Patent Application
20100158947
The invention relates generally to the field of virology. More particularly, the present invention relates to methods for determining the permissiveness of a cell for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
The invention further provides methods and compositions related to the generation of host cells permissive for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
Methods of using said cells thus identified or thus generated, in preparing a culture of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, as well as the use of said virus for the purpose of vaccine production or diagnosis, are also provided by the present invention.
A “mystery swine disease” appeared in the 1980's, and is present ever since in pig industry causing important economical damage worldwide (Neumann et al., 2005). The causative agent, designated porcine reproductive and respiratory syndrome virus (PRRSV), was first isolated in the Netherlands in 1991 and shortly after in the USA. It is a small enveloped positive-stranded RNA virus that is classified in the order Nidovirales, family Arteriviridae, genus Arterivirus together with equine arteritis virus, lactate dehydrogenase-elavating virus and simian hemorrhagic fever virus based on similar morphology, genomic organization, replication strategy and protein composition. In addition, they share a very narrow host tropism and a marked tropism for cells of the monocyte-macrophage lineage (Plagemann & Moennig, 1992). More specifically, in vivo PRRSV infects subpopulations of well-differentiated macrophages, with alveolar macrophages being the primary target cells, although in infected boars also testicular germ cells have been shown to allow PRRSV replication (Sur et al., 1997). In vitro, PRRSV replicates in primary cultures of alveolar macrophages and peripheral blood monocytes (PBMC), although PBMCs need treatments to improve infection (Delputte et al., 2007). Furthermore, African green monkey kidney cells and derivates thereof (Marc-145 and CL2621) have been shown to sustain PRRSV infection, although they are not from porcine origin and do not belong to the monocyte-macrophage lineage (Kim et al., 1993; Mengeling et al., 1995). Notwithstanding this very restricted cell tropism of PRRSV, the virus is able to replicate in several non-permissive cell-lines upon transfection of its viral RNA, indicating that cell tropism is determined by the presence or absence of specific receptors on the cell surface or other proteins involved in virus entry (Kreutz, 1998; Meulenberg et al., 1998).
So far, two PRRSV receptors were identified on macrophages, namely heparan sulphate (Delputte et al., 2002) and sialoadhesin (Vanderheijden et al., 2003; Wissink et al., 2003). In addition, Wissink et al. (2003) found a 150 kDa protein doublet to be involved in PRRSV infection of macrophages, however the identity of the N-glycosylated proteins is still unknown. In the current model for PRRSV infection of macrophages, PRRSV first binds to heparan sulphate most likely leading towards virus concentration. However, this first binding is rather unstable and is followed by binding to sialoadhesin and subsequent internalization (Delputte et al., 2005). Upon internalization, the virus is transported towards endosomes were a drop in pH is required for proper virus replication (Kreutz & Ackermann, 1996; Nauwynck et al., 1999). Despite this elegant research, the model is still incomplete. Transient expression of sialoadhesin in non-permissive PK-15 cells results in binding and internalization of the virus, but fusion and uncoating of the virus particles was not observed (Vanderheijden et al., 2003), indicating that other proteins are needed for virus disassembly, essential for virus replication.
PRRSV infection of Marc-145 cells makes use of a heparin-like molecule on the surface of Marc-145 cells (Jusa et al., 1997), resembling the initial step of PRRSV infection of macrophages. However, since sialoadhesin is absent from Marc-145 cells, virus entry will differ between the two cell-types. In Marc-145 cells, the intermediate filament vimentin has been described to bind to the PRRSV nucleocapsid protein and it has been suggested to interact with other cytoskeletal filaments to mediate transport of the virus in the cytosol (Kim et al., 2006). Recently, CD151 was found to interact specifically with PRRSV 3′ untranslated region (UTR) RNA (Shanmukhappa et al., 2007). CD151 was proposed to be possibly involved in fusion between the viral envelope and the endosome or to relocalize the ribonucleoprotein complexes to promote viral replication. Still, further research is needed to elucidate their precise molecular modes of action during PRRSV infection.
Recently, the scavenger receptor CD163 has been described to play a role in PRRSV infection of Marc-145 cells and to make some non-permissive cells somewhat susceptible to PRRSV upon expression (Calvert et al., 2007), where others remain unproductive upon infection, despite expression of CD163 (Calvert et al., 2007). Although the CD163 gene was originally isolated from macrophages, thus far no role for CD163 in PRRSV infection of its primary target cells has been shown. Also, the mechanism by which CD163 confers partial susceptibility of selected cell types to PRRSV infection was not elucidated.
We demonstrated that both sialoadhesin and CD163 are involved in PRRSV infection of macrophages. In addition, expression of recombinant forms of both CD163 and sialoadhesin in non-permissive cells renders all of them susceptible to PRRSV infection resulting in the production and release of infectious progeny virus. In contrast, when only CD163 is present, infection is clearly less efficient, and even absent in some cell types. In addition, viral adaptation that leads to antigenic differences in viral strains grown in cells only expressing CD163 when compared to the wild type viruses, has been reported.
Based on detailed analysis of the kinetics of PRRSV infection, both in primary macrophages and in cells expressing sialoadhesin and CD163, a role for CD163 in virus fusion and uncoating is proposed. Compared to the above mentioned systems, i.e. cells solely expressing CD163 or sialoadhesin, it has been found that the combination of CD163 and sialoadhesine expression in one cell provides permissive cells that are highly efficient to sustain viral replication, and which closely mimic the entry of the virus in the natural host, i.e. the known subpopulations of well-differentiated macrophages, in particular alveolar macrophages being the primary target cells of the virus. Such mimicry of the entry of the natural target cells will certainly reduce or avoid virus adaptation in cell culture and the associated genetic and antigenic changes that might result in viruses with altered epitopes. Such modified epitopes can have tremendous effects on the antigenicity of vaccine viruses produced on given cells, resulting in loss of induction of important neutralizing antibodies. Clearly, avoiding changes in epitopes associated with adaptation during cell culture will be beneficial for production of vaccine virus.
The results presented show that Sn and CD163 work synergistically, since co-expression of both molecules results in higher virus production compared to expression of either of the two receptors alone, and this in all cell types tested. In addition, the molecular basis of this synergistic effect was elucidated, being the receptors acting at different steps during virus entry. Sn is expressed on the surface of target cells and very efficiently captures the virus and internalizes it into the cell in endosomes. CD163 on the other does not interact with the virus on the cell surface and does not internalize the virus, but co-localizes with the virus in early endosomes, where it mediates virus uncoating, followed by release of the RNA genome in the cytoplasm. Once the genome is released in the cytoplasm, genome translation, transcription and virus replication can proceed.
The finding that CD163, which is also expressed on the cell surface, does not act on the cell surface during infection, but rather interacts with the virus in endosomes inside the cell, is quite surprising. Generally, cellular receptors act during virus attachment or internalization, or direct fusion at the cell surface. This model, in which the CD163 receptor is not active on the cell surface but acts on the virus in endosomes is surprising and explains the unanticipated and cooperative action of both Sn and CD163 during virus infection, resulting in very efficient virus infection and production of high titers of virus.
Thus, these results provide new means to generate a PRRSV permissive cell that allow for efficient viral replication, with less adaptation and accordingly solves the problems recognized in the art.
This invention is based on the characterization that both sialoadhesin and CD163 are not only involved in the permissivity of macrophages, the primary target cells, for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), but that these molecules also act at different steps of virus infection, thus allowing a cooperative effect during infection, resulting in enhanced virus production.
It has been found that non-permissive cells can be rendered permissive, or the permissivity of partially susceptible cells can be increased by directing said cells to express both sialoadhesin and CD163.
It is accordingly a first objective of the present invention to provide methods to identify the permissiveness of cells for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV); said method comprising determining CD163 and sialoadhesin expression in said cells; wherein cells having a both CD163 and sialoadhesin expression, are identified as permissive cells for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae.
In a second objective, the present invention provides a method to generate a cell(s) permissive for, or to increase the permissiveness of a cell(s) for a virus of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for PRRSV, said method comprising treating said cells to yield an expression of both CD163 and sialoadhesin.
In a further objective, the present invention provides a method for preparing a culture of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, said method comprising providing a cell line identified or obtained using any one of the aforementioned methods, infecting said cell line with virus and harvesting the virus from the cell culture.
Once the virus has been grown to high titres, it can be processed according to the intended use, for example in diagnosis or vaccine production, by means known in the art. For example, but not limited to, inactivating the harvested viruses with formalin, BPI, BEA or gamma-irradiation, for use in vaccines. In the alternative, the viral strain used in the infection, may be an attenuated strain for use in the production of live, attenuated vaccines.
Hence it is also an embodiment of the present invention to provide a vaccine comprising a viral strain/serotype obtained using the aforementioned method. As already mentioned hereinbefore, due to the synergetic effect of CD163 and sialoadhesin, there will be a reduction in viral adaptation and loss of altered epitopes. This taken together with the increased viral production has a tremendous effect on the antigenicity of vaccine viruses produced using the methods of the present invention. As is known to a person skilled in the art, the latter is also beneficial in isolating further viral strains from in vivo samples when diagnosing PRRSV infection in a subject.
It is clearly an objective of the present invention to provide cell lines identified or obtained using any one of the aforementioned methods. Cell lines identified using the methods of the present invention include primary cell cultures and continuous cell lines obtainable thereof, but for the natural host cells, i.e. the known subpopulations of well-differentiated macrophages, in particular the alveolar macrophages that are the primary target cells of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular of a PRRSV infection. In one embodiment said cells consist of non-permissive PRRSV cells, such as for example PK-15, CHO, BHK-21 and Hek293t cells, expressing both Sn and CD163; with in a particular embodiment the CHO cells stably expressing sialoadhesin and CD163 deposited on May 14, 2008 at the Belgian Coordinated Collections of Microorganisms as CHO—Sn/CD163 IC5; CHO—Sn/CD163 ID9 and CHO—Sn/CD163 IF3 with the respective accession numbers LMBP 6677CB; LMBP 6678CB; and LMBP 66779 CB respectively.
The cell lines identified or obtained using any one of the aforementioned methods, can also be used in a method of diagnosing a viral infection of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular of a PRRSV infection in a subject. It is accordingly a further object of the present invention to provide a method for diagnosing a viral infection of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular of a PRRSV infection in a subject, said method comprising contacting a cell line identified or obtained using any one of the methods of the present invention with a sample taken from said subject and determine whether viral replication occurs.
Alternatively, the viral infection is determined by assessing the presence of virus-specific antibodies in the sample taken from said subject. In this embodiment the cell line identified or obtained using any one of the methods of the present invention is infected with a virus of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular with PRRSV, and the reaction of the antibodies in a sample taken from said subject is done by means well known to the person skilled in the art.
In these diagnostic methods the sample taken from the subject, is typically a biological fluid; such as for example serum, colostrums, bronchoalveolar lavage fluids, saliva, urine or faeces; tissue or a tissue extract. The tissue or tissue extract to be analyzed includes those which are known, or suspected, to be permissive for the virus such as, for example PBMC (peripheral blood mononuclear cells), alveolar macrophages, lymphoid tissues such as lymph nodes, spleen, tonsils and thymus and non-lymphoid tissues such as lungs and liver.
In a final embodiment the cell lines identified or obtained using any one of the aforementioned methods, can be used in a method to identify anti-viral compounds, i.e. anti-viral compounds for a virus of the family Arteriviridae or Coronaviridae or Asfarviridae as defined herein, in particular for PRRSV. The invention accordingly provides in a further objective, a method to identify anti-viral compounds, said method comprising; —contacting a cell line infected with a virus of the Arteriviridae or Coronaviridae or Asfarviridae, with the compound to be tested; and determine the capability of said test compound to modulate the viral replication in said cell line.
The capability of a compound to modulate the viral replication can be determined by using amongst others, the presence of infectious viral particles in the media. The latter can be determined using any one of the available protein measurement techniques and is typically determined using late viral specific antibodies, in particular using (virus) specific antibodies as provided hereinafter.
In the immunoassays related thereto, the amount of viral protein produced can be quantified by any standard assay such as, for example, using a luminescence assay, a chemiluminesence assay, an enzyme-multiplied immunoassay technology (EMIT) assay, a fluorescence resonance excitation transfer immunoassay (FRET) assay, an enzyme channeling immunoassay (ECIA) assay, a substrate-labeled fluorescent immunoassay (SLFIA) assay, a fluorescence polarization assay, a fluorescence protection assay, an antigen-labeled fluorescence protection assay (ALFPIA), or scintillation proximity assay (SPA).
Alternatively, the effect of the compound on viral replication is determined by assessing the virus titres in the media, by quantifying numbers of infected cells by immunocytochemistry or by using a MTS cytotoxicity assay to determine the cytotoxic concentration of the viral particals in the media.
As already mentioned hereinbefore, the present invention is based on the observation that both sialoadhesin and CD163 are involved in the permissivity of macrophages, the primary target cells, for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In addition, and surprisingly, it was shown that CD163 does not act at the cell surface of susceptible cells during attachment and internalization, but rather acts during virus uncoating and genome release inside the cell in endosomes. This unexpected finding explains why CD163 acts synergistically with Sn during virus infection, since the latter interacts with the virus at the cell surface to allow virus attachment and internalization.
It is accordingly a first objective of the present invention to provide methods to identify the permissiveness of cells for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV); said method comprising determining CD163 and sialoadhesin expression in said cells; wherein cells having a both CD163 and sialoadhesin expression, are identified as permissive cells for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae.
Asfarviridae is a family of icosohedral enveloped viruses whose genome consists of a single molecule of linear double-stranded DNA of about 150000-190000 nucleotides long. The name of the family is derived from African Swine Fever And Related Viruses. African Swine Fever Virus (ASFV) is the type species of the Asfivirus genus and is the sole member of the family. Recently, porcine CD163 polypeptide has been surmised by implication to be the cellular receptor for African swine fever virus (ASFV) (Sanchez-Torres et al., 2003).
The Arteriviridae family is grouped with the Coronaviridae and Roniviridae to form the order Nidovirales. All members of the order have enveloped particles containing a single species of single-stranded RNA that encodes for a number of proteins by means of a series of nested (Latin Nido=nest) subgenomic RNAs. The family Arteriviridae contains those members with spherical virions 45-60 nm in diameter (those of the family Coronaviridae are more than 100 nm) and which infect mammals. Their genome consists of single-stranded RNA of size 12-16 kb and with a 3′-polyA tail. Two large, overlapping ORFs at the 5′-end of the genome encode the major non-structural proteins and are expressed as a fusion protein by ribosomal frameshift. Downstream are up to 9 other genes, mostly or entirely encoding structural proteins, and these are expressed from a 3′-coterminal nested set of subgenomic RNAs.
Viruses of the family of Arteriviridae includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). The Arterivirus having the greatest economic importance is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
Thus in one objective, the methods of the present invention are used to identify and/or modulate the permissivity of cells for a virus selected from the group consisting of African swine fever virus (ASFV), equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), simian hemorrhagic fever virus (SHFV) or porcine reproductive and respiratory syndrome virus (PRRSV), as well as variants thereof including orthologs and paralogs; in particular human orthologs. In a particular embodiment the methods of the present invention are used to identify and/or modulate the permissivity of cells for PRRSV.
As used herein, the terms “permissiveness of a cell(s)”, “permissivity of cell(s)” and “permissive cell(s)” refers to the ability in which a particular virus, i.e. a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, can complete its replication cycle in a given cell. This in contrast to “non-permissive” cells that do not support complete replication of a virus.
“CD163” is a member of the scavenger receptor cysteine-rich (SRCR) family of transmembrane glycoproteins, and is thought to be expressed exclusively on monocytes and macrophages. One identified role of CD 163 is to inhibit oxidative tissue damage following hemolysis by consuming hemoglobin:haptoglobin complexes by endocytosis. The subsequent release of interleukin-10 and synthesis of hemeoxygenase-1 results in antiinflammatory and cytoprotective effects. The human CD163 gene spans 35 kb on chromosome 12, and consists of 17 exons and 16 introns.
A number of isoforms of the CD163 polypeptide, including membrane bound, cytoplasmic and secreted types, are known to be generated by alternative splicing (Ritter et al., 1999). cDNA sequences that encodes a porcine CD 163 polypeptide (Genbank accession number AJ311716), a murine CD 163 polypeptide (Genbank access number AF274883), as well as multiple human variants, exemplified by Genbank access numbers AAH51281 and CAA80543, have been reported.
As used herein the “CD163” polypeptide is meant to be a protein encoded by a mammalian CD 163 gene, including allelic variants as well as biologically active fragments thereof containing conservative or non-conservative changes as well as artificial proteins that are substantially identical, i.e. 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the aforementioned CD 163 polypeptides. In a particular embodiment the CD 163 polypeptide is 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, (CO, 97%, 98%, or 99% identical to the porcine CD 163 (encoded by Genbank Accession No AJ311716 or bankit927381 EU016226).
By analogy, the “CD163” polynucleotide is meant to include allelic variants as well as biologically active fragments thereof containing conservative or non-conservative changes as well as any nucleic acid molecule that is substantially identical, i.e. 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the aforementioned CD163 encoding polynucleotides. In a particular embodiment the sialoadhesin polynucleotide is 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid molecule encoding for porcine CD163 (Genbank Accession No AJ311716 or bankit927381 EU016226).
Biologically active fragments of CD163 are meant to include fragments that retain the activity of the full length protein, such as the isoform with SwissProt accession number Q2VL90-2, the soluble form of CD163 (sCD163), or fragments containing at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the SRCR domain(s).
“Sialoadhesin” is a lectin-like adhesion shown to bind glycoconjugate ligands in a sialic acid-dependent manner and characterized in having conserved sialic acid binding sites. It is a transmembrane glycoprotein involved in cell-cell interactions and expressed only by a subpopulation of tissue macrophages.
cDNA sequences that encodes a porcine sialoadhesin polypeptide (Genbank accession number NM—214346), a murine sialoadhesin polypeptide (Genbank access number NM—011426), as well as a human variant (Genbank access number NM—023068), have been reported.
As used herein the “sialoadhesin” polypeptide is meant to be a protein encoded by a mammalian sialoadhesin gene, including allelic variants as well as biologically active fragments thereof containing conservative or non-conservative changes as well as artificial proteins that are substantially identical, i.e. 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the aforementioned sialoadhesin polypeptides. In a particular embodiment the sialoadhesin polypeptide is 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the porcine sialoadhesin (encoded by Genbank Accession No NM—214346).
By analogy, the “sialoadhesin” polynucleotide is meant to include allelic variants as well as biologically active fragments thereof containing conservative or non-conservative changes as well as any nucleic acid molecule that is substantially identical, i.e. 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the aforementioned sialoadhesin encoding polynucleotides. In a particular embodiment the sialoadhesin polynucleotide is 70%, 75%, 80%, 85%, 87%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid molecule encoding for porcine sialoadhesin (Genbank Accession No NM—214346).
Biologically active fragments of sialoadhesin are meant to include fragments that retain the activity of the full length protein, i.e. that retain the capability of binding a virus of the family of Arteriviridae includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV), in particular of binding PRRSV. Biologically active fragments include for example, the known soluble form of sialoadhesin, fragments containing at least 1, 2, 3 or 4 of the Ig-like domain(s); in particular the N-terminal domains; more in particular consisting of the N-terminal, variable, sialic acid-binding Ig-like domain.
As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably to refer polynucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs (e.g., inosine, 7-deazaguanosine, etc.) thereof. “Oligonucleotides” refer to polynucleotides of less than 100 nucleotides in length, preferably less than 50 nucleotides in length, and most preferably about 10-30 nucleotides in length. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
“Polypeptide” refers to any peptide or protein comprising amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
“Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications (see, for instance, Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Postranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182: 626-646 and Rattan et al., “Protein Synthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci (1992) 663: 4842).
The percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms which compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies/identities: BLAST, gapped BLAST, BLASTN and PSI-BLAST, which may be used with default parameters.
The algorithm GAP (Genetics Computer Group, Madison, Wis.) uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty=12 and gap extension penalty=4.
Another method for determining the best overall match between a nucleic acid sequence or a portion thereof, and a query sequence is the use of the FASTDB computer program based on the algorithm of Brutlag et al (Comp. App. Biosci., 6; 237-245 (1990)). The program provides a global sequence alignment. The result of said global sequence alignment is in percent identity. Suitable parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Suitable parameters to calculate percent identity and similarity of an amino acid alignment are: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter.
The “expression” generally refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the mRNA is subsequently translated into peptides, polypeptides or proteins. Hence the “expression” of a gene product, in the present invention of CD163 and sialoadhesin, can be determined either at the nucleic acid level or the protein level.
Detection can be by any appropriate method, including, e.g., detecting the quantity of mRNA transcribed from the gene or the quantity of nucleic acids derived from the mRNA transcripts. Examples of nucleic acids derived from an mRNA include a cDNA produced from the reverse transcription of the mRNA, an RNA transcribed from the cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified cDNA, and the like. In order to detect the level of mRNA expression, the amount of the derived nucleic acid should be proportional to the amount of the mRNA transcript from which it is derived. The mRNA expression level of a gene can be detected by any method, including hybridization (e.g., nucleic acid arrays, Northern blot analysis, etc.) and/or amplification procedures according to methods widely known in the art. For example, the RNA in or from a sample can be detected directly or after amplification. Any suitable method of amplification may be used. In one embodiment, cDNA is reversed transcribed from RNA, and then optionally amplified, for example, by PCR. After amplification, the resulting DNA fragments can for example, be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination. A specific amplification of differentially expressed genes of interest can be verified by demonstrating that the amplified DNA fragment has the predicted size, exhibits the predicated restriction digestion pattern and/or hybridizes to the correct cloned DNA sequence.
In hybridization methods a probe, i.e. nucleic acid molecules having at least 10 nucleotides and exhibiting sequence complementarity or homology to the nucleic acid molecule to be determined, are used. It is known in the art that a “perfectly matched” probe is not needed for a specific hybridization. A probe useful for detecting mRNA is at least about 80%, 85%, 90%, 95%, 97% or 99% identical to the homologous region in the nucleic acid molecule to be determined. In one aspect, a probe is about 50 to about 75, nucleotides or, alternatively, about 50 to about 100 nucleotides in length. These probes can be designed from the sequence of full length genes. In certain embodiments, it will be advantageous to employ nucleic acid sequences as described herein in combination with an appropriate label for detecting hybridization and/or complementary sequences. A wide variety of appropriate labels, markers and/or reporters are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. One can employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a signal that is visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
Detection of the level of gene expression can also include detecting the quantity of the polypeptide or protein encoded by the gene. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassay (RIA), ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS. One method to determine protein level involves (a) providing a biological sample containing polypeptides; and (b) measuring the amount of any immunospecific binding that occurs between an antibody reactive to the expression product of a gene of interest and a component in the sample, in which the amount of immunospecific binding indicates the level of the expressed proteins. Antibodies that specifically recognize and bind to the protein products of these genes are required for these immunoassays. These may be purchased from commercial vendors or generated and screened using methods well known in the art. See e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
In a second objective, the present invention provides a method to generate a cell(s) permissive for, or to increase the permissiveness of a cell(s) for a virus of the family Asfarviridae or Arteriviridae, in particular for PRRSV, said method comprising treating said cells to yield an expression of both CD163 and sialoadhesin.
CD 163 and sialoadhesin expression may be facilitated or increased by methods that involve the introduction of exogenous nucleic acid into the cell. Such a cell may comprise a polynucleotide or vector in a manner that permits expression of an encoded CD 163 or sialoadhesin polypeptide.
Polynucleotides that encode CD 163 or sialoadhesin may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein-coding region, or in a viral vector. Methods for introducing exogenous nucleic acid into the host cell well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Host cell systems of the invention include plant, invertebrate and vertebrate cells systems. Hosts may include, but are not limited to, the following: insect cells, porcine kidney (PK) cells, feline kidney (FK) cells, swine testicular (ST) cells, African green monkey kidney cells (MA-104, MARC-145, VERO, and COS cells), Chinese hamster ovary (CHO) cells, baby hamster kidney cells, human 293 cells, and murine 3T3 fibroblasts. Insect host cell culture systems may also be used for the expression of the polypeptides according to the invention. In another embodiment, the polypeptides are expressed using a drosophila expression system. Alternatively the polypeptides are expressed using plant-based production platforms such as for example described in Twyman R. M. et al., Molecular farming in plants: host systems and expression technology Trends Biotechnol. 21, 570-578.
The choice of a suitable expression vector for expression of the polypeptides according to the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pSport and pcDNA3 (Invitrogen), pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and modifier sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Rous sarcoma virus (RSV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.
Because CD163 sequences are known to exist in cells from various species, the endogenous gene may be modified to permit, or increase, expression of the CD 163 polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring CD163 promoter with all or part of a heterologous promoter, so that the cells express CD 163 polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous CD163 encoding sequences. [See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.] It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional cad gene, which encodes for carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the CD 163 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the CD163 coding sequences in the cells.
CD163 expression may also be induced by chemical treatment. Phorbol esters, especially phorbol myristyl acetate (PMA), activate one or more isozymes of the ubiquitous membrane receptor, protein kinase C (PKC) and are particularly preferred means of increasing CD163 expression. Other methods of intracellular calcium mobilization are also contemplated.
Sialoadhesin expression may also be induced by chemical treatment. It has been reported that IFN-α does increase and is even capable to induce sialoadhesin expression in the monocyte-macrophage lineage of cells. Thus IFN-α treatment is an alternative means of increasing/inducing sialoadhesin expression in a cell.
The cell lines identified or obtained using the methods of the present invention are clearly a further object of the present invention.
In a first embodiment these cell lines consist of primary cell cultures of cells identified as being permissive for a virus of the family Asfarviridae or Arteriviridae, in particular for PRRSV, provided said cells are not the natural host cells, i.e. the known subpopulations of well-differentiated macrophages, in particular the alveolar macrophages that are the primary target cells of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular of a PRRSV infection.
Cells that are cultured directly from an animal or person are known as “primary cells”. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability. Methods for growing suspension and adhesion cultures of primary cells are known to the person skilled in the art, such as for example described in “General Techniques of Cell Culture”, Maureen A. Harrison and Ian F. Rae, Cambridge University Press 2007.
As used in the methods of the present invention, the “primary cells” are derived from; cells that support the replication of said viruses, in particular of PRRSV. In one embodiment, said cells consist of the known subpopulations of differentiated cells of the monocyte/macrophage lineage, in particular the alveolar macrophages that are the primary target cells of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular of a PRRSV infection. In particular embodiments of the present invention, the “primary cells” are derived from the alveolar macrophages that are the primary target cells
This in contrast to “continuous cells” also known as “an established” or “immortalized” cell line that has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types.
In the context of the present invention, the continuous cells are either derived by immortalization from the primary cell cultures mentioned herein before or obtained from a well established continuous cell line treated, in particular by transfection with a nucleic acid sequence encoding CD163 and/or sialoadhesin, to yield a stable expression of both CD163 and sialoadhesin. In an alternative embodiment the CD163 expression may also be induced by chemical treatment in continuous cell lines stably expressing Sn. Phorbol esters, especially phorbol myristyl acetate (PMA), activate one or more isozymes of the ubiquitous membrane receptor, protein kinase C (PKC) and are particularly preferred means of increasing CD163 expression. Other methods of intracellular calcium mobilization are also contemplated. In analogy, continuous cells stably expressing CD163 may also be induced to express Sn by chemical treatment, using for example IFN-α.
Examples of continuous cell lines derived from the monocyte/macrophage lineage and useful in the context of the present invention are THP-1, MM-1, J774, SU-DHL, RAW264, 3D4, and others.
Several established methods exist for immortalizing mammalian cells in culture. Viral genes, including Epstein-Barr virus (EBV), Simian virus 40 (SV40) T antigen, adenovirus E1A and E1B, and human papillomavirus (HPV) E6 and E7 can induce immortalization by a process known as viral transformation. Although the process is reliable and relatively simple, these cells may become genetically unstable (aneuploid) and lose the properties of primary cells. For the most part, these viral genes achieve immortalization by inactivating the tumor suppressor genes that put cells into a replicative senescent state. The preferred method to immortalize cells is through expression of the telomerase reverse transcriptase protein (TERT), particularly those cells most affected by telomere length (e.g., human). This protein is inactive in most somatic cells, but when hTERT is exogenously expressed the cells are able to maintain telomere lengths sufficient to avoid replicative senescence. Analysis of several telomerase-immortalized cell lines has verified that the cells maintain a stable genotype and retain critical phenotypic markers.
The well established continuous cells used herein are typically selected from the group consisting of cells with leukocyte characteristics or non-leukocyte cells such as for example swine testicle cells, swine kidney cells (e.g. PK15 (CCL-33), SK-RST(CRL-2842)), epithelial cell cultures, skin keratinocytes (e.g. HEK001 (CRL-2404), CCD1102 (CRL-2310)), Vero cells (CCL-81), human fetal lung fibroblasts (e.g. HFL1 (CCL-153)), human embryonic lung cells (e.g. HEL299 (CCL-137)), Chinese Hamster Ovary cells (CHO) or human embryonic kidney cells (HEK).
Hence in a further embodiment the cell lines consist of continuous cells expressing both CD163 and sialoadhesin.
In a further objective, the present invention provides a method for preparing a culture of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, said method comprising providing a cell line identified or obtained using any one of the aforementioned methods, infecting said cell line with virus and harvesting the virus from the cell culture.
Once the virus has been grown to high titres, it can be processed according to the intended use, for example in diagnosis or vaccine production, by means known in the art.
For example, in case of vaccine production, the harvested viruses may be inactivated for example with formalin, BPI, BEA or gamma-irradiation, for use in vaccines. In the alternative, the viral strain used in the infection, may be an attenuated strain for use in the production of live, attenuated vaccines.
Hence it is also an embodiment of the present invention to provide a vaccine comprising a viral strain/serotype obtained using the aforementioned method.
Killed (inactivated) or live vaccines can be produced. To make a live vaccine, a viral isolate, or an attenuated or mutated variant thereof, is grown in cell culture. The virus is harvested according to methods well known in the art. The virus may then be concentrated, frozen, and stored at −70° C., or freeze-dried and stored at 4° C. Prior to vaccination the virus is mixed at an appropriate dosage, (which is from about 10 to 108 tissue culture infectious doses per ml), with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant.
The vaccine produced might also comprise an inactivated or killed vaccine comprising a PRRSV strain obtained by the methods of the invention. The inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines. The virus is then inactivated by treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), or other methods known to those skilled in the art. The inactivated virus is then mixed with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant. Examples of adjuvants include, but not limited to, aluminum hydroxide, oil-in-water and water-in-oil emulsions, AMPHIGEN, saponins such as QuilA, and polypeptide adjuvants including interleukins, interferons, and other cytokines.
Inactivation by formalin is performed by mixing the viral suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The virus-formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated virus mixture is then tested for residual live virus by assaying for growth on a suitable cell line.
Inactivation by BEI is performed by mixing the viral suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. The virus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours. The inactivated virus mixture is tested for residual live virus by assaying for growth on a suitable cell line.
The present invention now has as its object to provide the use of a virus, of an inactivated virus or of a vaccine of the present invention for preparing a medicament which is employed for the prophylactic and/or therapeutic treatment of PRRSV infection in animals, in particular in swine and piglets.
The vaccine used according to the invention advantageously is provided in a suitable formulation. Preferred are such formulations with a pharmaceutically acceptable carrier. This comprises, e.g., auxiliary substances, buffers, salts, preservatives.
In the diagnostic methods, the permissive cells of the present invention are contacted with a sample taken from an infected subject; the cells cultured to allow replication of the virus; the virus harvested from said cell culture and identified using art known procedures, such as for example using specific antibodies for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for PRRSV.
The (virus) specific antibody is in particular a monoclonal antibody or a derivative thereof, the latter preferably selected from the group of antibody fragments, conjugates or homologues, but also complexes and absorbates known to the skilled artisan. In a particular embodiment the (virus) specific antibodies are selected from the group consisting of PRRSV nucleocapsid-specific antibodies such as P3/27 and SDOW17 and WBE1, 4, 5 and 6.
In the alternative, the present invention provides the use of the viruses harvested from the above mentioned cell cultures in methods to determine a viral infection or a previous viral infection in a subject, by assessing the presence of virus-specific antibodies in the sample taken from said subject, i.e. detecting the binding of said virus-specific antibodies to the viruses harvested from the cell culture or the binding of the virus-specific antibodies to the viral protein-expressing infected cells.
A variety of techniques are available in the art to determine binding of the virus-specific antibodies to the virus. They include but are not limited to radioimmunoassay (RIA), ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, “competition” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
In a particular embodiment, the presence of virus-specific antibodies will be determined using a typical competition or sandwich assay. For example, in a sandwich assay the binding of the virus-specific antibodies is done using a secondary labeled antibody, which is reactive for the primary virus-specific antibody and preferably has the ability to react with multiple sites on the primary antibody. In a competition assay a standard amount of a labeled virus-specific antibody will compete with the antibodies present in the sample for binding to the virus.
Known labels are of the radioactive or fluorometric type, which are detected by instrumentation, and colorimetric labels, typically enzyme labels which cause the conversion of a corresponding substrate to colored form.
Enzymes have often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
It is accordingly an object of the present invention to provide a method to determine a viral infection or a previous viral infection in a subject, said method comprising;
In any one of the diagnostic methods, mentioned hereinbefore, the sample is typically a biological fluid; such as for example serum, colostrums, bronchoalveolar lavage fluids, saliva, urine or faeces; tissue or a tissue extract. The tissue or tissue extract to be analyzed includes those which are known, or suspected, to be permissive for the virus such as, for example PBMC (peripheral blood mononuclear cells), alveolar macrophages, lymphoid tissues such as lymph nodes, spleen, tonsils and thymus and non-lymphoid tissues such as lungs and liver.
This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
The following examples illustrate the invention. Other embodiments will occur to the person skilled in the art in light of these examples.
Primary alveolar macrophages were obtained from 4- to 6-week old conventional Belgian Landrace pigs from a PRRSV-negative herd as described by Wensvoort et al. (Wensvoort et al., 1991), and cultivated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids and 1 mM sodium pyruvate. Marc-145 cells were cultivated in Minimum Essential Medium with Earle's salts (MEM) supplemented with 5% FBS. PK-15 cells were grown in MEM supplemented with 10% FBS. BHK-21 cells were cultivated in MEM supplemented with 10% FBS, 1% nonessential amino acids and 1 mM sodium pyruvate. CHO-K1 cells were cultivated in F-12 medium supplemented with 10% FBS and 1 mM sodium pyruvate. All cells were grown in their specific medium supplemented with 2 mM L-glutamine and a mixture of antibiotics in a humified 5% CO2 atmosphere at 37° C. PK-15, BHK-21 and CHO-K1 cells were transfected respectively with lipofectamine (Invitrogen), lipofectamine 2000 (Invitrogen) and FuGENE 6 (Roche) according to the manufacturers' instructions.
A 13th passage on macrophages of the European prototype PRRSV strain, Lelystad virus (LV) (kindly provided by G. Wensvoort), was used (Wensvoort et al., 1991). The European PRRSV strain was first passaged on macrophages and subsequently cultivated on Marc-145 cells for 4 passages, while for the American prototype PRRSV strain, VR-2332, a 4th passage on Marc-145 cells was used which was never passaged on macrophages (Collins et al., 1992). From the Belgian isolate 94v360 a 5th passage on Marc-145 cells was used (Duan et al., 1997a).
CD163 was detected via mouse monoclonal anti-porcine CD163 antibody (mAb) 2A10 (Ab-Direct) (Bullido et al., 1997; Sanchez et al., 1999) or goat polyclonal anti-human CD163 antibody (pAb) (R&D Systems). For porcine sialoadhesin recognition, mAb 41D3 has been used (Duan et al., 1998b; Vanderheijden et al., 2003). Isotype-matched irrelevant mAb 13D12 directed against gD of pseudorabies virus (Nauwynck & Pensaert, 1995) and purified goat antibodies were used as negative controls. PRRSV was visualized via the nucleocapsid-recognizing mAb P3/27 (Wieczorek-Krohmer et al., 1996) or a polyclonal swine serum obtained from PRRSV infected pigs.
CD163 variants differing in their cytoplasmic tail have been described (Nielsen et al., 2006). Since these variations do not appear to determine PRRSV receptor function (Calvert et al., 2007), only one variant, the short one, has been cloned. Therefore, total cellular RNA was isolated from porcine macrophages via the RNeasy Mini Kit (Qiagen) and subsequently converted into cDNA via oligo dT primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen) followed by an RNase H (Gibco) treatment. The obtained single stranded cDNA served on its turn as template for PCR amplification of the CD163 sequence via the Platinum Pfx polymerase (Invitrogen) and following primers: forward primer 5′CAC CAT GGA CAA ACT CAG AAT GGT GCT ACA TGA AAA CTC T3′ and reverse primer 5′TCA TTG TAC TTC AGA GTG GTC TCC TGA GGG ATT 3′ (Invitrogen). The PCR fragment was then finally cloned in the pcDNA3.1D/V5-His-TOPO vector (Invitrogen).
Sialoadhesin was cloned into the same vector as was described by Vanderheijden et al. (Vanderheijden et al., 2003). All sequences were verified via restriction digestion and sequencing.
To construct a cell line co-expressing Sn and CD163, CHO-K1 cells were transfected with a plasmid containing the Sn cDNA and a geneticine resistance gene. After selection for geneticine resistance, cells were transfected with a plasmid containing the CD163 cDNA and a zeocin resistance gene, which allowed selection of cells expressing both Sn and CD163. Finally, 16 clones that co-expressed Sn and CD163 (CHOSn-CD163) were isolated. 10 clones in which 100% of the cells stably expressed Sn and CD163 were isolated, while the other 6 clones lost either Sn or CD163 receptor expression. After a first screening for susceptibility to PRRSV infection, three CHOSn-CD163 clones, i.e. IC5, ID9 & IF3 were selected for further research and deposited at the Belgian Coordinated Collections of Microorganisms as CHO—Sn/CD163 IC5; CHO—Sn/CD163 ID9 and CHO—Sn/CD163 IF3 with the respective accession numbers LMBP 6677CB; LMBP 6678CB; and LMBP 66779 CB respectively.
Inactivation with ultraviolet (UV) radiation was performed with a UV cross-linker (UVP, Inc). Purified virus (107 TCID50/ml) was radiated with UV light of different doses (0-100-1000-2000-3000 or 4000 mJ/cm2) (Darnell et al. 2004). Inactivation with binary ethyleneimine (BEI) was done by incubating purified virus (107 TCID50/ml) with 1 mM BEI (Aldrich) for several (0-6-12-24-48 or 72 hours) at 37° C. The reaction was stopped with 0,1 mM sodium thiosulfate (Sigma) (Mondal et al. 2005).
Twenty-four hours after seeding, macrophages were lifted from the cell culture plate by incubation with ice-cold PBS for 30 min at 4° C. immediately prior to immunostaining and flow-cytometric analysis. Cells were first fixed with 3% paraformaldehyde followed by washing and incubation with primary mAb 41D3, 2A10 or isotype-matched control antibodies at 4° C. Afterwards, cells were washed 3 times and subsequently incubated with FITC-labeled goat-anti-mouse Ab (Molecular Probes). Finally, cells were washed 3 times, resuspended in phosphate buffered saline (PBS) and analyzed with a Becton-Dickinson (San Jose, Calif.) FACScalibur. Twenty thousand cells were analyzed for each sample and three parameters were stored for further analysis: forward light scatter, sideward light scatter and green fluorescence.
To determine the titre of extracellular virus, supernatant was collected and centrifuged to remove cell debris. To determine the titre of intracellular virus, cells were washed, collected and lysed by 3 cycles of freeze-thawing. For titration on Marc-145 cells, cells were planted 3 days before inoculation. Then, they were inoculated with a 10 fold dilution series of the samples and incubated for 7 days at 37° C. followed by evaluation of the cytopathic effect (CPE). For titration on macrophages, cells were planted 1 day before inoculation followed by inoculation with a 10 fold dilution series of the samples, incubation for 3 days at 37° C. and finally evaluation. CPE was studied and furthermore infected cells were visualized via an immunoperoxidase monolayer assay (IPMA) (Wensvoort et al., 1991).
Transfected and/or infected cells were fixed with ice-cold methanol, unless pictures or colocalization studies were demanded. In those cases 3% paraformaldehyde was used and if needed, those cells were permeabilized with 0.1% Triton X-100. Fixation and permeabilization reagentia were removed via 3 times washing with PBS. Fixed cells were incubated with primary antibodies for at least 1 hr at 37° C., washed 3 times with PBS and further incubated with secondary antibodies for at least 1 hr at 37° C. Finally, cells were washed 3 times, embedded in glycerine-DABCO, mounted and analysed via fluorescence microscopy.
To quantitate colocalization between sialoadhesin and CD163 on the surface of macrophages, confocal images were taken and analysed via the programme CoLocalizer Pro. Prior to merging the two images, weak fluorescent background was substracted. Based on the overlay, different colocalization parameters were calculated according to the manual.
Treatment of Macrophages with Sialoadhesin and CD163 Specific Antibodies
Macrophages were seeded in 96-wells 24 hrs before the experiment was performed. A three-fold dilution series was prepared for different antibodies (2A10, CD163-pAb, 41D3, 13D12, purified goat antibodies) and the HbHp complex (Hb Ao H0267, Hp type 2-2 H9762 from Sigma-Aldrich), which was made by 15 min incubation of both components at room temperature. Macrophages were then incubated for one hour at 37° C. with the concentration gradient of antibodies/proteins followed by inoculation with PRRSV in the presence of antibodies/proteins for one hour at 37° C. After the treatment, cells were washed, further incubated for 9 hr at 37° C. and fixed with methanol. Infected cells were visualized via an immunoperoxidase staining with mAb P3/27 or the polyclonal swine serum as primary antibodies and respectively goat-anti-mouse HRP or rabbit-anti-swine HRP (Dako) as secondary antibodies. In control reactions, no difference in the percentage of infected cells was observed for the 2 different PRRSV-recognizing antibodies. After counting the percentage of infected cells, relative percentages of infection were calculated with cells without any antibody/protein treatment as reference value represented by the RPMI data.
For different infection experiments, a similar protocol was used as will be described here. Twenty-four hours post transfection of PRRSV non-target cells or 24 hrs post seeding of the macrophages, cells were washed once with RPMI followed by inoculation with PRRSV viral supernatant which was cleared from cell debris via centrifugation. Inoculated cells were incubated for 1 hr at 37° C. in the presence of the virus. After virus removal, macrophages were directly covered with medium unlike non-target cells, which were washed 5 times with RPMI before incubation in medium. The final wash solution was collected and titrated to determine the amount of background virus still present after removal of the inoculum. At different time points after inoculation, cells were fixed with ice-cold methanol or paraformaldehyde and intra- and extracellular virus was collected as described in virus titration.
For different infection experiments, a similar protocol was used as will be described here. Three different CHOSn-CD163 cell clones (IC5, ID9 and IF3) seeded at different densities (100 000, 200 000 or 300 000 cells/ml) are infected at different days post seeding (1, 2 or 3 days post seeding) with LV marc grown (moi 1) or with LV macrophage grown (moi 10). After 48 hours post inoculation the cells are fixed with methanol and stained with primary antibody P3/27 and secondary antibody goat-anti-mouse HRP. Afterwards AEC substrate is added. With a microscope infected cells of 3 fields with a 40× lens (500 cells per field) are counted. To enhance virus interaction with the sialoadhesin receptor which depends on interaction of virus linked sialic acids with the N-terminal, sialic acid binding domain of Sn, and subsequent infection, the cells were treated with neuraminidae (50 mU/ml of Vibirio cholerae neuraminidase) to remove cis-acting cellular sialic acids prior to infection with the PRRSV macrophage grown viruses.
1. Expression of PRRSV Receptors Sialoadhesin and CD163 on Macrophages, the in vivo Target Cells of PRRSV
Both PRRSV receptors sialoadhesin and CD163 are described to be restricted to monocytes-macrophages (Duan et al., 1998b; Sanchez et al., 1999). To further investigate their role in PRRSV infection, we first wanted to confirm their presence and analyse their expression pattern in a population of macrophages. Therefore, macrophages were cultivated for 24 hours, lifted from the cell culture plate, immunostained and analysed via flow cytometry or they were cultivated for 24 hours, fixed, immunostained and analysed via confocal microscopy. Similar to previous reports, the flow cytometry data (
To examine the role of CD163 in PRRSV infection of primary macrophages, the effect of a CD163-specific mAb and a pAb on PRRSV infection of macrophages was evaluated. Therefore, macrophages were pre-incubated with different concentrations of antibodies followed by inoculation of the macrophages with PRRSV in the presence of the antibodies for 1 hr, washing, further incubation for 9 hrs at 37° C., methanol-fixation and finally staining with PRRSV-specific antibodies. Relative percentages of infected macrophages are represented in
PRRSV displays remarkable genetic, antigenic, and clinical variability resulting in distinct groups of isolates within the same viral family (Goldberg et al., 2003), urging the need to investigate whether sialoadhesin and CD163 are involved in infection of different PRRSV strains (
Since the experiment with antibodies showed that both sialoadhesin and CD163 are involved in PRRSV infection of macrophages, we investigated their potential role in productive PRRSV infection in different non-susceptible cell-lines, either expressed separately or combined. PK-15, CHO-K1 and BHK-21 cells were transfected with sialoadhesin, CD163 or a combination of both and 24 hrs post transfection, cells were inoculated with the European prototype Lelystad virus or the American prototype VR-2332 virus. At 24 hours post inoculation, supernatant was collected and cells were fixed. The supernatant was titrated on Marc-145 cells to determine the amount of infectious virus produced in the transiently transfected cells (
In addition to PK-15, CHO-K1 and BHK-21 cells, HEK293t cells were tested for their susceptibility to PRRSV infection. Similar to the 3 other cell lines, HEK293t cells expressing sialoadhesin did not support productive PRRSV infection. HEK293t cells expressing CD163 alone supported productive PRRSV infection and 10-100 times more infected cells were observed in cells expressing both sialoadhesin and CD163 (data not shown).
Bottom line, cells only expressing sialoadhesin never showed infection, as was already noted by Vanderhijden et al. (2003). Infected cells were only observed when CD163 was present, alone or in combination with sialoadhesin (data not shown), but where CD163 alone is able to sustain PRRSV infection, 10-100 times more infected cells were observed in cells expressing both sialoadhesin and CD163.
In agreement with the results obtained via immunofluorescence microscopy, no extracellular virus was detected for cells only expressing sialoadhesin. Except for PK-15 cells, were some extracellular virus is present, however without showing infected cells. When only CD163 is expressed, all three cell-lines produce new infectious virus, but the virus titers are rather low probably because of low infection efficiency. When both sialoadhesin and CD163 are present, all three cell-lines produce new infectious virus with virus titers remarkably higher compared to cells with only CD163, especially for PK-15 and CHO-K1 cells. Comparison between the European and the American prototype PRRSV strain shows higher virus titers for the VR-2332 strain in PK-15 and CHO-K1 cells but not in BHK-21 cells. Furthermore, for one repetition of the experiment the titration was performed not only on Marc-145 cells but also on macrophages revealing the same virus titers. Thus, PRRSV non-target cells expressing CD163 or CD163 combined with sialoadhesin produce new virus that is infectious on both Marc-145 cells and on macrophages. Further support, to the fact that non-permissive PRRSV cells can be rendered permissive with high virus titers when expression both CD163 and Sialoadhesin, was given in assessing the infectivity of the European prototype PRRSV strain Lelystad, the American prototype PRRSV strain VR-2332, and the Belgian PRRSV isolate 94V360 on the PK-15, CHO-K1, BHK-21 and HEK293t cells. For all 4 cell lines, similar results were observed as described for the 2 prototype strains. 94V360 was not able to infect cells only expressing sialoadhesin. Cells expressing CD163 were able to sustain PRRSV infection, however 10-100 times more infected cells were observed in cells expressing both sialoadhesin and CD163.
Because the combination of sialoadhesin and CD163 efficiently supports PRRSV infection in non-permissive cells, we wanted to study the kinetics of PRRSV infection in those non-target cells. Therefore swine kidney PK-15 cells were transfected with a combination of sialoadhesin and CD163 and 24 hrs post transfection, cells were inoculated with Lelystad virus at a moi of 0.1 or 1. At different time points after infection, intra- and extracellular virus was collected to be titrated on Marc-145 cells, as shown in
Since PRRSV infection of macrophages and non-target cells is clearly dependent upon sialoadhesin and CD163, their specific molecular contributions to PRRSV infection need to be investigated. Results previously obtained in the lab indicate that sialoadhesin is important for the internalization of the virus (Vanderheijden et al., 2003). We want to confirm these results and study the role of CD163 during PRRSV infection. Therefore PK-15 cells were transfected with sialoadhesin, CD163 or a combination of both, which was followed by inoculation with Lelystad virus. At different time points after infection cells were fixed and PRRSV was visualized via immunofluorescence staining with the mAb P3/27 recognizing the PRRSV nucleocapsid protein represented in
6. Treatment of Macrophages with Sialoadhesin- and CD163-Specific Antibodies at 4° C.
In addition to sialoadhesin, CD163 is shown to be involved during PRRSV entry in macrophages. Sialoadhesin is known as PRRSV attachment and internalization receptor. Our data suggest a role for CD163 during PRRSV uncoating, however, further research is needed to unravel its exact functioning. Therefore we wanted to investigate whether CD163 is involved during PRRSV attachment.
Macrophages were seeded in 96-wells 24 hrs before the experiment was performed. A three-fold dilution series was prepared for different antibodies (pAb CD163, 41D3, 13D12, purified control goat antibodies). For treatment at 4° C., macrophages were preincubated for 30 min at 4° C. prior to incubation for 1 h at 4° C. with the ligands followed by inoculation with PRRSV in the presence of a new dilution series of ligands for 1 h at 4° C. After the inoculation, cells were washed, incubated for 10 hrs at 37° C. and fixed with methanol. Infected cells were visualized via an immunoperoxidase staining with mAb P3/27 or the polyclonal swine serum as primary antibodies and respectively HRP-labelled goat-anti-mouse or rabbit-anti-swine (Dako) as secondary antibodies. No difference in the percentage of infected cells was observed for the 2 different PRRSV-recognizing antibodies. Cells without ligand treatment are represented as control. For these untreated cells the average percentage of infected cells was calculated from 6 replicates. This average percentage was used as reference value in the calculation of the relative percentages of infection.
Monoclonal antibody 41D3 reduced PRRSV infection, contrasting with the pAb directed against CD163, which did not reduce PRRSV infection when administered at 4° C. (
CD163 is shown to be involved in PRRSV entry of macrophages, however not during attachment. Furthermore, CD163 enables PRRSV to disassemble and productively infect non-target cells, suggesting that CD163 acts during PRRSV entry. To test this hypothesis, we investigated the entry of PRRSV in macrophages via confocal analysis of immunofluorescence experiments showing PRRSV and CD163 at different time points after inoculation.
Twenty-four hours after seeding, macrophages were washed and incubated with PRRSV for 5, 10 or maximum 15 min at 37° C. 15 min post inoculation, the virus was replaced by medium and cells were further incubated at 37° C. At different time point post inoculation, cells were washed, fixed with paraformaldehyde, permeabilized with TX-100 and stained. Cells were first incubated with a mAb directed against GP5 (isotype IgG2a), followed by incubation with the secondary antibody goat-anti-mouse Texas Red. Cells were then again incubated with the GP5 recognizing antibody. Finally, CD163 was visualized via mAb 2A10, which was directly labeled with Alexa 488 via the mouse IgG1 specific zenon labelings kit (Invitrogen). Stainings were analysed via confocal microscopy.
PRRSV attaches to macrophages from 5 min post inoculation and first internalized virions were observed starting from 10 min post inoculation (data not shown). PRRSV bound to the surface of the macrophages did not colocalize with CD163. However, internalized virus particles ended up in CD163 positive endosomes. Starting from 45 min after inoculation, endosomes disappeared and PRRSV and CD163 started to separate. The surprising observation that PRRSV and CD163 colocalize in endosomes, this in contrast to Sn which colocalizes with PRRSV on the cell surface, further shows that CD163 is not involved in PRRSV attachment to macrophages, but rather has a role in virus fusion and uncoating inside the cell in endosomes.
From Day 2 onwards there is little difference in the degree of infectivity of the stable CHOSn-CD163 cells irrespective of the fact whether the PRRSV was grown on Marc-145 cells (
Three different CHOSn-CD163 cell clones (IC5, ID9 and IF3) were seeded at 200 000 cells/ml in a 24-well plate with insert. After 2 days the cells were inoculated with LV grown on marc cells or with inactivated LV grown on marc cells. The cells were fixed with methanol after 1 hour at 4° C. (binding, at 4° C. virus is not able to internalize), 1 hour at 37° C. (internalization), 5 hours at 37° C. (fusion, this means that virus particles are dismantled, as a consequence virus staining disappears), 12 hours at 37° C. (infection) and 24 hours at 37° C. (infection). The virus was stained with a primary antibody P3/27 and a secondary antibody goat-anti-mouse FITC. The virus particles were counted with a confocale microscope.
As shown in the aforementioned table, LV marc grown virus can perform a complete virus cycle in the three CHOSn-CD163 cell lines. First the virus particles bind to the cells, then virus particles inter the cells after which the particles are dismantled to release the genome. Finally infection occurs. Clearly, this shows that the infection route of PRRSV in these cells mimics the infection observed in the in vivo target cells, the macrophages. LV inactivated with BEI and UV show binding, internalization and fusion identical to the non-inactivated LV, but there is no infection, so these methods are good candidates for vaccine development. These results clearly demonstrate the use of the stable CHOSn-CD163 cell lines, in studying and optimizing viral inactivation processes as part of vaccine production, and accordingly provide an interesting alternative for the less accessible natural host cells.
Further evidence for the permissivity of the stable CHOSn-CD163 cells was shown when analysing the intracellular and extracellular virus production in said cells by titrating the produced virus not only on the CHOSn-CD163 cells, but also on the natural host (alveolar macrophages) and the Marc-145 cells known to sustain PRRSV infection. CHOSn-CD163 clone IC5 was infected 2 days post seeding with LV grown on marc cells, VR grown on marc cells, 94V360 grown on macrophages, similar to the infection procedures described hereinbefore. Pretreatment of the cells with neuraminidase was also included for infection with PRRSV 94V360 grown on macrophages. Titration of extracellular (extra) and intracellular (intra) was done at 3 days (Table 2) and 5 days (Table 3) post inoculation respectively and expressed as log10 units of the TCID50/ml.
Together, these results clearly show that virus is produced on CHOSn-CD163 cells and that this virus can infect not only CHOSn-CD163 cells, but also Marc-145 cells and primary macrophages, showing that no virus adaptation had occurred during infection of CHOSn-CD163 cells. It is in part interesting that for all the viruses produced in CHOSn-CD163 cells, highest levels were always detected by titration on macrophages, again showing that no adaptation during in vitro culture had occurred that would modify virus epitopes involved in infection of macrophages and induction of neutralizing antibodies. In conclusion these results show that the CHOSn-CD163 cells can be used for virus production for use in vaccines or diagnosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07014842.4 | Jul 2007 | EP | regional |
| 0811278.1 | Jun 2008 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/006045 | 7/23/2008 | WO | 00 | 1/13/2010 |
