX4: the diploid of the strain W3031B (ATCC 201238) having leu2-3 leu2-112 trp1-1 ura3-1 his3-11 his3-15 ade2-1 can1-100 was co-transformed by pESC-LEU-N (such as pCM103), pESC-TRP-P (such as pCM104), pESC-HIS-L (such as pCM105). This strain contains pCM403 (Measles alpha genome harboring eGFP and KNAMX4 markers). pCM403 plasmid was obtained by gap repair in yeast: pCM476 plasmid is digested with MscI/PflMI and pCM402 plasmid was digested by Not1 and then cotransformed in yeast to obtain pCM403. Purification of RNPs from yeast: Protocol 1: Overnight culture of strain W303-MV(GFP-KAN)-NPL, strain W303-MV(GFP-KAN), strain W303 in 400 ml of SD medium in a 1 liter flask and grow 14-18 hours to a final concentration of 8×106 cells/ml (0D=0.6-0.8). The cells were washed in 20 ml of sterile water and grown in 20 ml of sterile YG (YNB+Gal+raff+AA dropout) pH6-6.5 inducing medium at 30° C. with shaking for 4-6 h. Cells were centrifuged at 22° C. 1000-1200 g for 5 minutes. The cells were washed in 20 ml of sterile water and in 20 ml of 1 M sorbitol and resuspended in 20 ml of sterile 1 M sorbitol, 10 mM EDTA then transferred to 50 ml centrifuge tubes. 100_l of 2 M DTT and 5 U Lyticase per 106 cells of lyticase were added. The samples were incubated at 37° C. for 15 minutes then centrifuged at 200-300 g at 22° C. for 5 minutes. The spheroplasts were washed with 20 ml of 1 M sorbitol and with 20 ml of sterile 1 M sorbitol, 10 mM Tris pH 7.5 then centrifuged at 22° C., 200-300 g for 5 minutes. The spheroplasts were resuspended in 10 mM NaCl, 0.2% TritonX-100, 10 mM Tris pH7.5, 10 mM EDTA, a Protease Inhibitor Cocktail and RNAses inhibitors. The spheroplasts extract containing RNPs particles were centrifuged 5 mn at 1500 rpm. The extract were centrifuged through a 30% sucrose cushion in PBS, 3 h at 36 000 rpm. The RNPs were resuspended in 100 ul of Tris-EDTA and −80° C. (5 ug/ul). At the end we have 200 ug OD260 per 108 cells. By Bradford measures we obtain 0.4 ug RNP proteins/106 cells. (0.1 OD yeast=106 cells). Protocol 2: Overnight culture of strain W303-MV(GFP-KAN)-NPL, strain W303-MV(GFP-KAN), strain W303 in 400 ml of SD medium in a 1 liter fiask and grow 14-18 hours to a final concentration of 8×106 cells/ml (0D=0.8). The cells were washed in 20 ml of sterile water and grown in 20 ml of sterile YG (YNB+Gal+raff+AA dropout) pH6-6.5 inducing medium at 30° C. with shaking for 4-6 h. Cells were centrifuged 8×50 ml at 22° C. 1000-1200 g for 5 minutes in falcon tubes. The cells were washed in 20 ml of sterile water. The frozen or not yeast cell are lysed in lysis buffer containing, 10 mM NaCl, 0.2% TritonX-100, 10 mM Tris pH7.5, 10 mM EDTA, a Protease inhibitor Cocktail and RNAses inhibitors. A cold lysis buffer was added to an equal volume of glass beads and vortexed on ice. The yeast extract containing RNPs particles were centrifuged 5 mn, 1500 rpm. 10 mM Tris pH7.5, 1 mM EDTA was added then followed by centrifugation through a 30% sucrose cushion in PBS. 3 h at 36 000 rpm. Add 100 ul of Tris-EDTA and −80° C.
Transfection of Vero and 293T Cells.
Vero cells at 90% confluent in were grown in 3 ml plates of DMEM, 10% serum without antibiotic. 20 μg yRNPs were added to 375 μl DMEM w/o serum w/o antibiotic. 10 μl of Lipofectamine2000 were diluted in 375 μl DMEM w/o serum w/o antibiotic. The two solutions were mixed immediately and incubated for 20 mn at room temperature. The 750 μl mix were added to the cells in 3 ml plates. The medium were discarded after 16 h and replaced by fresh DMEM, 10% serum without antibiotic the cells were incubated for 6 days.
III Using Viral RNP as a New Formulation of Measles Vaccine,
Before the widespread use of live attenuated measles vaccine, measles was the single most lethal infectious agent. In the early 1960s, as many as 135 million cases of measles and over 6 million measles-related deaths are estimated to have occurred yearly (Clements C L, Hussey G D. Measles. In: Murray C J L, Lopez A D, Mathers C D, eds. Global Epidemiology of Infectious Diseases. Geneva: World Health Organization, 2004). The introduction of routine measles vaccination in most developing countries during the 1980s as part of the Expanded Programme on Immunization had a major effect on global measles mortality. By 1987, WHO estimated that the number of deaths from measles worldwide had been reduced to 1.9 million (Kejak K, Chan C, Hayden G, Henderson R H. Expanded Programme on Immunisation. World Health Stat Q 1988; 41: 59-63). During the 1990-1999 period, many industrialised countries introduced a second routine dose, usually at or around the time of school entry, to protect children who did not respond to the first dose (Henao-Restrepo A M, Strebel P, John Hoekstra E, Birmingham M, Bilous J. Experience in global measles control, 1990-2001. J Infect Dis 2003; 187 (suppl 1): S15-21). However, despite the availability of a safe, effective, and relatively inexpensive vaccine for over 40 years, measles remains a leading cause of childhood mortality, especially for children living in developing countries (Strebel P, Cochi S, Grabowsky M, et al. The unfinished measles immunization agenda. J Infect Dis 2003; 187 (suppl 1): S1-7). Most measles cases and deaths occur in developing countries, but outbreaks continue to occur in developed countries as well. In 2002, WHO and UNICEF began to implement a strategy for accelerated reduction in mortality due to measles by targeting 45 priority countries accounting for more than 90% of estimated global measles deaths. This program led to an important reduction in measles mortality. WHO claims that between 1999 and 2005, the mortality owing to measles was reduced by 60%, from an estimated 873 000 deaths (634 000-1 140 000) in 1999 to 345 000 deaths (247 000-458 000) in 2005 (L J Wolfson, P M Strebel, M Gacic-Dobo, E J Hoekstra, J W McFarland, B S Hersh. Has the 2005 measles mortality reduction goal been achieved A natural history modelling study. Lancet 2007; 369: 191-200).
Despite these vaccination campaigns, measles still remains the most common cause of vaccine-preventable death. The major reasons for the difficulty to control measles epidemics and outbreaks by routine vaccination are i) the failure to immunize efficiently children before the age of 9 months, mainly because of the presence of passive antibodies transmitted by the mother, and ii) problems with delivery and stability of the vaccine (live enveloped viral vaccines must be kept under 8° C.). Measles vaccine is given in developed countries between 12 and 15 months of age with seroconversion rates of 95%. In developing countries, many cases of measles occur in infants under the age of 1 year, and the vaccine is given at 9 months of age with seroconversion rates of 85% (Cutts, F T, Henao-Restrepo, A. & Olive, J M. (1999) Vaccine 17, Suppl. 3, S47-S52). In both situations, a second dose is necessary to establish sufficient herd immunity to interrupt endemic transmission (Centers for Disease Control, 2000. Morbid. Mortal. Wkly. Rep. 49, 1116-1118). A measles vaccine given before the age of 6 months despite the presence of maternal immunity and a formulation of the vaccine with a greater stability to temperature could improve measles control in many regions of the world.
To address these problems, we developed the possibility of using measles RNP as a new formulation of the vaccine. The viral glycoproteins H and F, which are targeted by neutralizing antibodies, are exposed on the surface of the viral envelope. Inside the viral particle, the RNP is composed of the negative strand RNA genome encapsidated by the nucleoprotein N and the polymerase complex P/L, involving a large number of viral proteins. FIG. 11A shows a schematic representation of MV RNP.
This viral RNPs complex contains all the information for the generation of replicating virus (full-length genome) but does not contain the surface glycoproteins. It should thus be insensitive to neutralisation by antibodies directed to the H and F glycoproteins. Using such RNPs complexes for immunisation could allow to circumvent the pre-existing neutralizing maternal immunity, at least for the first round of infection, and thus increase the uptake of the vaccine by younger infants. Moreover, the RNP formulation that does not contain the viral envelope and the surface glycoproteins should be more stable than the virus itself at higher temperatures.
Infectivity of MV RNP in Cell Culture
To first demonstrate the infectivity of MV RNPs and their capacity of initiating and spreading MV infection in cell culture, we purified MV RNPs from MV-infected cells and from a bulk vaccine batch (as a commercial product). The purification procedure consisted of cell lysis (freezing-thawing), viral membrane disruption using NP40 detergent, low-speed clarification, and centrifugation through a sucrose cushion. MV RNPs were obtained from Schwarz MV vaccine and from Vero cells infected with MV Schwarz strain. The yield was 100 μg (OD260) per 107 pfu. The infectivity of these RNPs was analyzed by transfecting Vero cells using lipofectamine. Table 1 shows that, using different conditions, MV RNPs were infectious for Vero cells after transfection, as detected by syncytia apparition in cell culture. Without lipofectamine, no infection was detected, demonstrating the absence of enveloped viral particles in the RNPs preparation. Infectivity was also tested using FUGENE reagent or calcium phosphate procedures.







TABLE 1







Transfection of Vero cells by MV RNP/lipofectamine









RNP μl
lipofect
Syncytia


(1 μg/μl)
μl
Vero (nb)












5
0
0


10
0
0


5
5
20


5
10
1200


5
20
170


5
50
0


10
20
600









Immunogenicity of MV RNPs in Mice

The best condition for in vitro infection (5 μl RNP+10 μl lipofectamine) was chosen for mice immunization. CD46+/− IFNAR−/− mice (susceptible to MV infection) were inoculated intraperitoneally with a mixture of RNPs/lipofectamine. To control for passive immunization, the same preparation previously UV inactivated (MV genome is UV sensitive) was also inoculated. FIG. 8B shows that after immunization with MV RNPs-lipofectamine, mice developed an immune response against measles, as detected by ELISA (Trinity Biotech, USA) 1 month after inoculation. The mice inoculated with the UV-inactivated RNPs remained MV negative, thus showing that the antibodies detected in other mice were not due to passive immunization.
MV RNP cannot be titrated directly because the infectivity is determined after transfection. However, the dose used in this experiment was estimated at 103-104 TCID50 which corresponds to the vaccine dose of standard measles vaccine. Immunization of the same mice with standard measles vaccine, is 5-10 times more efficient (as determined by ELISA). This difference should be reduced after a better formulation of RNPs. Moreover, higher doses of RNPs should be assayed in order to determine whether the same level of immunization than with standard vaccine can be obtained.
A similar experience was performed using RNPs purified from recombinant MV-sEWNV expressing the secreted form of the envelope E protein from West Nile virus (WNV). This recombinant virus was previously shown to protect mice from a lethal WNV challenge (Despres et al. 2005, J. of Infectious Diseases, 191, 207-214). Mice immunized with MV-sEWNV RNPs were challenged using lethal WNV doses. FIG. 8C shows that mice immunized with recombinant RNPs were partially protected from lethal infection.
In conclusion, these experiments demonstrated that MV RNP are infectious after lipofectamine transfection, and immunogenic in mice at a reasonable dose. Indeed, this new vaccination concept depends on the possibility to provide means allowing availability of RNPs on an industrial scale.
IV Genome-Wide Identification of Host Genes Affecting Replication and Transcription of a Negative-Strand RNA Virus
The engineered α/β minigenomes will be used to systematically identify host factors implicated in the replication and transcription of viral RNPs. Approximately 4500 yeast deletion strains from the Yeast Knock-out (YKO) deletion collection (more than 90% of yeast genes) can be screened (18). Each deletion strain will be transformed by the N, P, L and the derivatives α/β minigenomes in which KANMX4 gene will be replaced by a luciferase reporter gene. Luciferase expression, which is dependent on viral RNA replication and transcription, will be measured in yeast cells. This approach allows the identification of yeast genes whose absence inhibits or stimulates MV or any other negative strand RNA viruses replication/transcription. This functional genomics approach likely will reveal novel host genes required for MV or any other negative strand RNA viruses replication (FIG. 12A) and transcription processes (FIG. 12B).
The YKO deletion collection will be cotransformed in bulk by new vectors expressing N, P, L genes and the derivatives α/β minigenomes (or W303-MV-NPLc strain). To this end, we cloned the derivative 13 minigenome containing the CAN1 genes in the same plasmid expressing viral L polymerase and we generated a second plasmid expressing N and P genes from two distinct promoters. The growing yeast in the presence of Canavanin will be selected and the host genes affecting replication of the MV-minigenome will be identified. The CAN1 genes will be replaced by the Luciferase gene to obtain more quantitative results and measure the effects of each host gene in the replication of the negative-strand RNA virus.
V Genome-Wide Identification of Host Genes and Peptides Libraries Regulators of Min-MV Replication/Transcription in Yeast.
Yeast W303-NPLMV coexpressing MV N, P, L genes and the derivatives α and β minigenomes containing the luciferase or CAN1 genes under the control of viral transcription/replication machinery are transformed by DNA libraries coding for yeast/mammalian or peptides and the level of transcription/replication can be measured (FIG. 12A and FIG. 12B).
We generated a plasmid expressing the derivatives α and β minigenome containing the CAN1 genes and ADE2 selectable marker. The yeast strain W303-NPLMV coexpressing N, P, L and CAN1 genes will then be transformed by a yeast expression genomic DNA library. We performed gDNA library from yeast strain W303. Indeed, we partially digested the genomic DNA from the yeast strain W303 and cloned all the fragments (from 20 bp to 20 kb) in the expression GAL1 vector pYES2. This library is advantageous compared to the classical libraries because the DNA fragment from gDNA is not fused to any nuclear localization signal (NLS) or Tag/protein largely used in almost all genetic screens in yeast and notably yeast two hybrid screen. Thus we will be able to identify other factors required for MV replication. We cloned small fragment to identify small peptides expressed from theses short gDNA that could regulate MV replication.
We will perform cDNA library from human and screen for human cellular factors implicated in the MV replication.
VI Screening and Identification of Antiviral Compounds Inhibiting Viral Replication in Yeast.
Yeast W303-NPLMV coexpressing MV N, P, L genes and the derivatives α and β minigenomes containing CAN1 gene under the control of viral replication (FIG. 12A) and transcription (FIG. 12B) respectively are exposed to chemical compounds libraries and the antiviral active compounds identified by selecting colonies growing in the presence of canavanin. High-throughput screening of chemical compounds can be used to identify modulators of minigenomes replication/transcription (15).
The same strain used to identify factors required for MV replication will serve to screen and identify antiviral compounds inhibiting viral replication in yeast. Several chemical compounds libraries are under study.
Experimental Procedures
All the plasmids and yeast strains have been deposited at the CNCM on Jan. 31, 2008.
For the deposited yeast strains the culture medium is a synthetic complete drop out medium (SD): 13.4 g YNB with ammonium sulfate, 30 g Glucose, 4 g Dropout Amino Acid, 20 g Bacto-agar (Difco) and 1000 ml distilled water (final volume). Adjust the pH to 5.6 with 10 N NaOH and filter sterilize.
For the deposited plasmids, the culture medium is LB medium supplemented with ampicillin.
Construction of pESC-LEU-N Plasmid (pCM103) CNCM I-3897
The pESC series was purchased from Stratagene (#217455)
The N gene was amplified by PCR from pTM-MVSchw (19) plasmid using primers NSalI_5′CATGGTCGACAAGAGCAGGATTAGGGATAT3′ and NXhoI_SGCATCTCGAGTGGATGGTTGATGGGCTGGC3′ and was cloned in the same restriction sites of pESC-LEU (Stratagene, France) plasmid expression vector containing the LEU2 selectable marker, 2μ replication origin and GAL1 inducible promoter.
Construction of pESC-TRP-P Plasmid (pCM104) CNCM I-3898
The P gene was amplified by PCR from pTM-MVSchw plasmid using primers P2SalI_5′CATGGTCGACCAGGTCCACACAGCCGCCAG3′ and P2XhoI_5′GCATCTCGAGGGTCGACTGGCATGGGGTTG3′ and was cloned in the same restriction sites of pESC-TRP (Stratagene, France) plasmid expression vector containing the TRP1 selectable marker.
Construction of pESC-HIS-L Plasmid (pCM105) CNCM I-3899
The 6.7 kb SpeI/BglI blunt ended fragment containing L coding region from pTMMVSchw plasmid was transferred to SalI/XhoI blunt ended pESC-HIS plasmid (Stratagene, France) expression vector containing the HIS3 selectable marker.
Construction of pESC-LEU-NP Plasmid (pCM106) CNCM I-3900
The 1.66 kb Xho/SalI blunt ended fragment containing P coding region from pCM104 plasmid was transferred to NotI/SacI blunt ended pCM103 plasmid.
Construction of MV Schwarz Minigenomes (pCM112—CNCM I-3901, pCM113—CNCM I-3902, and pCM114 and pCM115),
The 1.1 kb DraI/EcoRV fragment containing KANMX4 coding region from pFA6a-KANM4 plasmid (1) was transferred to pTM-MVSchw, which contains a full-length infectious Schwarz MV antigenome/genome flanked by ribozymes sequences and NotI sites, digested by PflMI-MscI and blunt ended. The sequence corresponding to KANMX4 ORF was then cloned in sense (KANMX4 in frame with N promoter) and antisens (KANMX4 not in frame with N promoter) between the 5′Leader-N promoter and L terminator-Trailer sequences flanked by ribozymes sequences and NotI restriction sites.
This 1.7 kb NotI fragment containing the two minigenomes was cloned in the yeast plasmid expression vector pYES2 (Invitrogen, France) containing the URA3 selectable marker, 2μ replication origin and GAL1 inducible promoter, digested by NotI. In the one hand, the minigenome containing KANMX4 in frame with N promoter, was cloned in sens with GAL1 yeast promoter (expressing positive RNA minigenome, (3 construction or pCM113 plasmid), in the other hand, the minigenome containing KANMX4 which is not in frame with N promoter, was cloned in sense with GAL1 yeast promoter (expressing positive RNA minigenome, 6 construction or pCM115 plasmid)
The minigenome containing KANMX4 in frame with N promoter, was cloned in antisense with GAL1 promoter to obtain an intermediary plasmid containing a minigenome without ribozymes (pCM12). This construction was used to construct the a minigenome or pCM112 plasmid (expressing negative RNA minigenome). The overlapping primers below were used to obtain by PCR the a construction.
PCR from pCM 12 using the primers:







HHALPHA_1_5′ACCAGACAAAGCTGGGAATAGAAACTTCGTATTTTCAA


 


AGTTTTCTTTAATATATTGCAAATAATGCC3′


and


 


HDVALPHA1_5′GTCCCATTCGCCATTACCGAGGGGACGGTCCCCTCGGA


 


ATGTTGCCCAGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCA


 


AACAAAGTTGGG3′






Then the PCR fragment was used to make PCR with the following primers:







HHALPHA1_5′GACGGATCCAACTTTGTTTGGTCTGATGAGTCCGTGAGG


 


ACGAAACCCGGAGTCCCGGGTCACCAGACAAAGCTGGGAATAG3′


and


 


HDVALPHA2/2_5′CGAGCTGCTCGAGTCCCATTCGCCATTACC3′






Then the PCR fragment was used to make PCR with the following primers:









HHALPHA2_5′GAAGCTTGACGGATCCAACTTTGTTTGGTCTG3′



and



 



HDVALPHA2/2_5′CGAGCTGCTCGAGTCCCATTCGCCATTACC3′






The PCR fragment was digested by BamHI/XhoI and cloned in the same restriction sites of the pYES2 vector to obtain pCM112 plasmid. The same strategy was used to obtain pCM114 plasmid. It is remarkable that pCM 12 plasmid confers G418 resistance in the yeast strain W303-NPLMV growing in medium containing G418.
Construction of MV Schwarz Minigenomes Containing ADE2 Based Minigenome (pCM226—CNCM I-3906 and pCM227—CNCM i-3907)
The ADE2 gene was amplified by PCR from yeast genomic DNA plasmid using primers ADE2NheI_5′CCATGCTAGCCGAGAATTTTGTAACACC and ADE2ApaI_5′GGCATGGGCCCTTGCTTCTTGTTACTGG and was cloned in the same restriction sites of the pCM112 and pCM113 plasmids. We obtained respectively pCM322 and pCM325 plasmids.
The pCM322 and pCM325 plasmids were digested by KpnI/SacI, blunt ended and ligated to eliminate extraminigenomic SacI site to obtain pCM226 plasmid (a minigenome) and pCM227 plasmid (β minigenome) respectively.
Construction of MV Schwarz Minigenomes Containing CAN1 Based Minigenome (pCM224—CNCM I-3904 and pCM225—CNCM I-3905)
The CAN1 gene was amplified by PCR from yeast genomic DNA plasmid using primers CAN1SacI_5′GAATTCGAGCTCATGACAAATTCAAAAG and CAN1NcoI_5′CTACTGCCATGGACTATGCTACAACATTC, digested with SacI/NcoI and cloned in the pCM226 and the pCM227 digested with SacI/NcoI to obtain pCM224 and pCM225 respectively.
Construction of pESC-URA3-MV Plasmid (pCM101-CNCM I-3896)
The 16.2 kb NotI fragment containing full-length MV genome from pTM-MVSchw plasmid was transferred to NotI pESC-URA plasmid (Stratagene, France) expression vector containing the URA3 selectable marker.
Construction of pCM101-CAN1 Plasmid (pCM201—CNCM I-3903)
The CAN1 gene was amplified by PCR from yeast genomic DNA plasmid using primers CAN1NotI_GCTCGCGGGCCGCATGACAAATTCAAAAGA and CAN1NheI_CCATGGGCTAGCACTATGCTACAACATTCC, digested with NotI/NheI and was cloned in pCM105 plasmid digested by NotI/SpeI.
Generation of Yeast Strain W303-NPLMV 
The strain W303-1B (=ATCC 201238) with the genotype MATalpha leu2-3 leu2-112 trp1-1 ura3-1 his3-11 his3-15 ade2-1 can1-100 was co-transformed by pESC-LEU-N (such as pCM103), pESC-TRP-P (such as pCM104), pESC-HIS-L (such as pCM105) and one of the α or β or γ or δ minigenome constructions. When the minigenome was the alpha one (pCM112), the yCM112 recombinant yeast strain was obtained. It is deposited at the CNCM on Jan. 31, 2008 under N0 I-3908. When the minigenome was the β one (pCM113), the yCM113 recombinant yeast strain was obtained. It is deposited at the CNCM on Jan. 31, 2008 under N0 I-3909.
Generation of Yeast Strain W303-MV-NPLc 
The strain W303-1B (=ATCC 201238) with the genotype MATalpha leu2-3 leu2-112 trp1-1 ura3-1 his3-11 his3-15 ade2-1 can1-100 was co-transformed by pESC-LEU-N (such as pCM103), pESC-TRP-P (such as pCM104), the priming plasmid pESC-HIS-L-CAN1 (such as pCM201) and one of the ADE2 (pCM226/pCM227) based minigenomes constructions. When the minigenome was the alpha one (pCM112), the yCM226 recombinant yeast strain was obtained. It is deposited at the CNCM under N0 I-3910.
Yeast Culture Conditions
The yeast strain W303 was grown in YPD medium before plasmid transformation. W303-NPLMV was grown at 30° C. for 24 hours in 25 ml of defined medium to an optical density at TO of 0.5 or 5 106 cells/ml (8 h in 2% Raffinose, we do not wash away the raffinose medium before the induction for 16 h in 2% Galactose+1% Raffinose) and were pelleted. The yeasts were cultured at 30° C. in defined drop out medium, with selected nutrients omitted (tryptophan, histidin, leucin, uracil, adenin) to provide selection for DNA plasmids. The Synthetic Complete drop-out Medium Mix was enriched 2 times for YNB (Yeast Nitrogen Base with Ammonium Sulfate and without Amino Acids) (Difco, France) and 4 times for amino-acids (Sigma, France). Galactose-inducible expression of KANMX4 was obtained by using a mix of 2% of galactose (Sigma, France) and 1% of raffinose (Sigma, France). KANMX4 expression was selected by growth in medium supplemented with 100 mg/I G418 geneticin (Invitrogen, France). CAN1 based plasmid was eliminated by growth in medium supplemented with 200 mg/I L-Canavanine sulfate salt (C9758, Sigma). The pH of medium was adjusted to 5.6 or 6.5. All plasmids were introduced into yeast by the transformation method described in Gietz et al (20).
Yeast Culture Media
YPD medium (growing yeast without plasmids before transformation): 20 g yeast extract (Difco), 40 g Peptone (Difco), 30 g Glucose, 200 mg adenine hemisulphate, 20 g Bacto-agar (Difco) and 1000 ml distilled water (final volume), filter sterilize.
Synthetic complete drop-out medium (SG): 13.4 g YNB with ammonium sulfate, 10 g Galactose, 20 g Raffinose (Sigma R7630), 4 g Dropout AA, 20 g Bacto-agar (Difco) and 1000 ml distilled water (final volume). Adjust the pH to 5.6 or 6.5 with 10 N NaOH and filter sterilize.
Synthetic complete drop-out medium (SD): 13.4 g YNB with ammonium sulfate, 30 g glucose, 4 g Dropout AA, 20 g Bacto-agar (Difco) and 1000 ml distilled water (final volume). Adjust the pH to 5.6 or 6.5 with 10 N NaOH and filter sterilize.
Synthetic Complete Drop Out Mix: 2 g Arginine, 2 g Threonine, 2 g Cysteine, 2 g Isoleucine, 2 g Tyrosine, 2 g Glutamate, 2 g Lysine, 6 g Valine, 2 g Glutamine, 2 g Methionine, 2 g Alanine, 2 g Glycine, 3 g Phenylalanine, 2 g Aspartate, 2 g Proline, 2 g Serine and 2 g Asparagine.
The different metabolites used in medium complementations are 100× concentrated and filter sterilized: Adenine 2.0 mg/ml, Uracil 2.0 mg/ml, Histidine HCl 4.0 mg/ml, Leucine 6.0 mg/ml, Tryptophan 6.0 mg/ml. One ml of metabolites stock was added per plate containing 25 ml medium.
Transformation of Yeast
Yeast was inoculated into 15 ml liquid medium (2×YPD or 2×SD selection medium) and incubated overnight on a shaker at 200 rpm and 30° C. The day after, cells were diluted to an OD600=0.5 in same medium and incubated under stirring (200 rpm) at 30° C. for 3-4 hours, until OD600 reaches 1. Cells were harvested by centrifugation (3000 g for 5 min), washed two times in 25 ml and 1 ml of sterile water and centrifuged for 15 sec. to collect cell pellet. Transforming plasmid mixtures prepared according to table were added to cell pellets.













Reagents



















PEG 3500 50% w/v
240 μl 



LiAc 1.0M
36 μl



Boiled SS-carrier DNA
50 μl



Plasmid DNA plus Water
34 μl



Total
360 μl 










PEG (Sigma P3640), LiAc (Sigma L6883), SS-carrier DNA (DNA Sodium Salt Type III from Salmon Testes, Sigma D1626).
Cells are resuspended by mixing vigorously and incubated at 42° C. for 40 min. The transformation mixture was removed by centrifugation and cells were washed with 1 ml sterile water before plating appropriate dilutions onto SD selection medium. After 3 to 4 days incubation at 30° C., the number of transformants was determined.
RNA Expression Analysis by Reverse Transcription and Real-Time PCR Assay.
The yeasts were grown at 30° C. for 24 hours in 25 ml of defined medium and were pelleted. We isolated total RNA using Trizol method (Invitrogen) followed by RNEASY® (Qiagen), a silica-membrane spin column-based RNA purification kit, and prepared cDNA using SuperScript II reverse transcriptase (Invitrogen, France) (21). Quantitative PCR analysis was done using SYBR PCR Mix (Applied Biosystems, France) and the Abiprism 7000 machine (Applied Biosystems, France). Quantification is described in Miled et al (22). In all quantitative PCR calculations, the amount of nucleic acid material was standardized using oligonucleotide primers for yeast 18S RNA genes. All quantification data are presented as the standardized values, mean±standard deviation of triplicates.
Oligonucleotides Used for qRT-PCR












Genes
Forward
Reverse







Yeast Sc18S
5′GAATAAGGGTTCGATTCCGGAG
5′CTGCCTTCCTTGGATGTGGTAG


 


Virus MvSsN
5′CCCTGGAGATTCCTCAATTACCA
5′CCAATTAACCTCACCAACCGG


 


Virus MvSsP
5′CAGACGCGAGATTAGCCTCATT
5′GGTTGCACCACCTGTCAATAAAG


 


Virus MvSsL
5′TGCTTATGAGAGCGGAGTAAGGA
TACGGCTATGGTCTGATTGTCCC









Primers are purchased from Sigma-Proligo, France.
RNPs Purification from Yeast
The frozen or unfrozen yeast cells were lysed in lysis buffer containing, 10% Glycerol, 0.2% TritonX-100 150 mM NaCl, 25 mM Tris(pH7.5), 1 mM EDTA, a Protease Inhibitor Cocktail and RNAases inhibitors. A cold lysis buffer was added to an equal volume of glass beads and vortexed on ice. The yeast extract containing RNPs particles was filtered to remove cellular debris and followed by centrifugation through a 30% sucrose cushion. The resulting preparation containing purified viral RNPs may be adjuvanted with any available adjuvant.
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## Claims

1. A recombinant yeast strain which expresses infectious non-segmented negative-strand RNA virus Ribonucleocapsids (RNPs) or infectious RNPs-like, wherein the yeast is transformed with the following expression vectors:
(i) a first plasmid genome vector comprising, as an insert operatively linked with expression control sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding the (+) strand full-length sequence (antigenome) of said non-segmented negative-strand RNA virus and wherein said cDNA is flanked, in the cloned DNA molecule, by autocatalytic ribozyme sequences enabling the recovery of mRNA transcripts and of antigenomic RNAs of said non-segmented negative strand or derivatives thereof;
(ii) a second plasmid genome vector comprising, as an insert under the control of regulatory expression sequences functional in yeast, a cloned DNA molecule which comprises a cDNA encoding part of the antigenome of said non-segmented negative-strand RNA virus including in the 5′ to 3′ orientation, a viral Terminator sequence, a polynucleotide which codes a selectable marker cloned in sense orientation with respect to the cis-acting sequences of said virus and the Leader sequence of said virus; and
(iii) one or more trans-complementation plasmid vectors comprising, under the control of regulation expression sequences functional in yeast, nucleotide sequences which enable said vector(s) to collectively express the proteins necessary for the synthesis of the viral transcriptase complex of said non-segmented negative-strand RNA virus, and enable assembly of the ribonucleocapsid (RNPs) of said non-segmented negative-strand virus or assembly of RNPs-like comprising recombinant RNA derived from viral RNA of a non-segmented negative-strand RNA virus, wherein the RNPs or RNPs-like are functional for the replication and transcription, each of said vector(s) further comprises, under the control of regulatory expression sequences functional in yeast, a selectable marker, wherein in said vectors all the selectable markers are different from each other, and wherein the vector encoding the viral L polymerase of said non-segmented negative-strand RNA virus is a priming plasmid harboring an auxotrophy marker gene.

2. The recombinant yeast strain according to claim 1, wherein the non-segmented negative-strand RNA virus is selected from the group consisting of: Rhabdoviridae, Paramyxoviridae, Filovihdae, and Bomaviridae.

3. The recombinant yeast strain according to claim 2, wherein the non-segmented negative-strand RNA virus is selected from the group consisting of: Measles virus, RSV, HPIV2, and HPIV3.

4. The recombinant yeast strain according to claim 1;
wherein the non-segmented negative-strand RNA virus is a measles virus (MV);
wherein the one or more trans-complementation plasmid vectors are capable of expressing the nucleoprotein (N), the Phosphoprotein (P) and the Polymerase (L) or derivatives thereof as functional ribonucleoproteins (RNPs) comprising the transcriptase complex; and
wherein the first or second plasmid genome vector comprises a cloned molecule which comprises a cDNA encoding the full-length (+) strand antigenome of said measles virus and wherein said cDNA is framed by autocatalytic ribozyme sequences.

5. The recombinant yeast strain according to claim 1; wherein the non-segmented negative-strand RNA virus is a measles virus; and
wherein the one or more trans-complementation plasmid vectors and the first and second plasmid genome vectors are further characterized as follows:
(a) the one or more trans-complementation plasmid vectors are capable of collectively expressing the nucleoprotein (N), the Phosphoprotein (P) and the Polymerase (L) or functional derivatives thereof which enable assembly of functional ribonucleoproteins (RNPs) or RNPs-like comprising the transcriptase complex; and
(b) the first or second plasmid genome vectors comprises, in an insert, a cloned DNA molecule which comprises a cDNA encoding a fragment of the (+)strand (antigenome) of said virus, including the cis-acting Leader and Trailer sequences, and furthermore one or more coding sequences, or ORF(s), heterologous to said virus, the expression of which is sought.

6. The recombinant yeast strain according to claim 4, wherein the nucleoprotein (N), the phosphoprotein (P) and the polymerase (L) are expressed by several plasmid expression vectors, said expression vectors comprising cloned polynucleotides consisting of viral coding sequences for one of the N, P or L proteins, under the control of a promoter suitable for expression in yeast.

7. The recombinant yeast strain according to claim 4, wherein the nucleoprotein (N), and the phosphoprotein (P) are expressed by a single expression vector, and the polymerase (L) is expressed by another expression vector said expression vectors comprising cloned polynucleotides consisting of viral coding sequences for the N and P proteins or for the L protein respectively, under the control of a promoter suitable for expression in yeast.

8. The recombinant yeast strain according to claim 1, wherein in the first and second plasmid genome vectors the cloned molecule is cloned in a plasmid under the control of expression control sequences suitable for expression in yeast, including a promoter and a transcription terminator sequence in sense or in antisense orientation.

9. The recombinant yeast strain according to claim 1, wherein in at least one of the first and second plasmid genome vectors the cDNA in the cloned DNA molecule comprises at least one of the following polynucleotides:
(a) a Leader and/or Trailer sequence of measles virus (MV);
(b) an additional Promoter sequence derived from the MV, selected from the group consisting of: the promoter of the nucleoprotein (N), phosphoprotein (P) and polymerase (L) of an MV;
(c) an additional Terminator sequence derived from the MV, selected from the group consisting of: the terminator of the polymerase (L) or of the nucleoprotein (N), and the phosphoprotein (P) of an MV; and
(d) the cDNA of the cloned molecule is framed by different or identical autocatalytic ribozymes selected among hammerhead ribozyme and hepatitis delta virus ribozyme.

10. The recombinant yeast strain according to claim 9, wherein the measles virus is an attenuated measles virus.

11. The recombinant yeast strain according to claim 10, wherein the measles virus is a Schwarz MV.

12. The recombinant yeast strain according to claim 1, wherein at least one of said plasmid vectors is selected from pCM101, pCM103, pCM104, pCM105, pCM106, pCM112, pCM113, pCM201, pCM224, pCM225, pCM226, pCM227, pCM402, pCM476, pCM503, and pCM603 deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) under No. I-3896, I-3897, I-3898, I-3899, I-3900, I-3901, I-3902, I-3903, I-3904, I-3905, I-3906, I-3907, I-4117, I-4118, I-4119, and I-4120, respectively.

13. The recombinant yeast strain according to claim 1, wherein in the second plasmid genome vector the cDNA encoding part of the antigenome of said non-segmented negative-strand RNA virus is devoid of all the viral genes or is devoid of all the viral coding sequences.

14. The recombinant yeast strain according to claim 1, wherein in at least one of the first and the second genome vectors the cDNA of the cloned molecule is a recombinant cDNA which comprises a coding sequence of a reporter gene.

15. The recombinant yeast strain according to claim 1, wherein in at least one of the first and the second genome vectors the cDNA of the cloned molecule is a recombinant cDNA which comprises a coding sequence of a cellular protein.

16. The recombinant yeast strain according to claim 1, wherein in at least one of the first and the second genome vectors the cDNA of the cloned molecule is a recombinant cDNA which comprises a coding sequence of an antigen or an epitope, suitable for eliciting an immune response in a host in need thereof.

17. The recombinant yeast strain according to claim 1, which is a strain of Saccharomyces Cerevisiae.

18. The recombinant yeast strain according to claim 1, which is a strain of Pichia Pastoris or Saccharomyces Pombe.

19. The recombinant yeast strain according to claim 1, which is the strain yCM112, yCM113, yCM226, or yCM403 deposited at the CNCM under No. I-3908, I-3909, I-3910, and I-4121, respectively.

20. A set of RNPs of a non-segmented negative-strand RNA virus or a set of RNPs-like of a non-segmented negative-strand RNA virus, which is expressed from a recombinant yeast strain according to claim 1.

21. The set of RNPs or RNPs-like according to claim 20, wherein the RNPs or RNPs-like are formulated with a transfectant agent.

22. An immunogenic composition comprising RNPs or RNPs-like according to claim 20.

23. A system for the preparation of RNPs or RNPs-like from a non-segmented negative-strand RNA virus by reverse genetics in yeast strains, wherein said system comprises:
(a) the recombinant yeast strain according to claim 1; and
(b) a culture medium for said yeast strain, which comprises an adequate culture medium for a yeast which is devoid of the components which are expressed by the selectable markers contained in complementation vectors of said recombinant yeast.

24. The recombinant yeast strain according to claim 6, wherein the promoter suitable for expression in yeast is an inducible promoter.

25. The recombinant yeast strain according to claim 7, wherein the promoter suitable for expression in yeast is an inducible promoter.

26. The recombinant yeast strain according to claim 1; wherein the one or more trans-complementation plasmid vectors each independently comprise a selectable auxotrophy marker.

27. A process for the preparation of infectious RNPs of a non-segmented negative-strand RNA virus or infectious RNPs-like, wherein said RNPs or RNPs-like are expressed from yeast after:
(a) transforming a yeast strain with vectors according to claim 1;
(b) growing said recombinant yeast strain; and
(c) recovering the produced infectious virus RNPs or infectious RNPs-like.

28. A process for preparation of RNPs or RNPs-like of a non-segmented negative-strand RNA virus characterized in that it comprises the steps of:
(a) obtaining recombinant yeasts expressing RNPs or RNPs-like according to claim 1; and
(b) recovering the RNPs or RNPs-like from said yeasts.

29. A method for preparing an immunogenic composition comprising infectious RNPs or RNPs-like, which comprises seeding a culture with the recombinant yeast strain according to claim 1, and isolating infectious RNPs or RNPs-like.