EP2788477A2 - Identification d'un virus porcin de type paréchovirus et applications - Google Patents

Identification d'un virus porcin de type paréchovirus et applications

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Publication number
EP2788477A2
EP2788477A2 EP12797926.8A EP12797926A EP2788477A2 EP 2788477 A2 EP2788477 A2 EP 2788477A2 EP 12797926 A EP12797926 A EP 12797926A EP 2788477 A2 EP2788477 A2 EP 2788477A2
Authority
EP
European Patent Office
Prior art keywords
spelv
polynucleotide
sequence
virus
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12797926.8A
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German (de)
English (en)
Inventor
Marc Eloit
Justine Cheval
Virginie Sauvage
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur de Lille
Ecole Nationale Veterinaire dAlfort
PATHOQUEST
Original Assignee
Institut Pasteur de Lille
Ecole Nationale Veterinaire dAlfort
PATHOQUEST
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Filing date
Publication date
Application filed by Institut Pasteur de Lille, Ecole Nationale Veterinaire dAlfort, PATHOQUEST filed Critical Institut Pasteur de Lille
Publication of EP2788477A2 publication Critical patent/EP2788477A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32511—Parechovirus, e.g. human parechovirus
    • C12N2770/32521—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32511—Parechovirus, e.g. human parechovirus
    • C12N2770/32522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32511—Parechovirus, e.g. human parechovirus
    • C12N2770/32534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Picornaviruses are non-enveloped, positive-stranded RNA viruses with an icosahedral capsid. They are usually grouped in the Picornaviridae family, which encompasses a great number of different genera and includes many important pathogens of humans and animals.
  • the picornavirus genome RNA comprises a large open reading frame encoding a large polypeptide precursor and is bordered by two non-coding sequences. The polyprotein precursor then gives different mature viral capsid proteins by proteolytic cleavage.
  • the 5' non-coding sequence is important for viral replication, while the role of the 3' non-coding region is not well characterized.
  • Parechovirus is a viral gen us in the fami ly Picornaviridae.
  • the gen us is composed of two species: Human parechovirus and Ljungan virus.
  • h u man parech ovi ru s h ave been id entified : h u man parechovirus 1 (formerly echovirus 22), human parechovirus 2 (formerly echovirus 23), and human parechoviruses 3 to 14 (Benshop KS et al, J. Gen Virol 2010).
  • They display the characteristic structure of the viruses of the family Picornaviridae. In particular, they have a single-stranded RNA genome of positive polarity encoding a polyprotein precursor, which is then matured into the capsid proteins VP1 to VP3 (Stanway and Hyypia, J. Virol., 1999; Williams et al., J. Gen. Virol., 2009).
  • LV has been implicated with diabetes and intrauterine fetal death in human (Niklasson B, birth Defects Res. A. Clin. Mol. Teratol 2007; Niklasson B, Int. J. Exp. Diabesity. Res. 2003).
  • LV infection has been linked with intrauterine fetal death (IUFD), malformations, placental inflammation, myocarditis, encephalitis, and Guillain-Barre syndrome (Niklasson B. et al, Forensic. Sci. Med. Pathol. 2009; Niklasson B. et al, Birth Defects Res. A. Clin. Mol. teratol. 2009; Samsioe A.
  • Figure 1 A to I depicts the mapping of the 9 contigs of SEQ ID NO:29 to 37 with the amino acid reference sequence of LV (NCBI accession:21309880 AF327922 - Protein id: AAM46081 )(NCBI BlastX). As can be seen, large regions of each coding gene have been sequenced.
  • Figures 2 and 3 depict SPeLVI VP1 nucleotide alignment with the closest known VP1 from HPeVs and LV.
  • VP1 s have been extracted from the best BLASTP hit between SPeLVI polyprotein and other parechoviruses and Ljungan Virus fully annotated polyproteins (except for H PeVI O and H PeV1 1 were only VP 1 was available).
  • Fig u re 4 represents the phylogenetic tree of the SPeLVI with in the Picornaviridae family.
  • SPeLVI Single Parechovirus-l i ke vi ru s 1
  • HPEV1 human parechovirus type 1
  • the present invention thus provides an isolated polynucleotide wherein said polynucleotide is a virus genome sharing at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequence represented by SEQ ID NO: 1 .
  • the said polynucleotide is the SPeLVI virus genome, represented by SEQ ID NO: 1 .
  • the present invention also relates to the SPeLV virus genome, in a recombinant form (meaning that the virus displays at least one modification from naturally occurring virus), to the SPeLV virus genome in the form of complementary sequence of SEQ I D NO: 1 , or even to the SPeLV virus genome in the form of complete cDNA of SEQ ID NO: 1 .
  • the invention also encompasses parts of said SPeLV virus genome as defined above comprising at least 100, 200 or 500 consecutive nucleotides of the sequence as defined above (SEQ ID NO: 1 , complementary sequence thereof or cDNA thereof).
  • the invention also relates to an SPeLV viral particle containing the said SPeLV virus genome.
  • the genome of the parechoviruses contains a large open reading frame encoding a polyprotein precursor.
  • the present invention thus relates to a polynucleotide comprising an open reading frame encoding the large polyprotein of the SPeLVI genome.
  • said open reading frame comprises a sequence sharing at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequence represented by SEQ ID NO: 2.
  • the said polynucleotide encodes a polyprotein precursor displaying at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the polyprotein precursor of SPeLVI , whose sequence is represented by SEQ I D NO: 3.
  • the said polynucleotide encodes the SPeL1 polyprotein precursor of SEQ ID NO: 3.
  • the present invention provides the large polyprotein precursor of SPeLVI , said polyprotein precursor being encoded by a polynucleotide sequence sharing at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequence represented by SEQ ID NO: 2.
  • the present invention provides the large polyprotein precursor of SPeLVI , said polyprotein precursor comprising an amino acid sequence displaying at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99% , 99.5%, 99.9% identity with the polyprotein of SPeLVI , whose sequence is represented by SEQ ID NO: 3.
  • the said polyprotein precursor comprises an amino acid sequence of SEQ ID NO: 3.
  • the polyprotein precursor of parechoviruses is matured by proteolytic cleavage into 3 viral capsid proteins, designated VP1 , VP2 and VP3.
  • the present invention also relates to the capsid protein VP1 of SPeLVI and to a polynucleotide sequence encoding said VP1 protein.
  • VP1 is specifically important for tropism as, within the Picornaviridae family, it mediates interactions with the cell receptor(s).
  • VP1 is also a major target of antibodies, including neutralizing and opsonizing antibodies and thus is fundamental for medical application as it is the main candidate for the development of vaccines and serological tests.
  • the said VP1 capsid protein is encoded by a polynucleotide sequence displaying at least 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90% , 92.5% , 95% , 96% , 97% , 98% , 99% , 99.5% , 99.9% i d entity with th e polynucleotide sequence represented by SEQ I D NO: 4.
  • the polynucleotide has the sequence of SEQ ID NO: 4.
  • the invention also relates to a polynucleotide sequence encoding a VP 1 protein, said polynucleotide sequence displaying at least 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the polynucleotide sequence represented by SEQ ID NO: 4.
  • the polynucleotide has the sequence of SEQ ID NO: 4.
  • the said a VP1 capsid protein comprises a sequence displaying at least 50 %, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the amino acid sequence represented by the sequence SEQ ID NO: 5.
  • the invention also relates to a polynucleotide sequence encoding a VP 1 protein, said polynucleotide sequence displaying at least 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the polynucleotide sequence represented by SEQ ID NO: 4.
  • the polynucleotide has the sequence of SEQ ID NO: 4.
  • the present invention also relates to the VP1 protein in association with at least one protein obtained by proteolytic cleavage from the polyprotein.
  • the said protein is VP2.
  • the said protein is VP3.
  • the VP1 protein is associated with both the VP2 and the VP3 protein.
  • sequence identity refers to the identity between two peptides or between two nucleic acids. I dentity between sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of sequence identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are shared between these sequences. Since two polypeptides may each (i) comprise a sequence (i.e.
  • a portion of a complete polynucleotide sequence) that is similar between two polynucleotides, and (ii) may further comprise a sequence that is divergent between two polynucleotides, sequence identity comparisons between two or more polynucleotides over a "comparison window" refers to the conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference nucleotide sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence or a first nucleic acid sequence for optimal alignment with the second amino acid sequence or second nucleic acid sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.
  • sequences can be the same length or can be different in length.
  • Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 1981 ), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol, 1972), by the search for similarity via the method of Pearson and Lipman (Proc. Natl. Acad. Sci. U.S.A., 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 575, Science Drive, Mad ison , Wisconsin) or by inspection. The best alignment (i.e. resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected.
  • sequence identity means that two polynucleotide or polypeptide sequences are identical (i.e. on a nucleotide by nucleotide or an amino acid by amino acid basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, U , or 1 ) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity.
  • the same process can be applied to polypeptide sequences.
  • the percentage of sequence identity of a nucleic acid sequence or an amino acid sequence can also be calculated using BLAST software (Version 2.06 of September 1998) with the default or user defined parameter.
  • sequence similarity means that amino acids can be modified while retaining the same function. It is known that amino acids are classified according to the nature of their side groups and some amino acids such as the basic amino acids can be interchanged for one another while their basic function is maintained.
  • the SPeLV virus of the invention is likely to infect human beings, especially young children. This is all the more relevant since the virus of the invention appears to be present in the general pig population. It is therefore important to be capable of detecting the presence or not of the SPeLV virus of the invention in pig as well as in human beings, in particular in young children. Detection of the SPeLV virus in pregnant women would be also crucial in order to monitor the risk of vertical transmission from the mother to the child.
  • the invention thus relates to an in vitro method of detection of a SPeLV virus in a subject, comprising the step a) of determining the presence of the SPeLV virus in a biological sample of the said subject.
  • a “biological sample” may be any sample that may be taken from a subject, and thus includes, but is not limited to, for example, blood, serum, plasma, sputum, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, breast milk, and tears.
  • a sample can be obtained by an oropharyngeal swab, nasopharyngeal swab, throat swab, nasal aspirate, nasal wash, fluid collected from the ear, eye, mouth, or respiratory airway, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal central nervous system tissue.
  • a sample can also be a pool of individual samples, especially those made during the process of manufacturing of biological samples obtained from h umans (blood or urine derived products, for exam ple), or any intermediate product sampled during the manufacturing of such products. Such sample must allow the determination of the presence of SPeLV through the methods of the invention.
  • the presence of the SPeLV virus may be determined by any technology known to a person skilled in the art.
  • the SPeLV virus may be detected at the genomic and/or nucleic and/or protein level.
  • the method according to the invention may thus comprise another preliminary step, between the taking of the sample from the patient and step a) as defined above, corresponding to the transformation of the biological sample into a genomic DNA sample, or into an mRNA (or corresponding cDNA) sample, or into a protein sample, which is then ready to use for in vitro detection of SPeLV in step a).
  • the detection of the SPeLV virus may be performed, depending on the type of transformation and the available ready-to-use sample, either at the genomic DNA (i .e. based on the presence of at least one sequence consisting of at least a part of the SPeLV genome as defined above), mRNA (i.e. based on the mRNA content of the sample) or at the protein level (i.e. based on the protein content of the sample).
  • Methods for detecting a genomic nucleic acid in a biological sample include inter alia hybridization with a labeled probe, genomic PCR, nucleic microarrays, high- throughput sequencing, and all other methods known to the person of skills in the art.
  • the amount of nucleic acid transcripts can be measured by any technology known by the skilled person . I n particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a person skilled in the art, including nucleic microarrays, quantitative PCR, and hybridization with a labeled probe.
  • mRNA messenger RNA
  • cDNA retrotranscribed complementary DNA
  • the presence of the said virus is determined by hybridization of probes specific for the said virus or parts thereof with the biological sample.
  • amplification and/or sequencing of the SPeLV sequences is performed in order to assess the presence of the said virus.
  • the presence of the SPeLV virus is determined by detecting the polyprotein produced by the single open reading frame, or the VP1 capsid protein.
  • Another object of the invention therefore relates to a probe capable of hybridizing to the genomic DNA of SPeLV. By "probe capable of hybridizing", one should understand that the said probe is substantially complementary to at least part of the SPeLV virus genome.
  • the said probe comprises a nucleotide sequence displaying at least 50 %, 55 %, 65 %, 70 %, 75 %, 80 %, 85 %, 87.5 %, 90 %, 92.5 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.5 %, 99.9 % identity with at least a part of a sequence of the genomic DNA of SPeLV (SEQ ID NO:1 ).
  • the probe of the invention comprises at least 12 nucleotides, more preferably at least 15 nucleotides, even more preferably at least 20 nucleotides.
  • the method of the invention is performed by hybridization with the probes of the invention. Detection of a hybridization signal is thus indicative of the presence of a SPeLV virus in the biological sample. It is advantageous to use labeled probes in this embodiment.
  • the present invention also includes primers specific for the SPeLV virus.
  • the said primers have the sequences as laid out in SEQ ID NOs: 6-29.
  • the said primers can be used for amplification of specific regions of the SPeLV virus of the invention .
  • the amplification may be carried out directly on genomic DNA, on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art.
  • the said primers of the invention can also be used for sequencing the SPeLV virus. Alternatively, the said SPeLV virus is detected by high-throughput sequencing.
  • An embodiment of the present invention thus provides a method of detection of an SPeLV virus comprising a step of amplification and/or sequencing of the said virus using the primers of the invention.
  • amplification or sequencing of nucleic acid using the primers of the invention is indicative of the presence of the SPeLV virus in the said sample.
  • sequencing it is within the scope of the invention to detect SPeLV in samples, or to screen for SPeLV in biological materials, with deep sequencing technics, such as pyro-sequencing.
  • the said method of detection can be used for detecting a virus homologous to SpeLV in a species other than pig, in particular in man.
  • the SPeLV virus When the SPeLV virus is detected at the protein level , it may be notably performed using specific antibodies.
  • the invention thus also encompasses antibodies directed against the SpeLV virus and their use in detecting the said virus.
  • the antibodies of the invention are capable of recognizing specifically the SPeLV VP1 protein.
  • the SPeLV protein has the sequence represented by SEQ ID NO: 5.
  • the antibodies of the invention can be used for detecting of the SpeLV virus by using in particular well known technologies such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry.
  • suitable techniques include FRET or BRET, single cell microscopic or histochemistery methods using single or multiple excitation wavelength and applying any of the adapted optical methods, such as electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g.
  • the invention also provides recombinant vectors comprising at least the polynucleotide of the invention as defined above.
  • the polynucleotide of the invention may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • Various vectors are publicly available.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Thus, it is within the scope of the invention to provide a vector for expressing any of the above defined polypeptides.
  • the polynucleotides encoding said proteins are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational sequences.
  • Expression vectors include plasmids, YACs, cosmids, retrovirus, adenovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of the protein of interest.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • Polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable host cell. Transformation can be performed by any known method for introducing polynucleotides into a cell host. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNA into nuclei. Therefore, the invention also encompasses a host cell comprising a vector of the invention.
  • the said host cell is a bacterial cell; more preferably, it is a eukaryotic cell; even more preferably it is a mammalian cell.
  • the nature of the host cell will be dictated by the intended use of the vector of the invention .
  • a cloning vector will usually be maintained and propagated in bacterial cells.
  • stable expression is preferred.
  • cell lines which stably express the protein of the invention may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the SPeLV protein of the invention.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the said SPeLV protein.
  • the proteins of the invention may be prepared by growing a culture transformed host cells under culture conditions necessary to express the desired protein . The resulting expressed protein may then be purified from the culture medium or cell extracts. Soluble forms of the protein of the invention can be purified from conditioned media. Membrane-bound forms of protein of the invention can be purified by preparing a total membrane fraction from the expressing cell and extracting the membranes with a non-ionic detergent such as Triton X-100.
  • the proteins can be purified using methods known to those skilled in the art.
  • the proteins of the invention can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the concentrate can be applied to a purification matrix such as a gel filtration medium.
  • a purification matrix such as a gel filtration medium.
  • an anion exchange resi n can be em ployed , for exam ple, a matrix or su bstrate havi n g pen da nt diethylaminoethyl (DEAE) or polyetheyleneimine (PEI) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed .
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred (e.g. , S-Sepharose B columns).
  • the purification of the MU-1 protein from culture supernatant may also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-Toyopearl or Cibacrom blue 3GA Sepharose B; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity ch romatography.
  • RP-HPLC reverse-phase h igh performance liqu id chromatography
  • Affinity columns including antibodies to the protein of the invention can also be used in purification in accordance with known methods. Some or all of the foregoing purification steps, in various combinations or with other known methods, can also be employed to provide a substantially purified isolated recombinant protein.
  • the isolated protein of the invention is purified so that it is substantially free of other mammalian proteins. It is thus also an aspect of the invention to provide a method for producing a recombinant SPeLV protein of the invention.
  • the method of the invention comprises the steps of: (a) introducing a nucleic acid encoding the recombinant SPeLV protein into one of the host cell described above;
  • SPeLV protein it is herein referred to a protein directly encoded by the open reading frame of the swine virus of the invention, or a cleavage product thereof.
  • the SpeLV protein of the invention can be the polyprotein precursor, or any of the capsid proteins VP1 , VP2, or VP3.
  • the SPeLV protein of the invention is a protein having the amino acid sequence represented by SEQ ID NO: 3, or a cleavage product thereof, such as for example the protein having the sequence of SEQ ID NO:5.
  • Proteins of the invention may be used to screen for agents which are capable of binding to the said protein. Binding assays using a desired binding protein, immobilized or not, are well known in the art and may be used for this purpose using the protein of the invention. Purified cell based or protein based (cell free) screening assays may be used to identify such agents.
  • the SpeLV virus is the etiological agent of a number of human pathological conditions, including intrauterine fetal death (I U F D), malformations, placental inflammation, myocarditis, encephalitis, Guillain-Barre syndrome, and diabetes mellitus type 1 .
  • the present invention also provides a method of i nd ucing neutral izing antibodies against the SPeLV virus in a mammal, comprising the administration of a vaccinal SPeLV composition.
  • the present invention is drawn to a vaccinal SPeLV composition, for its use inducing neutralizing antibodies against the SPeLV virus in a mammal.
  • the vaccinal SpeLV composition of the invention is for preventing intrauterine fetal death (I UFD), malformations, placental inflammation, myocarditis, encephalitis, Guillain-Barre syndrome, and diabetes mellitus type 1 .
  • the vaccinal SpeLV composition of the invention is for preventing diabetes mellitus type 1 .
  • the present invention also relates to a vaccinal S PeLV composition .
  • vaccinal SpeLV composition it is herein referred to a composition comprising vaccinal SPeLV virus and/or at least one SpeLV protein of the invention and/or at least one polynucleotide encoding an SPeLV protein of the invention.
  • a "vaccinal SPeLV virus” is an SPeLV virus which is capable of inducing one or more immune responses against SpeLVI , as well as any other existing, but not yet identified, SpeLV serotype.
  • Immune responses according to the present invention include the humoral immune response and the cellular immune response.
  • a vaccinal SPeLV virus according to the invention is capable of inducing neutralizing . opsonizing antibodies or antibodies mediating Antibody Dependant Cell Cytotoxicity against SpeLVI , as well as any other existing, but not yet identified, SpeLV serotype.
  • vaccinal SPeLV virus viruses such as inactivated SPeLV virus, attenuated SpeLV virus, and chimeric SPeLV virus.
  • An SPeLV virus is "inactivated” if it is unable to replicate to any significant degree in cells permissive for replication of wild-type SPeLV virus.
  • An SPeLV virus which can replicate in a permissive cell but only to a degree significantly lower than a wild-type SPeLV virus is an "attenuated SPeLV virus”.
  • a "chimeric SPeLV virus” is a non- SPeLV virus which has been genetically engineered to express one or more SPeLV envelope proteins.
  • the vaccinal SPeLV composition of the invention may also comprise an SPeLV protein of the invention.
  • the vaccinal SPeLV composition of the invention may comprise the polyprotein precursor or the capsid proteins, VP1 , VP2 or VP3, or any combination of these 4 polypeptides.
  • the vaccinal SPeLV composition of the invention may also comprise at least one polynucleotide encoding an SPeLV polypeptide. It is possible to administer the polynucleotide of the invention to the said subject using gene therapy techniques.
  • the vaccinal composition of the invention may contain the SPeLV virus.
  • th e vacci n al com position of th e i nvention may com prise th e polynucleotide of the invention carried by a vector suitable for administration to a patient.
  • Such vectors may be either derived from a virus or from a non-viral origin.
  • Non-viral vectors include plasmids.
  • a plasmid may be a conditionally replicating plasmid that is incapable of replicating in the patients for safety reasons.
  • These plasm ids may be based on the plasmids described i n the patent PCT applications WO 97/10343 and WO 2009/027351.
  • Naked plasmid DNA can be directly injected into muscle cells (Wolff et al, Science, 1990) or attached to gold particles that are bombarded into the tissue (Cheng et al, Proc. Natl. Acad. Sci. U.S.A, 1993). Though not very efficient, this can result in prolonged low level expression in vivo.
  • the plasmid DNA can also be transfected into the cell with the use of non-viral gene delivery vectors, termed "self-assembled" systems, based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids (Eliyahu et al., Molecules, 2005).
  • the vector is a viral vector.
  • the recombinant viral vectors can transduce the cell type it would normally infect.
  • the nonessential genes are provided in trans, either integrated into the genome of the packaging cell line or on a plasmid.
  • viruses such as poxvirus, adenovirus, adeno-associated virus (AAV), lentivirus, or herpes si mplex virus 1 (HSV1 ), are available for gene therapy. All of them are encompassed within this invention.
  • Adenoviral vectors are currently the most frequently used viral vectors in gene therapy in humans.
  • third-generation (or "gutless") adenoviral vectors (Lindermann and Thomasler, Thromb. Haemost., 2009) is preferred for the use in the present invention. Said vectors need not be detailed here, since the skilled person is fully aware of the characteristics and uses of said adenoviral vectors.
  • the AAV used for treating a neuromuscular disease according to the invention is preferentially an AAV1 , i.e. its capside is of the serotype 1 .
  • AAV1 has been shown to be the most efficient for muscle cells transduction.
  • the sequences of a viral origin, and in particular the ITRs, associated to the transgene are preferably of AAV2 origin .
  • the resulting AAV-based vector of the invention has, preferentially, a 2/1 pseudotype.
  • AAV6, AAV8 or AAV9 also effectively transduce striated muscle cells, while AAV5 is highly efficient in transducing neural cells in the brain (Markakis et al., Molecular Therapy, 2010); all of them can therefore be used successfully in the context of the invention.
  • AAV-based vectors Like adenoviral vectors, the AAV-based vectors have already been used extensively by the skilled person for gene therapy purposes (see e.g. Michelfelder and Trepel, Adv Genet., 2009); there is thus no need for detailing methods for constructing and using the said AAV vectors.
  • the skilled person may use a lentiviral vector to deliver the proteins of the invention.
  • the said lentiviral is a self-inactivating (SI N) lentivirus.
  • the lentiviral vector genome comprises, as an inserted c/s-acting fragment, at least one polynucleotide consisting in the DNA flap (Zennou et al ., Cell, 101 : 1 73-185, 2000; WO 99/55892; WO 01 /27304; WO 2009/019612) or containing such DNA flap.
  • the DNA flap is inserted upstream of the polynucleotide of interest, advantageously but not necessarily to be located in an approximate central position in the vector genome.
  • any lentiviral vector can be used in the context of the present invention .
  • the construction and the manipulation of lentiviral vectors are well known to the skilled person.
  • poxvirus refers to a virus belonging to the Poxviridae family.
  • poxvirus according to the invention may be obtained from canarypox (e.g. ALVAC as described in WO 95/27780), fowlpox (e.g. TROVAC as described in Paoletti et al., Dev. Biol. Stand. 1995) or vaccinia virus, the latter being preferred.
  • Suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., Virol. 1990a and 1990b; Johnson et al., Virol. 1993), the Wyeth strain, NYVAC (see WO 92/15672 and Tartaglia et al., Virol. 1992) and the highly attenuated modified Ankara (MVA) strain (Mayr et al., Infection 1975).
  • the basic techniques for inserting the nucleic acid molecule and associated regulatory elements required for expression in a poxviral genome are already available to the person of skills in the art (Paul et al., Cancer Gene Ther. 2002; Piccini et al., Methods Enzymol 1987; US 4,769,330; US 4,772,848; US 4,603,1 12; US 5,100,587; and US 5,179,993).
  • a "vaccinal composition” is a composition comprising an immunoeffective quantity of an antigen sufficient to induce a specific immune response against a pathogen in an immunocompetent mammal.
  • the immune response according to the invention comprises the synthesis of neutralizing antibodies
  • mammal comprises individuals of the mammalian family, including cow, dogs, horse, primates, pigs, rabbits, cats, and humans.
  • the mammals of the invention are humans.
  • the protein or polynucleotide or SPeLV virus is preferably formulated in the vaccinal composition in an effective amount.
  • An "effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result, i.e., to immunize effectively the patient.
  • An effective amount as meant herein should also not have any toxic or detrimental severe effects.
  • the vaccinal SPeLV compositions of the invention comprise, in addition to the SPeLV protein or SPeLV polynucleotide or SPeLV virus, one or more pharmaceutically acceptable excipients.
  • Suitable excipients are well known in the art. Suitable excipients are typically large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et a/., Vaccine, 19: 21 18, 2001 ), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art.
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20.sup.th edition.
  • the vaccinal SPeLV composition of the invention may contain, in addition to the carrier and SPeLV protein or SPeLV polynucleotide or SPeLV virus, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • the vaccinal SPeLV composition of the invention may optionally comprise one or more adjuvants to enhance the immunogenicity of the said composition in a mammal.
  • Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes.
  • the adjuvant may be an oil-in-water emulsion adjuvants (EP 0 399 843B), as well as combinations of oil in water emulsions and other active agents (WO 95/1721 0; WO 98/56414; WO 99/12565; WO 99/1 1241 ).
  • oil emulsion adjuvants have been described, such as water-in-oil emulsions (U.S. Pat. No. 5,422, 109; EP 0 480 982 B2) and water-in-oil-in- water emulsions (U .S. Pat. No. 5,424,067; EP 0 480 981 B1 ).
  • the adjuvant examples include MF59, AF03, AF04, AF05, AF06 and derivatives thereof.
  • the adjuvant may be a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide, an alkyl glucosamide phosphate, an oil in water emulsion or combinations thereof.
  • saponins include Quil A and purified fragments thereof such as QS7 and QS21 .
  • the protein or polynucleotide or SPeLV virus of the invention is administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above.
  • administrations may include injection via the intramuscular, intraperitoneal, intradermal, transcutaneous or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
  • Th us one aspect of the present disclosure is a method of immunizing a human host against a disease caused by infection of SpeLV virus, which method comprises administering to the host an immunoprotective dose of the vaccinal SPeLV composition of the present disclosure.
  • the present invention also includes kits for achieving immunization against SpeLV-related conditions, e.g. intrauterine fetal death (IUFD), malformations, placental inflammation, myocarditis, encephalitis, Guillain-Barre syndrome, or diabetes mellitus type 1 .
  • the kit according to the invention comprises at least one vaccinal SPeLV composition as described above.
  • the kit of the invention therefore comprises at least one container holding the at least one vaccinal SPeLV composition.
  • the kit of the invention may also advantageously contain an explanatory brochure including useful information for administration of the said compositions.
  • the vaccinal compositions wh ich may be used in the kit accordi ng to the invention i nclude the vacci nal compositions described herein in relation to the method of immunization according to the invention. If the vaccinal compositions are provided in lyophilized form, the kit will advantageously comprise at least one additional container holding a solution which can be used to reconstitute a lyophilized vaccinal composition suitable for administration by intradermal , transcutaneous, subcutaneous , or i ntram uscu lar ad m i n istration . Pharmaceutically acceptable diluents and carriers may be used for reconstitution.
  • the examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.
  • Nucleic acid samples extracted from feces taken from two healthy pigs have been screened. These nucleic acids have been amplified by the bacteriophage phi29 polymerase based multiple displacement amplification (MDA) assay using random primers. The ligation and amplification were performed with the QuantiTect® Whole Transcriptome kit (Qiagen) according essentially to the manufacturer's instructions. This provides concateners of high molecular weight DNA. Resulting DNAs from the two pigs were pooled.
  • MDA multiple displacement amplification
  • Sequencing was conducted by an lllumina HiSeq 2000 sequencer. 5 ⁇ g of high molecular weight DNA resulting from isothermal amplification was fragmented into 200 to 350 nt fragments, to which were ligated adapters. 27146966 reads of 96 nt were derived from the sample.
  • Sorting out the flow of l llumina sequences was first done by a subtractive database comparison procedure. To this end, the whole host genome sequence (Sus scrofa, ref: susScr2) was scanned with the reads using SOAPaligner (remaining 24834733 reads). A number of assembly programs dedicated to short or medium- sized reads (Velvet, SOAPdenovo, CLC) have been tested for their efficiency in our pipeline. Optimal parameters have been set. The comparison of the single reads and contigs with already known genomic and taxonomic data was done on generalist databases maintained locally (nt, nrprot). The aforementioned databases were scanned using BLASTN and BLASTX. Binning (or taxonomic assignment) was based on the best hits among reads with a significant e-value. 2. Results
  • SPeLVI SPeLV type 1
  • a set of 12 primer pairs encompassing the whole target genome was defined (table 1 ).
  • the primers allowed to amplify 6057 nt of the genome by PCR, corresponding to around 80 % of the genome, which was sequenced by a conventional Sanger method.
  • the figure 3 shows the SPeLVI sequenced genome. The quasi-full length sequence of the large polyprotein was obtained.
  • the polyprotein is encoded by a single open reading frame that encompasses all the RNA genome, except the 5' and 3' untranslated regions (UTRs).
  • the figure 4 shows the nucleotide sequence of the major part of the polyprotein of SPeLVI .
  • the figure 5 corresponds to the deduced amino acid sequences of the polyprotein of SPeLV 1 .
  • the figure 6 shows the seq uence of VP 1 of SPeLVI translated from its nucleotide sequence: the probable NH2 and COOH ends have been deduced by homology with the other viruses of the Parechovirus genus. Nucleotide and amino acid alignments with the closest known VP1 from the HPeVs and LV are shown in figure 7 and 8 respectively.
  • the sequence identity with LV is 45.6% (nt) or 29.1 % (aa.).
  • the sequence identity of SPeLVI with the closest HPeV type is 44.7% (nt -HpeV2-) or 26.7% (aa.-HPeV1 -) (table 2). Alignment of this protein with other representatives of the H PeVs and LV by MUSCLE using curation by G-Blocks allowed to generate a phylogenetic tree using PhymL (Phylogeny package)(figure 9).
  • the SPeLVI VP1 is devoid of RGD motif at the C terminus of the protein.
  • a RGD motif is present in HPeV1 , 2,4,5 and 6 but is lacking in HPeV3, 7 and 8. This motif is important for virus entry in cells through interaction with alpha v beta 5 and alpha v beta 3 integrins.
  • the receptor of HPeV 3, 7 and 8 is yet unknown (Seitsonen J, J. of Virology, 2010).
  • VP1 of SPELV1 could reflect similar tropism than HPeV 3, which is the main HPeV found in Central Nervous Disease-related disease (Verboon-Maciolek MA, Ann. Neurol.2008, Abed Y., Emerg. Infect. Dis.2006).
  • VP1s have been extracted from the best blastp hit between SPeLVI polyprotein and other parechoviruses and Ljungan Virus fully annotated polyproteins (except for HPeVIOand HPeV11 were only VP1 was available).
  • herd # 03 another herd for which stools samples were available.

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Abstract

La présente invention concerne le SPe LV, un virus de type paréchovirus porcin apparenté au virus Ljungan (LV) et au paréchovirus humain (HPeV). La présente invention concerne en outre de nouvelles protéines codées par le SPeLV, qui présentent une certaine homologie avec les protéines de capside du LV et du HPeV. La présente invention concerne en outre des procédés destinés à monter une réponse immunitaire contre le SPeLV chez un sujet.
EP12797926.8A 2011-12-07 2012-12-07 Identification d'un virus porcin de type paréchovirus et applications Withdrawn EP2788477A2 (fr)

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