FIELD OF INVENTION

The present invention relates to the field of immunology, in particular to the field of production of antibodies. The present invention specifically relates to production of recombinant anti-canine parvovirus antibody.

BACK GROUND OF THE INVENTION

Canine parvovirus (CPV) is one of the most highly contagious and fatal disease in dogs which causes severe gastroenteritis and is often associated with a relative lymphopenia (Perrish, C. R. 1990. Emergence, natural history, and variation of canine, mink, and feline parvoviruses. Adv. Virus Res. 38: 403-450) in juvenile dogs and myocarditis in neonatal puppies upto 16 weeks of age and causes a high mortality rate of 20- 100% (Appel, M, F. W. Scott, and I, R. Carmichael.1979. Isolation and immunization studies of a canine parvovirus like virus from dogs with haemorrhagic enteritis. Vet. Rec. 105: 156-159).

CPV is an autonomously replicating member of feline parvovirus which belongs to the family parvoviridae (Ranz, A. I., J. J. Manclus, E. Diaz, 1. Casal.1989. Porcine parvovirus. DNA sequence and genome organization. J. Gen Virol. 62: 1 13-1 15) and is the cause of an important disease in dogs which was identified first in 1978 (Burtonboy, G., F. Coignoul, N. Delferrier, and P. P. Pastoret. 1979. Canine haemorrhagic enteritis: detection of viral particles by electron microscopy. Arch. Virol. 61 : 1 - 1 1 ). The disease has become endemic in almost all populations of wild and as well in domestic dogs throughout the world thereby causing a panzootic disease. CPV is genetically and antigenically very closely related to feline and mink enteritis virus (Parrish, C. R., C. F. Aquadro, and L. E. Carmichael; 1988; Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink and raccoon parvoviruses. Virology 166: 293-307). CPV has a single stranded DNA genome encapidated by a non- enveloped icosahedral capsid composed of three structural proteins VP1, VP2 and VP3. The capsid is formed mainly of the major structural protein VP2 (Paradiso,P.R., Rhode, S.L., and Singer, S.L.1982. Canine parvovirus. A biochemical and ultrastructural characterization. J. Gen. Virol. 62, 1 13-1 15) the N-terminus of which codes for a single B-cell epitope that is immunodominant and produces neutralizing antibodies (Strassheim, M.L., Gruenberg, A., Veijalainen, P., Sgro, J.Y., and Parrish., C.R. 1994. Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid. Virology 198, 175-184). CPV replicates very actively in dividing tissues (Perrish, C. R. 1990. Emergence, natural history, and variation of canine, mink, and feline parvoviruses. Adv. Virus Res. 38: 403-450) and dogs that do not have antibodies against the virus succumb to the infection. The presence of circulating antibodies help control parvoviral infections (Meunier, P. C, B. J. Cooper, M. J. G. Appel, M. E. Launieu, and D. Slauson. 1985. Pathogens of canine parvovirus enteritis: sequential virus distribution and passive immunization studies. Vet.Pathol. 22: 617-624).

Currently available vaccines against CPV are based on either live or attenuated viruses and they are widely used for controlling parvoviral infections. In pups, vaccination with current vaccine formulations is hampered because of the amount of antigen used are too low to overcome the interference of maternally derived antibodies and does not give guaranteed protection as the presence of maternal antibodies blocks the virus replication. Insufficient attenuation or incomplete inactivation always constitutes a risk for animal health.

US Patent number 5316764 discloses a vaccine comprising a novel canine parvovirus strain and having the property of being able to break through the maternally derived antibody levels persistent in 9-12 week old pups, and even to immunize the majority of pups at the age of 6 weeks in the presence of maternally derived antibodies.

US Patent number 6187759 discloses use of a DNA vaccine for the manufacture of a medicament for use in eliciting an immune response against parvovirus in a maternally derived antibody positive animals. The DNA vaccine comprises a p!asmid vector and at least one isolated nucleotide sequence encoding a parvovirus immunogenic polypeptide, and transcriptional regulatory sequences operably linked to the isolated nucleotide sequence. Several conventional monoclonal antibodies (MAb) have been generated against different viruses and their utilization tends to be limited to clinical applications because of viral contamination and high cost involved in MAb preparations. To overcome these problems, generation of single chain antibody fragments (ScFv) by utilizing recombinant DNA technology in the expression of antibody fragments has been widely used. As the antibody fragments can be readily produced from the genes encoding antibody variable domains, which can be derived either from hybridomas or from bacteriophage displaying antibody fragments (McCafferty, J., A. D. Griffiths, G. Winter, and D. J. Chiswell. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552-554.) ScFvs consign antigen binding sites with in a single gene was well established in antibody engineering (DalPAcqua, W., P. Carter. 1998. Antibody engineering. Curr. Opin. Struct. Biol. 8, 443-450). Use of monoclonal antibodies for therapeutic purpose involves production of antibodies using conventional technologies, a process which is expensive and requires complex bio- processes. The use of recombinant antibody fragments such as monovalent antibodies will overcome these limitations and provide homogenous, pure and large scale reagents necessary for canine parvovirus.

Single chain variable fragment consists of variable heavy and light chain domains tethered by a flexible peptide linker which retains the antigen binding site in a single linear molecule. Their design, construction and expression in Escherichia coli demonstrated their structure-function relationship and antigen-antibody interactions thus making the scFv useful in both clinical and medical application (Condra JH, Sardana VV, Tomassini JE, Schlabach AJ, Davies ME, Lineberger DW, Graham DJ, Gotlib L, Colonno RJ. Bacterial expression of antibody fragments that block human rhinovirus infection of cultured cells. J Biol Chem. 1990 265:2292-5).

However, there is no report of anti-canine parvovirus antibody comprising scFv. Therefore, there is much felt need for anti- canine parvovirus antibody useful for therapeutic purposes against canine parvoviruses, which is both economical and immunologically very effective.

SUMMARY OF THE INVENTION An aspect of the present invention relates to a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5.

Another aspect of the present invention relates to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 encoding a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Yet another aspect of the present invention relates to a DNA expression cassette comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Still another aspect of the present invention relates to a composition comprising the antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to a method of treating or preventing canine parvovirus infection in a subject, the method comprising administering an effective amount of the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 to the subject.

Yet another aspect of the present invention relates to a method for detecting the presence of canine parvovirus in a sample, the method comprising contacting a sample with the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant antibody and canine parvovirus using conventional methods, wherein the formation of complex confirms presence of the canine parvovirus in the sample.

Still another aspect of the present invention relates to a kit for treating or preventing canine parvovirus infection in a subject, the kit comprising the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Another aspect of the present invention relates to a kit for detecting the presence of canine parvovirus in a sample, the kit comprising the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 shows the vector map showing cloned single chain variable fragment (scFv).

Figure 2 shows vector construct comprising single chain variable fragment (scFv).

Figure 3A shows electrophoretic analysis of PCR amplified heavy chain variable and light chain variable. M represents molecular weight markers; VH represents heavy chain variable and VL represents light chain variable.

Figure 3B shows electrophoretic analysis of scFv fragment generated by SOE PCR. M represents molecular weight markers and ScFv represents single chain variable fragment.

Figure 4 shows SDS-PAGE analysis of purified scFv. Lane M represents molecular marker and Lane 1 represents purified scFv.

Figure 5 shows immunoblot analysis of purified monovalent scFv under reducing conditions which was probed with 6x Histidine HRP. Lane M represents pre-stained molecular marker and Lane 1 represents purified scFv.

Figure 6 shows the binding activity of purified scFv to CPV VLP antigen (Ag) by indirect ELISA. A represents Ag + Purified scFv + Anti-His HRP; B represents Purified scFv + Anti-His HRP; C represents Ag + Anti-His HRP; D represents Ag + Purified CPV VLP Mab + Anti-Mouse HRP; E represents Purified CPV VLP Mab + Anti-Mouse HRP; F represents Ag + Anti-Mouse HRP; G represents Ag + E.coli Lysate + Anti-His HRP; and H represents E.coli Lysate + Anti-His HRP.

Figure 7 shows the image of microtitre plate showing the Hemagglutination-inhibition test for CPV using scFv. Wells A l to A 12 and B l to B 12 represent serially diluted scFv (starting with l mg/ml concentration); Wells C I to C 12 and Dl to D12 represent serially diluted Positive control serum obtained from dog immunised with CPV vaccine; Wells E l to E10 and F l to F10 represent serially diluted Negative control serum; Wells E l l , E 12, F l 1 and F 12 represent RBC control.

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO: 1 shows nucleotide sequence of Heavy chain variable (357 bp)

SEQ ID NO: 2 shows nucleotide sequence of Light chain variable (318 bp)

SEQ ID NO: 3 shows the nucleotide sequence of linker (45 bp)

SEQ ID NO: 4 shows nucleotide sequence of single chain variable fragment (720 bp) SEQ ID NO: 5 shows amino acid sequence of single chain variable fragment (240 a. a.) SEQ ID NO: 6 shows M 13 forward primer ( 16 bp)

SEQ ID NO: 7 shows M 13 reverse primer ( 16 bp)

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and methods referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

As used in the specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used in this specification, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The terms "polynucleotide", "nucleotide", "DNA", "gene" and "nucleic acid" are used interchangeably.

The terms "primer" and "oligonucleotide" used herein are used interchangeably.

"Nucleotide" means a building block of DNA or RNA, consisting of one nitrogenous base, one phosphate molecule, and one sugar molecule (deoxyribose in DNA, ribose in RNA).

"Oligonucleotide" means a short string of nucleotides.

"Primer" means a short strand of oligonucleotides complementary to a specific target sequence of DNA, which is used to prime DNA synthesis.

The term "antibody" is used to refer to any antibody like molecule that has an antigen binding region and comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.

Light chains are classified as either kappa or lambda. Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (V _L ) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH I , CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

The invention also encompasses portions of antibodies that comprise sufficient variable region sequence to confer antigen binding. Portions of antibodies include, but are not limited to Fab, Fab', F(ab') ₂ , Fv, SFv, scFv (single-chain Fv), whether produced by proteolytic cleavage of intact antibodies, such as papain or pepsin cleavage, or by recombinant methods, in which the cDNAs for the intact heavy and light chains are manipulated to produce fragments of the heavy and light chains, either separately, or as part of the same polypeptide.

The term ''antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable region".

"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

The terms "scFv" and "antibody" used herein are used interchangeably.

"VLP" or "VLPs" mean(s) virus-like particle or virus-like particles. As used herein, the term "linker" refers to a portion of or functional group on a building block that can be employed to or that does (e.g., reversibly) couple the building block to a support, for example, through covalent link, ionic interaction, electrostatic interaction, or hydrophobic interaction.

As used herein, the term "monovalent" means that a given domain antibody can bind only a single molecule of its target. Naturally-occurring antibodies are generally divalent, in that they have two functional antigen-binding loops, each comprising a VH and a VL domain. Where steric hindrance is not an issue, a divalent antibody can bind two separate molecules of the same antigen. In contrast, a "monovalent" antibody has the capacity to bind only one such antigen molecule. The antigen-binding domain of a monovalent antibody can comprise a VH and a VL domain.

The term "test sample" or "sample" refers to material obtained from a biological source, environmental source or a processed sample. The processed sample may include extraction of genetic material from the sample. The terms "test sample" or 'sample' are used interchangeably.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "recombinant host cell" or "host cell", as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as E. coli, yeast cells, plant cells or mammalian cells such as CHO cells, NS/0 cells, and lymphocytic cells.

The term "therapeutically effective amount" or "effective amount" is an amount of the antibody that is effective, upon single or multiple dose administration to a subject, in inhibiting CPV infection, disease, or sequelae thereof, in a subject. A therapeutically effective amount of the antibody or antibody fragment may vary according to factors such as the disease state, CPV strain or isolate, and the ability of the antibody or antibody portion to elicit a desired response in the subject.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

The term "treating" or treatment refers to the administration of an effective amount of a composition of the present invention to a subject, who has obesity, overweight and other metabolic disorders, or a symptom or a predisposition of such diseases, with the purpose to cure, alleviate, relieve, remedy, or ameliorate such diseases, the symptoms of them, or the predispositions towards them.

As used herein, the term "subject" includes cat and any member of the canidae family such as dogs, dogs, wolves, foxes, jackals, coyotes etc.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.

The present invention provides a recombinant monovalent murine anti-canine parvovirus antibody and a method for production of the antibody. The present invention further provides a recombinant vector and host cells comprising the antibody. Further, the present invention provides a composition comprising the antibody.

The present invention also provides a method of treating or preventing infection with canine parvovirus in a subject using the recombinant murine anti-canine parvovirus antibody. The present invention further provides a method for detecting the presence of canine parvovirus in a sample using the recombinant murine anti-canine parvovirus antibody. The murine anti-canine parvovirus antibody disclosed in the present invention recognizes canine parvovirus (CPV) or canine parvovirus Virus like Particles (CPV VLPs).

An embodiment of the present invention provides recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5.

In an embodiment of the present invention there is provided a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is encoded by a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4.

In another embodiment of the present invention there is provided a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a monovalent antibody.

In yet another embodiment of the present invention there is provided a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a Fab fragment or a single chain antibody (scFv).

In still another embodiment of the present invention there is provided a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a Fab fragment.

In an embodiment of the present invention there is provided a recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a single chain antibody (scFv). Another embodiment of the present invention provides a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 encoding a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Yet another embodiment of the present invention provides a DNA expression cassette comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Still another embodiment of the present invention provides a recombinant vector comprising the DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4.

Another embodiment of the present invention provides a recombinant vector comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

In another embodiment of the present invention there is provided a recombinant vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide is operably linked to a promoter.

In yet another embodiment of the present invention there is provided a recombinant vector comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter.

Still another embodiment of the present invention provides a recombinant host cell comprising the recombinant vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4.

Another embodiment of the present invention provides a recombinant host cell comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

In yet another embodiment of the present invention there is provided a recombinant host cell comprising the recombinant vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the host cell is selected from the group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

In another embodiment of the present invention, there is provided a a recombinant host cell comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the host cell is selected from the group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

An embodiment of the present invention provides a composition comprising recombinant antibody having a binding specificity for canine parvovirus, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and a pharmaceutically acceptable carrier.

Another embodiment of the present invention provides a method of treating or preventing canine parvovirus infection in a subject, the method comprising administering an effective amount of the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 to the subject.

In yet another embodiment of the present invention there is provided a method of treating or preventing canine parvovirus infection in a subject, the method comprising administering an effective amount of the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 to the subject, wherein the recombinant antibody is administered intracutaneously, intravenously, intramuscularly, intraarticularly, intraarterialy, intrasynovialy, intrasternaly, intrathecaly or intralesionaly. In still another embodiment of the present invention there is provided a method of treating or preventing canine parvovirus infection in a subject, the method comprising administering an effective amount of the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 to the subject, wherein the subject is selected from the group consisting of dog, cat, mink, raccoons, wolves, jackals and foxes.

An embodiment of the present invention provides a method for detecting the presence of canine parvovirus in a sample, the method comprising contacting a sample with the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant antibody and canine parvovirus using conventional methods, wherein the formation of complex confirms presence of the canine parvovirus in the sample.

In another embodiment of the present invention there is provided a method for detecting the presence of canine parvovirus in a sample, the method comprising contacting a sample with the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant antibody and canine parvovirus using conventional methods, wherein the formation of complex confirms presence of the canine parvovirus in the sample, wherein the sample is selected from the group consisting of feces, blood, vomit material and heart tissue.

Yet another embodiment of the present invention provides kit for treating or preventing canine parvovirus infection in a subject, the kit comprising the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Still another embodiment of the present invention provides a kit for detecting the presence of canine parvovirus in a sample, the kit comprising the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and reagents.

In the present invention, the recombinant monovalent murine anti-canine parvovirus single chain variable fragment (scFv) is generated from mouse hybridoma against canine parvovirus virus like proteins (CPV VLPs). Total RNA is isolated from the hybridoma cells secreting anti-CPV VLPs and cDNA is synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). The RT-PCR amplified DNA is used as template for the amplification of variable domains of the antibody with commercial primers from Amersham Biosciences (Figure 3A).

The nucleotide sequence of the heavy chain variable (VH) and light chain variable (VL) is as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. These amplified variable domains are assembled to form a single chain variable fragment by splicing by overlap extension polymerase chain reaction (SOE PCR) (Figure 3B). scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker. The nucleotide sequence of scFv is as set forth in SEQ ID NO: 4 and the amino acid sequence of scFv is as set forth in SEQ ID NO: 5. The scFv (SEQ ID NO: 4) was cloned into TOPO-TA vector for sequencing analysis.

The scFv of the present invention is expressed as a recombinant protein using vectors for transforming the appropriate host cells. The host cells comprising the polynucleotide (SEQ ID NO: 4) of the invention can be prokaryotic or eukaryotic host cells and allows the production of the recombinant antibody. Methods for producing such antibody include culturing host cells transformed with the expression vectors comprising their coding sequences under conditions suitable for protein expression and recovering the protein from the host cell culture. The vectors should include a promoter, a ribosome binding site (if needed), a selectable marker gene, and the start/stop codons. The vectors should allow the expression of the recombinant antibody in the prokaryotic or eukaryotic host cells. For eukaryotic hosts (e.g. yeasts, plant cells or mammalian cells), different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine Papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast ga!4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for the transient (or constitutive) repression and activation and for modulating gene expression.

In accordance with the present invention, host cells can be either prokaryotic or eukaryotic. Amongst prokaryotic host cells, the preferred one is E. coli. Amongst eukaryotic host cells, the preferred ones are yeast, insect, plant or mammalian cells. In particular, cells such as human, monkey, mouse, insect (using baculovirus-based expression systems) and Chinese Hamster Ovary (CHO) cells, provide post- translational modifications to protein molecules, including correct folding or certain forms of glycosylation at correct sites. Also yeast cells can carry out post-translational peptide modifications including glycosylation. Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C I 27, 3T3, BHK, HEK 293, Per.C6, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.

The scFv (SEQ ID NO: 4) is cloned into pET 28a bacterial expression vector. Figure 1 shows the vector map showing cloned single chain variable fragment (scFv). Figure 2 shows vector construct comprising single chain variable fragment (scFv). Selected scFv plasmids are transformed into Escherichia coli BL21 (DE3) for production of soluble scFv fragments (Figure 4 and Figure 5).

The purification of the recombinant antibody of the invention can be carried out by any of the conventional methods known for this purpose, i.e. any procedure involving extraction, precipitation, chromatography, or the like. In particular, methods for antibody purification can make use of immobilized gel matrices contained within a column, exploiting the strong affinity of antibodies for substrates such Protein A, Protein G, or synthetic substrates, or for specific antigens or epitopes. After washing, the protein is eluted from the gel by a change in pH or ionic strength. Alternatively, HPLC (High Performance Liquid Chromatography) can be used. The elution can be carried out using a water-acetonitrile-based solvent commonly employed for protein purification.

The purified scFv is checked for their antigen binding activity against CPV VLPs. The scFv shows strong binding activity towards CPV VLPs. No reactivity was observed with a lysate of E. coli indicating the binding specificity of scFv (Figure 6). Hemagglutination inhibition (HI) test was also performed for CPV using scFv. CPV virus with known HA activity (8 HA units) was used in the HI test. The last dilution of serum that fully inhibits the hemagglutination is the hemagglutination titer. The HI titer of the scFv was found to be 128 (table 2). The result indicates that the scFv can bind to the virus and prevent hem-agglutination which is a property of CPV neutralizing antibodies (Figure 7). Thus, the anti-CPV antibody disclosed in the present invention neutralizes CPV.

The recombinant antibody of the present invention has single antigen binding site that is reactive against the VP2 of CPV. The antibody is devoid of constant regions and therefore provides high binding avidity and specificity to the target antigens similar to the parent antibody. The antibody of the present invention is used for detecting, treating, inhibiting, preventing, and/or ameliorating CPV infection. To this purpose, the antibody can be used for preparing diagnostic, therapeutic, or prophylactic compositions for the management of CPV infection. The recombinant antibody of the present invention can be over-expressed in prokaryotic or eukaryotic cells and thus can be produced in large quantities at low cost to guarantee a consistent supply of well- characterized specific antibody for use in therapeutic purpose against CPV.

The antibody of the present invention can be further used for detection of CPV in a sample. The method comprises contacting a sample with the recombinant antibody of the present invention under conditions which allows formation of a complex between the recombinant antibody and the CPV. The complex can also from between the recombinant antibody and CPV VLPs. The formed complex is detected using conventional methods, wherein the formation of complex confirms the presence of canine parvovirus in the sample. The conventional methods include but are not limited to immunoassays, radio-immunoassays, competitive-binding assays, western blot analysis, ELISA (enzyme linked immunosorbent assay) assays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, immunofluorescence assays and protein A immunoassays.

The antibody of the present invention can also be used as a reagent for titration of the virus in production to replace HA tests. The antibody can also be used for passive immunization of pups infected with CPV. This antibody can serve as an alternative for monoclonal antibodies for preparation of antibodies involving large production cost, high batch to batch variation etc. Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Example 1

Construction of scFv fragment

Hybridoma was generated by immortalizing immune mouse B cells mediated by a mouse hybridoma (mouse X mouse). The hybridoma was outsourced by Indian Immunologicals Limited, Gachibowli, Hyderabad - 500032, Andhra Pradesh, India to a contract research organization and the antigen used to develop the hybridoma was CPV Virus-like-particles (VLP's) formed as a result of expressing VP2 (major coat protein of CPV) in the baculovirus expression system.

Total RNA was isolated from the hybridoma cells secreting anti-CPV VLPs (Virus Like Particles) using Trizol reagent (Invitrogen) and resuspended in nuclease free water. The RNA was subjected to cDNA synthesis by RT-PCR using Thermoscript reverse transcriptase (RT)-PCR kit (Invitrogen) according to the manufacturer's instructions. The RT-PCR amplified DNA was used as a template for amplification of variable domains of an antibody using the commercially available primers supplied by Amersham Biosciences. The primer sequences are proprietary material and owned by Enzon Inc. and licensed to Amersham Biosciences. However, a person skilled in the art can design the sequence of forward and reverse primers used for amplification of heavy chain variable and light chain variable based on the nucleotide sequence of heavy chain variable and light chain variable as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The PCR reactions were set up in a total volume of 50 containing 5 of cDNA, 5 //L of 10 x Taq DNA polymerase reaction buffer, 1 μ\, of l OmM dNTP's, 1.25 units of Taq DNA polymerase enzyme and 1 //L (10 pmol/ L) of each forward and reverse primers (Commercial primers from Amersham Biosciences) for heavy chain variable region and for light chain variable region. The reaction mixture was cooled on ice, and transferred to a master cycler (Eppendorf) and cycled 34 times at 60 seconds at 92°C, 30 seconds at 63°C and 60 seconds at 72°C. Finally the extension cycle was increased to 10 minutes at 72°C. Amplifications were repeated several times to obtain the final products of a fragment of 357 bp (as set forth in SEQ ID NO: 1 ) which corresponds to DNA encoding heavy chain variable (V _H ) and another fragment of 318 bp (as set forth in SEQ ID NO: 2) which corresponds to DNA encoding light chain variable (V _L ). Figure 3 A shows electrophoretic analysis of PCR amplified heavy chain variable and light chain variable. M represents molecular weight markers; V _H represents heavy chain variable and VL represents light chain variable.

Assembly of variable heavy and light chain to form single chain variable fragment by SOE PCR

The final PCR products i.e. DNA encoding heavy chain variable (V _H ) (SEQ ID NO: 1 ) and DNA encoding light chain variable (VL) (SEQ ID NO: 2) were pooled and gel purified using the commercial kit from QIAGEN. 200 ng of gel purified VH and VL DNA were used in PCR for 14 cycles without primers. The amplified PCR product was used as a template for amplification of the 720 bp scFv product using the commercially available forward light chain primer having an EcoRl restriction site and commercially available reverse heavy chain primer having a No/1 restriction site. The primer sequences are proprietary material and owned by Enzon Inc. and licensed to Amersham Biosciences. However, a person skilled in the art can design the sequence of forward and reverse primers used for amplification based on the nucleotide sequence of single chain variable fragment as set forth in SEQ ID NO: 4. The amplification was carried out for 34 cycles at 94°C for 60 seconds, 63°C for 60 seconds, 68°C for 2 minutes. The amplified scFv PCR product was gel purified using gel extraction kit Qiagen (Hilden, Germany). The nucleotide sequence of the scFv fragment is as set for the in SEQ ID NO: 4. The amino acid sequence of the scFv fragment is as set for the in SEQ ID NO: 5. Figure 3B shows electrophoretic analysis of scFv fragment generated by SOE PCR. M represents molecular weight markers and ScFv represents single chain variable fragment.

Example 2

Analysis of scFv sequences

The scFv fragment having nucleotide sequence as set forth in SEQ ID NO: 4 was cloned into TOPO-TA vector according to the manufacturers instructions (TOPO-TA cloning kit, Invitrogen). Positive clones which showed release of 720 bp fragment after digestion of plasmid with EcoRl of were sequenced using the M l 3 forward primer (as set forth in SEQ ID NO: 6) and M l 3 reverse primer (as set forth in SEQ ID NO: 7). The obtained sequence was confirmed using NCBI blast search. The NCBI blast search confirmed the presence of 357 bp of heavy chain variable (SEQ ID NO: 1), 318 bp of light chain variable (SEQ ID NO: 2) and 45 bp of linker DNA (SEQ ID NO: 3) which formed the single chain variable fragment of 720 bp (SEQ ID NO: 4).

Example 3

Production of soluble scFv fragments

Cloning of scFv into pET vector

The scFv (SEQ ID NO: 4) was cloned into pET 28a bacterial expression vector. The vector pET 28a and insert scFv referred to monovalent antibody fragment of size 720 bps were digested with EcoRl and Noil, respectively by incubating at 37°C for 12 hours. The digested products were purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1 :3 and 1 :6) cohesive end ligation and incubated at 22°C for 2 hours. The ligated product was further incubated for 20 minutes at 65°C in order to inactive the enzyme. Figure 1 shows the vector map showing cloned single chain variable fragment (scFv). Figure 2 shows vector construct comprising single chain variable fragment (scFv). The pET 28a vector carries T7 promotor, LacZ, ribosome binding site, N-terminal His Tag, T7 Tag, heavy chain variable, linker, light chain, C-terminal His tag sequence and T7 terminator. The recombinant plasmids were chemically transformed into XL-Blue strain of E. coli cells.

E. coli transformation

Overnight grown E. coli XL-Blue strain were sub-cultured and grown at 37°C with shaking until the OD of the culture reached to 0.6 at 600nm. The culture was harvested by centrifuging at 5000 x rpm for 10 minutes at 4°C and re-suspended in ice-cold 0.1 mM CaCl ₂ and incubated overnight on the ice, before proceeding for transformation.

The chemically competent E. coli XL-Blue cells thus prepared were incubated with recombinant plasmid comprising the nucleotide sequence of the scFv as set forth in SEQ ID NO: 4 for 30 minutes on ice. The cells were transformed by heat shock treatment at 42°C for 90 seconds and immediately transferred on ice for 2 minutes before the media was added to cells. The cells were incubated for one hour at 37°C for recovery and plated onto LB agar plates containing l OOmg/ml of ampicillin. The plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to digestion with EcoRl and No/I. The positive clones were sequence verified before the plasmid was transformed into E. coli BL21 (DE3) cells for soluble expression of the antibody gene. The recombinant plasmid fro the transformed E. coli BL21 (DE3) was isolated and further transformed in E. coli BL21 (DE3) as described above. Cultures of E. coli BL21 (DE3) were grown till the OD reached 0.6 at 600 nm and induced by addition of I mM isopropyl-P-D-thiogalactopyranoside (IPTG) and incubated for 4 hours at 30°C. The bacterial pellet was harvested by centrifugation at 5000 x g for 20 minutes at 4°C, resuspended in Tris buffer and sonicated for cell lysis. The supernatant was collected after centrifugation at high speed and purified by immobilized metal affinity chromatography (IMAC). The Nickel-Nitrilotriacetate agarose beads (5mL) was equilibrated with 10 column volumes of 50 mM Tris-HCl, 155 mM NaCl, pH 7.6 (equilibration buffer). The supernatant was loaded to the column at a flow rate of 1 mL/minute and washed with 20 column volumes of washing buffer containing equilibration buffer with 30 mM imidazole, pH 7.6. Bound scFv was eluted with 3 column volumes of elution buffer containing equilibration buffer with 300 mM imidazole, pH 7.6, as fractions of l mL each. The purified protein eluted fractions of scFv fragments were analyzed by SDS-PAGE under reducing conditions after staining with coomassie brilliant blue. Figure 4 shows SDS-PAGE analysis of purified scFv. Lane M represents molecular marker and Lane 1 represents purified scFv. The purified protein was detected by staining with coomassie brilliant blue. Figure 5 shows immunoblot analysis of purified monovalent scFv under reducing conditions which was probed with 6x Histidine HRP. Lane M represents pre-stained molecular marker and Lane 1 represents purified scFv. A major band with an apparent molecular weight of 30 kDa was detected. The eluted fractions were screened for antigen binding by ELISA.

Example 4

Determination of ability of scFv to bind to CPV VLP antigen by Indirect ELISA

A micro titer well plate was coated with CPV VLP antigen ( l OOng/well) in carbonate buffer by incubating overnight at 4°C. The wells were washed thrice with PBST and the unbound sites in the wells were blocked with 1% bovine gelatin. Purified scFv, CPV VLP monoclonal antibody (MAb) and E.coli lysate were serially diluted, added into wells and incubated for 1 hour at 37°C (please confirm). E.coli lysate was used as a negative control. After incubation, the wells were washed and binding was detected by adding His-Probe, anti-mouse HRP and a chromogenic substrate TMB. The plate was incubated at 37°C for 10 minutes and the reaction was stopped by addition of 1.25M H2SO4 The absorbance was measured at 450 nm using a microplate reader (BIO-TE , US). The result is provided in Figure 6. Figure 6 shows the binding activity of purified scFv to CPV VLP antigen (Ag) by indirect ELISA. A represents Ag + Purified scFv + Anti-His HRP; B represents Purified scFv + Anti-His HRP; C represents Ag + Anti-His HRP; D represents Ag + Purified CPV VLP Mab + Anti- Mouse HRP; E represents Purified CPV VLP Mab + Anti-Mouse HRP; F represents Ag + Anti-Mouse HRP; G represents Ag + E.coli Lysate + Anti-His HRP; and H represents E.coli Lysate + Anti-His HRP. Titration of purified scFv against CPV VLP antigen revealed a concentration dependent reduction of the optical density values. scFv was able to bind to CPV VLP antigen. No reactivity was observed with E.coli lysate indicating the binding specificity of scFv.

Example 5

Hemagglutination-inhibition (HI) assay for CPV using scFv

Hemagglutination-inhibition (HI) assay was carried out for CPV using scFv. Positive and Negative serum samples were inactivated by keeping at 56°C for 30 minutes. 20% kaolin solution was prepared by dissolving kaolin in normal saline. 0.1 ml of each serum sample including positive and negative control was added to 0.9 ml of 20% kaolin solution. All samples were vortexed on vertex mixer for 20 minutes at room temperature and then the samples were centrifuged at 2000 rpm for 10 minutes and supernatant was collected. To this supernatant, 0.1 ml of 50% pig RBC was added and kept for 1 hour at temperature in the range of 2°C to 8°C. After incubation, the mixer was centrifuged at 2000rpm for 10 minutes and supernatant was collected referred as treated serum. A microtiter plate was taken and 25μΙ of saline was dispensed in each well. Subsequently, 25 μΐ of scFv ( l mg/ml) was added to A l and B l wells. Similarly 25 μΐ treated positive control serum was added to C I and D l wells and 25 μΐ treated negative control serum was added to E l and F l wells. Wells E l l , E l 2, F l 1 , and F 12 contain only 25 μΐ of saline and were treated as RBC control. All the samples were 2 fold serially diluted except RBC control in E l 1, E 12, Fl 1 , and F 12 wells. 25 μΙ of 8 HA CPV virus was added to all wells except El l , E 12, F l l and F 12. The samples were incubated at 37°C for 30 minutes and transferred at a temperature in the range of 2°C to 8°C for 15 minutes. Further, 50 μΐ of saline was added to RBC control wells i.e. El l , E12, F l l and F 12. Further, 50 μΐ of 1% pig RBC was added to all the wells including RBC control. The plate was then kept a temperature in the range of 2°C to 8°C for 2 hours. After 2 hours, the plate was observed on plate reading mirror. Figure 7 shows the image of microtitre plate showing the Hemagglutination-inhibition test for CPV using scFv. Wella A l to A 12 and B l to B 12 represent serially diluted scFv (starting with l mg/ml concentration); Well CI to C 12 and Dl to D12 represent serially diluted Positive control serum obtained from dog immunized with CPV vaccine; Wells E l to E 10 and Fl to F 10 represent serially diluted Negative control serum; Wells E l l , E l 2, Fl 1 and F 12 represent RBC control.

The last dilution of serum that fully inhibits the hemagglutination is the hemagglutination titer. Table 1 provides the dilution of antibody and serum sample in HI assay. The result is provided in Table 2. Table 2 provides the HI titer of various samples. As inferred from table 2, the HI titer of the scFv is 128.

Table 1 : Antibody/serum dilutions in HI assay

Table 2: HI titers of various samples

Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.