Patent Application
20130129764
The invention relates to compositions and methods for the detection of various infectious organisms, including heartworm (Dirofilaria immitis), Ehrlichia Canis, Anaplasma phagocytophilum, and Borrelia burgdorferi. More particularly, this invention relates to antibodies that bind to a heartworm antigen, the E. Canis gp36 polypeptide, the A. phagocytophilum p44 polypeptide, the B. burgdorferi OspA, OspC, OspF, p39, p41 and VLsE polypeptides, and uses thereof.
Infectious diseases that affect dogs, cats and other animals having close interactions with humans are important not only from a veterinary standpoint, but also because of the risk to public health. An infectious disease is caused by the presence of organisms such as viruses, bacteria, fungi, or parasites (either animalian or protozoan). Most of these diseases are spread directly from animal to animal, while others require a vector such as a tick or mosquito. Certain infectious diseases are a concern from a public health standpoint because they are zoonoses (transmittable to humans).
Heartworm is a dog parasitoid. It is hard to eliminate and can be fatal; prevention, however, is easily achieved using medication. As the name suggests, an infected mosquito injects a larva into the dog's skin, where it migrates to the circulatory system and takes up residence in the pulmonary arteries and heart, growing and reproducing to an alarming degree. The effects on the dog are quite predictable, cardiac failure over a year or two, leading to death. Treatment of an infected dog is difficult, involving an attempt to poison the healthy worm with arsenic compounds without killing the weakened dog, and frequently does not succeed. Prevention is much the better course, via heartworm prophylactics which contain a compound which kills the larvae immediately upon infection without harming the dog. Often they are available combined with other parasite preventives. The definitive host for heartworm is dog but it can also infect cats, wolves, coyotes, foxes and other animals, such as ferrets, sea lions and even, under very rare circumstances, humans.
There are several species of Ehrlichia, but the one that most commonly affects dogs and causes the most severe clinical signs is E. canis. This species infects monocytes in the peripheral blood. Two conserved major immunoreactive antigens, gp36 and gp19, are the first proteins to elicit an E. canis-specific antibody response, while gp200 and p28 elicit strong antibody responses later in the acute phase of the infection. Recombinant polypeptides gp36, gp19, and gp200 (N and C termini) exhibited 100% sensitivity and specificity for immunodiagnosis by the recombinant glycoprotein enzyme-linked immunosorbent assay (ELISA) compared with the results obtained by an indirect fluorescent-antibody assay (IFA) for the detection of antibodies in dogs that were naturally infected with E. canis. Cárdenas et al. (2007) Clin. Vacc. Immunol. 14:123-128.
A. phagocytophilum is a Gram negative, obligate bacterium of neutrophils. It is also known as the human granulocytic ehrlichiosis (HGE) agent, Ehrlichia equi, and Ehrlichia phagocytophila, and is the causative agent of human granulocytic anaplasmosis, tick-borne fever of ruminants, and equine and canine granulocytic anaplasmosis. See la Fuente et al. (2005) J. Clin. Microbiol. 43:1309-1317. A. phagocytophilum binds to fucosylated and sialylated scaffold proteins on neutrophil and granulocyte surfaces. A type IV secretion apparatus is known to help in the transfer of molecules between the bacterium and the host. The most studied ligand is PSGL-1 (CD162). The bacterium adheres to PSGL-1 (CD162) through 44-kDa major surface protein-2 (Msp2 or P44). After the bacteria enters the cell, the endosome stops maturation and does not accumulate markers of late endosomes or phagolysosomes. Because of this the vacuole does not become acidified or fused to lysosomes. A. phagocytophilum then divides until cell lysis or when the bacteria leaves to infect other cells. See Dumler et al. (2005) Emerging Infec. Dis. 11.
B. burgdorferi is a species of Gram negative bacteria predominant in North America, but also exists in Europe, and is the agent of Lyme disease. Lyme disease clinical features include the characteristic bull's eye rash and erythema chronicum migrans (a rash which spreads peripherally and spares the central part), as well as myocarditis, cardiomyopathy, arrythmias, arthritis, arthralgia, meningitis, neuropathies and facial nerve palsy.
A variety of serologic tests, such as IFA staining methods, Western blot analysis, and ELISAs, have been used to verify past or current infections of B. burgdorferi and A. phagocytophilum infections. Although sensitivities and specificities of these assays were considered acceptable, there is potential for false positive reactions when whole-cell antigens are used because heatshock, flagellin, or other proteins of these pathogens may be shared with other bacteria. Recent advances in the production and use of purified recombinant antigens (i.e., fusion proteins) in ELISAs to detect antibodies in human, dog, horse, and bovine sera have improved laboratory analyses. See IJdo et al. (1999) J. Clin. Microbiol. 37:3540-3544; Magnarelli et al. (2001) Eur. J. Clin. Microbiol. & Infect. Dis. 20:482-485; Magnarelli et al. (2001) J. Med. Microbiol. 50:889-895; Magnarelli et al. (2001) Am. J. Vet. Res. 9:1365-1369; Magnarelli et al. (2002) J. Med. Microbiol. 51:326-331; Magnarelli et al. (2002) J. Med. Microbiol. 51:649-655. The B. burgdorferi OspA, OspC, OspF, p39, p41 and VLsE antigens and A. phagocytophilum p44 antigen have been all shown to have some, but not 100%, seropositivity. Magnarelli et al. (2004) J. Wildlife Dis. 40:249.
The current invention is directed to various polypeptide antigens from infectious organisms including heartworm, E. Canis, A. phagocytophilum, and B. burgdorferi, the polynucleotides encoding them, and the antibodies against them. The current invention is also directed to methods of detecting the various polypeptide antigens and antibodies, and use thereof for the detection of infections by these organisms. Further provided are methods of combination detection which are capable of detecting infections by multiple organisms.
Therefore, in one aspect, provided herein is an A. phagocytophilum p44 polypeptide comprising amino acids 222-236 of SEQ ID NO:1 (P44-2 disclosed in U.S. Pat. No. 6,436,399 B1), wherein said polypeptide comprises at least one mutation. Additionally, provided herein is an A. phagocytophilum p44 polypeptide that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to amino acid 222-236 of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:1, wherein said polypeptide is not a wild-type P44 protein, and wherein said polypeptide binds to an antibody that is specific for a wild-type P44 protein. Also provided herein is an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 or an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 that comprises at least one mutation. Additionally, provided herein is an A. phagocytophilum p44 polypeptide that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, wherein said polypeptide is not a wild-type P44 protein, and wherein said polypeptide binds to an antibody that is specific for a wild-type P44 protein.
In some embodiments, the polypeptide may comprise 1 to 10, preferably 3-7, mutations. In some embodiments, the mutations may be selected from the group consisting of a substitution, an insertion and a deletion. Some of the exemplary mutations are: Gly222(Del), His 223→Asn, Ser224→Thr, Ser225→Thr, Val227→Ala, Thr228→Ser, Gln229→Asn, Leu233→Val, Leu233→Thr, Phe234→Leu, Ser235→Thr, and Thr236→Ser. In some embodiments, the polypeptide may comprise at least 1, 2, 3, 4, 5, 10 or 12 of the exemplary mutations.
In some embodiments, the polypeptide may further comprise a second polypeptide comprising amino acids 237-247 of SEQ ID NO:1. In some embodiments, the second polypeptide may comprise at least 1, or 1 to 5, preferably 2 to 3, mutations. Some of the exemplary mutations are: Thr240→Ser, Gln229→Asn, Ile243→Val, Glu245→Asp, Glu245→Asn, Asp246→Lys, and Asp246→Glu. In some embodiments, the polypeptide may comprise at least 1, 2, 3, 4, 5 or 7 of the exemplary mutations.
In some embodiments, the polypeptide may comprise the amino acid sequence selected from the group consisting of SEQ ID NOs:3-6, or a multimer, a combination, or a chimera of the polypeptides. In some embodiments, the polypeptide may further comprise a tag sequence. In some embodiments, the polypeptide may further comprise an amino acid linker between the polypeptides. In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:7, which may further comprise a tag sequence.
Further provided herein is a kit for detecting an antibody that specifically binds to an A. phagocytophilum p44 polypeptide, which kit comprises, in a container, the polypeptide disclosed above.
Also provided herein is a polynucleotide which encodes the A. phagocytophilum p44 polypeptide disclosed above, or a complimentary strand thereof. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. An exemplary codon-optimized polynucleotide that encodes an A. phagocytophilum p44 polypeptide comprising amino acids 222-247 of SEQ ID NO:1 comprises the sequence GGTCACTCCAGCGGCGTTACCCAGAATCCGAAACTGTTCAGTACCTTTGTTGATACC GTTAAAATCGCAGAAGATAAA (SEQ ID NO:34). In some embodiments, the organism may be a virus, a bacterium, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:8.
Further provided herein is polynucleotide which encodes an A. phagocytophilum p44 polypeptide having the amino acid sequence of SEQ ID NO:1, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type P44 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:2 under moderately or highly stringent conditions. Further provided herein is polynucleotide which encodes an A. phagocytophilum p44 polypeptide having the amino acid sequence of 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, or a complimentary strand thereof. In some embodiments, said polynucleotide is not a wild-type P44 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO:34 or SEQ ID NO:37. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:34 or SEQ ID NO:37 under moderately or highly stringent conditions.
Further provided herein is a vector comprising the A. phagocytophilum p44 polynucleotide disclosed above. In some embodiments, the polynucleotide may comprise a promoter sequence. In some embodiments, the polynucleotide may comprise a poly-A sequence. In some embodiments, the polynucleotide may comprise a translation termination sequence. In some embodiments, the polynucleotide may further encode a tag sequence.
Further provided herein is a non-human organism transformed with the vector disclosed above. In some embodiments, the organism may be a virus, a bacterium, a yeast cell, an insect or an insect cell, or a non-human mammal or a mammalian cell. In some embodiments, the organism may be used in a method for recombinantly making an A. phagocytophilum p44 polypeptide, which method comprises culturing the organism, and recovering said polypeptide from said organism. In some embodiments, the method may further comprise isolating the polypeptide, optionally by chromatography. Additionally, provided herein is a polypeptide produced by the method disclosed above. In some embodiments, the polypeptide may comprise post-translational modifications, e.g., a native glycosylation pattern and/or a native phosphorylation pattern.
Further provided herein is a method for detecting an antibody that specifically binds to an A. phagocytophilum p44 polypeptide in a sample, which method comprises contacting the polypeptide disclosed above with said sample and detecting a polypeptide-antibody complex formed. In some embodiments, the sample may be from a subject selected from the group consisting of dog, cat, human and horse. In some embodiments, the method may be used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease. In some embodiments, the disease may be granulocytic anaplasmosis. In some embodiments, the sample may be selected from the group consisting of a serum, a plasma and a blood sample. In some embodiments, the sample may be a clinical sample. In some embodiments, the antibody may be a monoclonal or polyclonal antibody or antibody fragment. In some embodiments, the polypeptide-antibody complex may be assessed by a sandwich or competitive assay format, optionally with a binder or antibody. In some embodiments, the binder or antibody may be attached to a surface and functions as a capture binder or antibody. In some embodiments, the capture binder or antibody may be attached to the surface directly or indirectly. In some embodiments, the capture binder or antibody may be attached to the surface via a linker, e.g., a biotin-avidin (or streptavidin) linking pair. In some embodiments, at least one of the binders or antibodies may be labeled. In some embodiments, the polypeptide-antibody complex may be assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay. In some embodiments, the polypeptide-antibody complex may be assessed in a homogeneous or a heterogeneous assay format.
In a second aspect, provided herein is a polynucleotide which encodes a B. burgdorferi OspC polypeptide having the amino acid sequence of SEQ ID NO:15, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspC polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:16. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:16 under moderately or highly stringent conditions. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect, an insect cell, a non-human mammal and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:17.
Further provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi OspC polypeptide in a sample, which method comprises contacting the polypeptide having the amino acid sequence of SEQ ID NO:15 encoded by the polynucleotide which is not a wild-type OspC polynucleotide with said sample and detecting a polypeptide-antibody complex formed.
In a third aspect, provided herein is a polynucleotide which encodes a B. burgdorferi OspF polypeptide having the amino acid sequence of SEQ ID NO:18, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspF polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:19 under moderately or highly stringent conditions. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect, an insect cell, a non-human mammal and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:20.
Further provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi OspF in a sample, which method comprises contacting the polypeptide having the amino acid sequence of SEQ ID NO:18 encoded by the polynucleotide which is not a wild-type OspF polynucleotide with said sample and detecting a polypeptide-antibody complex formed.
In a fourth aspect, provided herein is a polynucleotide which encodes a B. burgdorferi p39 polypeptide having the amino acid sequence of SEQ ID NO:21, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type p39 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:22. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:22 under moderately or highly stringent conditions. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect, an insect cell, a non-human mammal and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:23.
Further provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi p39 polypeptide in a sample, which method comprises contacting the polypeptide having the amino acid sequence of SEQ ID NO:21 encoded by the polynucleotide which is not a wild-type p39 polynucleotide with said sample and detecting a polypeptide-antibody complex formed.
Further provided herein is a vector comprising the B. burgdorferi OspC, OspF and p39 polynucleotide disclosed above. In some embodiments, the polynucleotide may comprise a promoter sequence. In some embodiments, the polynucleotide may comprise a poly-A sequence. In some embodiments, the polynucleotide may comprise a translation termination sequence. In some embodiments, the polynucleotide may further encode a tag sequence.
Further provided herein is a non-human organism transformed with the vector comprising the B. burgdorferi OspC, OspF and p39 polynucleotide disclosed above. In some embodiments, the organism may be a virus, a bacterium, a yeast cell, an insect, insect cell, a non-human mammal or a mammalian cell. In some embodiments, the organism may be used in a method for recombinantly making a B. burgdorferi OspC, OspF and p39 polypeptide, which method may comprise culturing the organism, and recovering said polypeptide from said organism. In some embodiments, the method may further comprise isolating the OspC, OspF and p39 polypeptide, optionally by chromatography. Additionally, provided herein is a B. burgdorferi OspC, OspF and p39 polypeptide produced by the method disclosed above. In some embodiments, the polypeptide may comprise a post-translational modification, e.g., a native glycosylation pattern and/or a native phosphorylation pattern.
In a fifth aspect, provided herein is an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE. In some embodiments, the antigenic composition does not consist of an OspA polypeptide and an OspC polypeptide. In some embodiments, the antigenic composition does not consist of an OspA polypeptide and an OspF polypeptide. In some embodiments, the antigenic composition does not consist of an OspC polypeptide and an OspF polypeptide. In some embodiments, the antigenic composition does not consist of an OspA polypeptide, an OspC polypeptide and an OspF polypeptide. In some embodiments, the antigenic composition may comprise at least 3, 4, or all 5 of said B. burgdorferi polypeptides. In some embodiments, the OspC polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:15 encoded by the polynucleotide which is not a wild-type OspC polynucleotide. In some embodiments, the OspF polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:18 encoded by the polynucleotide which is not a wild-type OspF polynucleotide. In some embodiments, the p39 polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:21 encoded by the polynucleotide which is not a wild-type p39 polynucleotide. In some embodiments, the fusion peptide of p41 and VLsE may comprise an amino acid sequence of SEQ ID NO:24. In some embodiments, the polypeptides may form a fusion molecule.
Also provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method may comprise contacting the antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of OspA, OspC, OspF, p39 polypeptide and a fusion peptide of p41 and VLsE disclosed above with said sample and detecting a polypeptide-antibody complex formed. In some embodiments, the sample may be from a subject selected from the group consisting of cat, dog, human and horse. In some embodiments, the method may be used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease. In some embodiments, the disease may be Lyme disease. In some embodiments, the method may be used to distinguish between infection by a Lyme disease pathogen and exposure to a Lyme disease vaccine. In some embodiments, the method may be used to distinguish between exposure to a Nobivac™ Lyme vaccine and exposure to another vaccine. In some embodiments, the sample may be selected from the group consisting of a serum, a plasma and a blood sample. In some embodiments, the sample may be a clinical sample. In some embodiments, the antibody may be a monoclonal or polyclonal antibody or antibody fragment. In some embodiments, the polypeptide-antibody complex may be assessed by a sandwich or competitive assay format, optionally with a binder or antibody. In some embodiments, the binder or antibody may be attached to a surface and functions as a capture binder or antibody. In some embodiments, the capture binder or antibody may be attached to the surface directly or indirectly. In some embodiments, the capture binder or antibody may be attached to the surface via a linker, e.g., a biotin-avidin (or streptavidin) linking pair. In some embodiments, at least one of the binders or antibodies may be labeled. In some embodiments, the polypeptide-antibody complex may be assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay. In some embodiments, the polypeptide-antibody complex may be assessed in a homogeneous or a heterogeneous assay format.
Further provided herein is a method of classifying Borrelia burgdorferi infection of a mammal, e.g., an animal, the method comprising: calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VLsE using a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method may comprise contacting the antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of OspA, OspC, OspF, p39 polypeptide and a fusion peptide of p41 and VLsE disclosed above with said sample and detecting a polypeptide-antibody complex formed; calculating reference values of the levels of the antibodies; and determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values.
Also provided herein is a kit for detecting an antibody that specifically binds to a B. burgdorferi polypeptide, which kit comprises, in a container, an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE. In some embodiments, the antigenic composition does not consist of an OspA polypeptide and an OspC polypeptide. In some embodiments, the antigenic composition does not consist of an OspA polypeptide and an OspF polypeptide. In some embodiments, the antigenic composition does not consist of an OspC polypeptide and an OspF polypeptide. In some embodiments, the antigenic composition does not consist of an OspA polypeptide, an OspC polypeptide and an OspF polypeptide.
In an sixth aspect, provided herein is a composition for detecting multiple disease antigens and/or antibodies, which composition comprises at least two, preferably three of the following reagents: a) an antibody against a Dirofilaria immitis antigen, b) an E. Canis gp36 polypeptide, c) an A. phagocytophilum p44 polypeptide, and d) an antigenic composition comprising a B. burgdorferi polypeptide selected from the group consisting of OspA, OspC, OspF, p39 and a fusion peptide of p41 and VLsE. In some embodiments, the composition may comprise all four of the reagents. In some embodiments, the reagent a) may be a chicken polyclonal antibody. In some embodiments, the chicken polyclonal antibody may be produced by immunizing chickens with a canine heartworm antigen. In some embodiments, the reagent b) may comprise a polypeptide having an amino acid sequence of SEQ ID NO:26, which may further comprise a tag sequence. In some embodiments, the reagent c) may comprise an A. phagocytophilum p44 polypeptide comprising amino acids 222-236 of SEQ ID NO:1, wherein said polypeptide comprises at least one mutation. In some embodiments, the reagent d) may comprise an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE.
Also provided herein is a kit for detecting multiple infectious organisms, which kit may comprise, in a container, the composition disclosed above. Further provided herein is a method for detecting multiple disease antigens and/or antibodies in a sample, which method may comprise: a) contacting said sample with the composition for detecting multiple disease antigens and/or antibodies, which composition may comprise at least two, preferably three of the following reagents: an antibody against a Dirofilaria immitis antigen, an E. Canis gp36 polypeptide, an A. phagocytophilum p44 polypeptide, and an antigenic composition comprising a B. burgdorferi polypeptide selected from the group consisting of OspA, OspC, OspF, p39 and a fusion peptide of p41 and VLsE; and b) detecting a polypeptide-antibody complex formed. In some embodiments, the method may be used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease. In some embodiments, the disease may be selected from the group consisting of a heartworm disease, ehrlichiosis, granulocytic anaplasmosis, and Lyme disease.
In a seventh aspect, provided herein is a computer readable medium containing executable instructions that when executed perform a method of classifying Borrelia burgdorferi infection of a mammal, e.g., an animal, the method comprising: calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VLsE using a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method may comprise contacting the antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of OspA, OspC, OspF, p39 polypeptide and a fusion peptide of p41 and VLsE disclosed above with said sample and detecting a polypeptide-antibody complex formed; calculating reference values of the levels of the antibodies; and determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values.
Further provided herein is a system for classifying Borrelia burgdorferi infection of a mammal, e.g., an animal comprising the computer readable medium disclosed herein and an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
As used herein, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “a” dimer includes one or more dimers.
The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY, which is the major antibody type in avian species such as chicken, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
As used herein, the term “specific binding” refers to the specificity of an antibody such that it preferentially binds to a target antigen, such as a polypeptide antigen, or a heartworm (Dirofilaria immitis) antigen. Recognition by an antibody of a particular target in the presence of other potential interfering substances is one characteristic of such binding. Preferably, antibodies or antibody fragments that are specific for or bind specifically to a target antigen bind to the target antigen with higher affinity than binding to other non-target substances. Also preferably, antibodies or antibody fragments that are specific for or bind specifically to a target antigen avoid binding to a significant percentage of non-target substances, e.g., non-target substances present in a testing sample. In some embodiments, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred. For example, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of non-target substances. In other embodiments, antibodies or antibody fragments of the present disclosure avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non-target substances.
As used herein, the term “specific binding” also refers to the specificity of a polypeptide such that it preferentially binds to a target antibody, such as a target antibody in a testing sample, e.g., antibodies against an Ehrlichia Canis gp36 polypeptide, an Anaplasma phagocytophilum p44 polypeptide or a Borrelia burgdorferi OspA, OspC, OspF, p39, p41 and/or VLsE polypeptide. Recognition by a polypeptide of a particular target antibody in the presence of other antibodies or substances is one characteristic of such binding. Preferably, a polypeptide that is specific for or binds specifically to an antibody binds to the target antibody with higher affinity than binding to other non-target antibodies or substances. Also preferably, a polypeptide that is specific for or binds specifically to a target antibody avoids binding to a significant percentage of non-target antibodies or substances, e.g., non-target antibodies present in a testing sample. In some embodiments, polypeptides of the present disclosure avoid binding greater than about 90% of non-target antibodies or substances, although higher percentages are clearly contemplated and preferred. For example, polypeptides of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of non-target antibodies or substances. In other embodiments, polypeptides of the present disclosure avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non-target antibodies or substances.
As used herein, the term “antigen” refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. The antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies. Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.
As used herein, the term “epitope” refers to a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may, for example, comprise nearly the full-length of the antigen sequence, or even a fusion protein comprising two or more epitopes from the target antigen. An epitope for use in the subject invention is not limited to a peptide having the exact sequence of the portion of the parent protein from which it is derived, but also encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (conservative in nature).
As used herein, a “tag” or an “epitope tag” refers to a sequence of amino acids, typically added to the N- and/or C-terminus of a polypeptide. The inclusion of tags fused to a polypeptide can facilitate polypeptide purification and/or detection. Typically a tag or tag polypeptide refers to polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of chimeric polypeptide to which it is linked. The tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues. The tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture. Such tags are well known and can be readily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification and include His tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody. See, e.g., Field et al. (1988) Mol. Cell. Biol. 8:2159-2165; Evan et al. (1985) Mol. Cell. Biol. 5:3610-3616; Paborsky et al. (1990) Protein Engineering 3:547-553.
The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, e.g., at least 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ to P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, “caps,” substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
It will be appreciated that, as used herein, the terms “nucleoside” and “nucleotide” will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.
“Nucleic acid probe” and “probe” are used interchangeably and refer to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
As used herein, “complementary or matched” means that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. “Complementary or matched” also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition(s).
As used herein, “substantially complementary or substantially matched” means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition(s).
In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M EDTA. Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).
Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See Kanehisa (1984) Nucleic Acids Res. 12:203-215.
The terms “homologous”, “substantially homologous”, and “substantial homology” as used herein denote a sequence of amino acids having at least 50%, 60%, 70%, 80% or 90% identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.
As used herein, “vector (or plasmid)” refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well known within the skill of the artisan. An expression vector includes vectors capable of expressing DNA's that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eucaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, “a promoter region or promoter element” refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters, and the like.
As used herein, “operatively linked or operationally associated” refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5′ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5′ of the start codon and may enhance expression. See, e.g., Kozak (1991) J. Biol. Chem. 266:19867-19870. The desirability of (or need for) such modification may be empirically determined.
As used herein, “biological sample” refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom. Also included are soil and water samples and other environmental samples, viruses, bacteria, fungi, algae, protozoa and components thereof.
The terms “level” or “levels” are used to refer to the presence and/or amount of protein, and can be determined qualitatively or quantitatively. A “qualitative” change in the protein level refers to the appearance or disappearance of a protein spot that is not detectable or is present in samples obtained from normal controls. A “quantitative” change in the levels of one or more proteins of the profile refers to a measurable increase or decrease in the protein levels when compared to a healthy control.
A “healthy control” or “normal control” is a biological sample taken from an individual who does not suffer from an infectious disorder. A “negative control,” is a sample that lacks any of the specific analyte the assay is designed to detect and thus provides a reference baseline for the assay.
It is understood that aspects and embodiments of the invention described herein include “consisting” and/or “consisting essentially of” aspects and embodiments.
Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.
As discussed above, the present invention is concerned with compositions and methods for detecting infectious organisms including heartworm, E. Canis, A. phagocytophilum, and B. burgdorferi. The diseases caused by these organisms include, but are not limited to, a heartworm disease, ehrlichiosis, granulocytic anaplasmosis, and Lyme disease. These infectious organisms may cause diseases in mammalian subjects such as dogs, cats, horses, humans, etc.
In one aspect, provided herein are polypeptides, antibodies and antigenic compositions for detecting infectious organisms including heartworm, E. Canis, A. phagocytophilum, and B. burgdorferi in a subject. An antigenic composition may comprise a combination of antibodies and antigenic polypeptides that are specific for one or several infectious organisms.
Therefore, provided herein is an A. phagocytophilum p44 polypeptide comprising amino acids 222-236 of SEQ ID NO:1, wherein said polypeptide comprises at least one mutation. Additionally, provided herein is an A. phagocytophilum p44 polypeptide that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:1, wherein said polypeptide is not a wild-type P44 protein, and wherein said polypeptide binds to an antibody that is specific for a wild-type P44 protein. In some embodiments, the polypeptide may further comprise a second polypeptide comprising amino acids 237-247 of SEQ ID NO:1. Also provided herein is an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 or an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 that comprises at least one mutation. Additionally, provided herein is an A. phagocytophilum p44 polypeptide that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, wherein said polypeptide is not a wild-type P44 protein, and wherein said polypeptide binds to an antibody that is specific for a wild-type P44 protein. In some embodiments, the polypeptide may comprise the amino acid sequence selected from the group consisting of SEQ ID NOs:3-6, the amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, or a multimer, a combination, or a chimera of the polypeptides. In some embodiments, the polypeptide may further comprise a tag sequence. In some embodiments, the polypeptide may further comprise an amino acid linker between the polypeptides. In one embodiment, the polypeptide may comprise the amino acid sequence of SEQ ID NO:7, which may further comprise a tag sequence.
In addition, provided herein is an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE, wherein said antigenic composition does not consist of a) and b). In some embodiments, the antigenic composition may comprise at least 3, 4, or all 5 of said B. burgdorferi polypeptides. In some embodiments, the OspA polypeptide may be a multimer of a partial or full-length sequence, which may have a molecular weight of about 85 kDa. In some embodiments, the OspA polypeptide may comprise a sequence tag, e.g., a His tag. In some embodiments, the OspA polypeptide may be commercially available, e.g., OspA from Meridian Life Science, Inc. (Catalog #: R8A131), which contains multiple copies of the B. burgdorferi OspA sequence and a 6-HIS epitope tag. In some embodiments, the OspC polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:15 encoded by the polynucleotide which is not a wild-type OspC polynucleotide. In some embodiments, the OspF polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:18 encoded by the polynucleotide which is not a wild-type OspF polynucleotide. In some embodiments, the p39 polypeptide may comprise the polypeptide having the amino acid sequence of SEQ ID NO:21 encoded by the polynucleotide which is not a wild-type p39 polynucleotide. In some embodiments, the fusion peptide of p41 and VLsE may comprise an amino acid sequence of SEQ ID NO:24. In some embodiments, the polypeptides may form a fusion molecule.
In some embodiments, the at least 3, 4, or all 5 of said B. burgdorferi polypeptides form a fusion molecule. The fusion molecule, which may be a fusion protein, may include linkers that separate the individual polypeptides. One or multiple copies of each polypeptide may exist in the fusion molecule, and may exist in any order.
The polypeptide may include the addition of an antibody epitope or other tag, to facilitate identification, targeting, and/or purification of the polypeptide. The use of 6×His and GST (glutathione S transferase) as tags is well known. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other amino acid sequences that may be included in the polypeptide include functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions. The polypeptide may further include one or more additional tissue-targeting moieties.
Epitope tags are well known to those of skill in the art. Moreover, antibodies specific to a wide variety of epitope tags are commercially available. These include but are not limited to antibodies against the DYKDDDDK epitope, c-myc antibodies (available from Sigma, St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSV epitope, the His4, His5, and His6 epitopes that are recognized by the His epitope specific antibodies (see, e.g., Qiagen), and the like. In addition, vectors for epitope tagging proteins are commercially available. A polypeptide can be tagged with the FLAG® epitope (N-terminal, C-terminal or internal tagging), the c-myc epitope (C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal) epitopes.
In some embodiments, the A. phagocytophilum p44 polypeptide, the B. burgdorferi polypeptides, or the fusion protein, may contain a tag sequence, either at the N-terminus, or C-terminus, or both. Tag sequences that may be used are set forth in SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:33.
Polypeptides may possess deletions and/or substitutions of amino acids relative to the native sequence. Sequences with amino acid substitutions are contemplated, as are sequences with a deletion, and sequences with a deletion and a substitution. In some embodiments, these polypeptides may further include insertions or added amino acids.
Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly to increase its efficacy or specificity. Substitutions of this kind may or may not be conservative substitutions. Conservative substitution is when one amino acid is replaced with one of similar shape and charge. However, if used, conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Changes other than those discussed above are generally considered not to be conservative substitutions. It is specifically contemplated that one or more of the conservative substitutions above may be included as embodiments. In other embodiments, such substitutions are specifically excluded. Furthermore, in additional embodiments, substitutions that are not conservative are employed in variants.
In addition to a deletion or substitution, the polypeptides may possess an insertion of one or more residues.
The variant amino acid sequence may be structurally equivalent to the native counterparts. For example, the variant amino acid sequence forms the appropriate structure and conformation for binding targets, proteins, or peptide segments.
The following is a discussion based upon changing of the amino acids of a polypeptide to create a mutant molecule. For example, certain amino acids may be substituted for other amino acids in a polypeptide without appreciable loss of function, such as ability to interact with an antibody or a target peptide sequence. Since it is the interactive capacity and nature of a polypeptide that defines that polypeptide's functional activity, certain amino acid substitutions can be made in a polypeptide sequence and nevertheless produce a polypeptide with like properties.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive function on a protein is generally understood in the art. See Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (2.3); phenylalanine (−2.5); tryptophan (−3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. However, in some aspects a non-conservative substitution is contemplated. In certain aspects a random substitution is also contemplated. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In some embodiments, the A. phagocytophilum p44 polypeptide may comprise 1 to 10, preferably 3-7, mutations. In some embodiments, the mutations may be selected from the group consisting of a substitution, an insertion and a deletion. Some of the exemplary mutations are: Gly222(Del), His223→Asn, Ser224→Thr, Ser225→Thr, Val227→Ala, Thr228→Ser, Gln229→Asn, Leu233→Val, Leu233→Thr, Phe234→Leu, Ser235→Thr, and Thr236→Ser. In some embodiments, the polypeptide may comprise at least 1, 2, 3, 4, 5, 10 or 12 of the exemplary mutations.
In some embodiments, the second A. phagocytophilum p44 polypeptide may comprise at least 1, or 1 to 5, preferably 2 to 3, mutations. Some of the exemplary mutations are: Thr240→Ser, Gln229→Asn, Ile243→Val, Glu245→Asp, Glu245→Asn, Asp246→Lys, and Asp246→Glu. In some embodiments, the polypeptide may comprise at least 1, 2, 3, 4, 5 or 7 of the exemplary mutations.
Further provided herein is a composition for detecting multiple disease antigens and/or antibodies, which composition comprises at least two, preferably three of the following reagents: a) an antibody against a Dirofilaria immitis antigen, b) an E. Canis gp36 polypeptide, c) an A. phagocytophilum p44 polypeptide, and d) an antigenic composition comprising a B. burgdorferi polypeptide selected from the group consisting of OspA, OspC, OspF, p39 and a fusion peptide of p41 and VLsE. In some embodiments, the composition may comprise all four of the reagents. In some embodiments, the reagent a) may be a chicken polyclonal antibody. In some embodiments, the chicken polyclonal antibody may be produced by immunizing chickens with a canine heartworm antigen. In some embodiments, the chicken polyclonal antibody may be a type IgY antibody, e.g., IgY antibody isolated from chicken egg yolk or serum. In some embodiments, the reagent b) may comprise a polypeptide having an amino acid sequence of SEQ ID NO:26, which may further comprise a tag sequence. In some embodiments, the reagent c) may comprise an A. phagocytophilum p44 polypeptide comprising amino acids 222-236 of SEQ ID NO:1, wherein said polypeptide comprises at least one mutation. In some embodiments, the reagent d) may comprise an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE.
In another aspect, provided herein are polynucleotides, vectors and methods for the production of the polypeptides disclosed above, including the A. phagocytophilum P44 polypeptide. Polypeptides, vectors and methods for the production of the B. burgdorferi OspC, OspF and p39 polypeptides are also provided.
Therefore, provided herein is a polynucleotide which encodes the A. phagocytophilum p44 polypeptide disclosed above, or a complimentary strand thereof. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. An exemplary codon-optimized polynucleotide that encodes an A. phagocytophilum p44 polypeptide comprising amino acids 222-247 of SEQ ID NO:1 comprises the sequence GGTCACTCCAGCGGCGTTACCCAGAATCCGAAACTGTTCAGTACCTTTGTTGATACC GTTAAAATCGCAGAAGATAAA (SEQ ID NO:34). In some embodiments, the organism may be a virus, a bacterium, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:8.
Further provided herein is polynucleotide which encodes an A. phagocytophilum p44 polypeptide having the amino acid sequence of SEQ ID NO:1, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type P44 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:2 under moderately or highly stringent conditions. Further provided herein is polynucleotide which encodes an A. phagocytophilum p44 polypeptide having the amino acid sequence of 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, or a complimentary strand thereof. In some embodiments, said polynucleotide is not a wild-type P44 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO:34 or SEQ ID NO:37. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:34 or SEQ ID NO:37 under moderately or highly stringent conditions.
Further provided herein is a polynucleotide which encodes a B. burgdorferi OspC polypeptide having the amino acid sequence of SEQ ID NO:15, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspC polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:16. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:16 under moderately or highly stringent conditions. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:17.
Further provided herein is a polynucleotide which encodes a B. burgdorferi OspF polypeptide having the amino acid sequence of SEQ ID NO:18, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspF polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:19 under moderately or highly stringent conditions. In some embodiments, the polynucleotide is codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:20.
Further provided herein is a polynucleotide which encodes a B. burgdorferi p39 polypeptide having the amino acid sequence of SEQ ID NO:21, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type p39 polynucleotide. In some embodiments, the polynucleotide may exhibit at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:22. In some embodiments, the polynucleotide may hybridize to the nucleotide sequence of SEQ ID NO:22 under moderately or highly stringent conditions. In some embodiments, the polynucleotide may be codon-optimized for expression in a non-human organism. In some embodiments, the organism may be selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell. In some embodiments, the polynucleotide may be DNA or RNA. In some embodiments, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:23.
Further provided herein is a vector comprising the A. phagocytophilum p44 polynucleotide or the B. burgdorferi OspC, OspF and p39 polynucleotide disclosed above. In some embodiments, the vector may comprise a promoter sequence. In some embodiments, the vector may comprise a poly-A sequence. In some embodiments, the vector may comprise a translation termination sequence. In some embodiments, the vector may further encode a tag sequence.
Further provided herein is a non-human organism transformed with the vector comprising the A. phagocytophilum p44 polynucleotide or the B. burgdorferi OspC, OspF and p39 polynucleotide disclosed above. In some embodiments, the organism may be a virus, a bacterium, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the organism may be used in a method for recombinantly making an A. phagocytophilum p44 or B. burgdorferi OspC, OspF and p39 polypeptide, which method may comprise culturing the organism, and recovering said polypeptide from said organism. In some embodiments, the method may further comprise isolating the P44, OspC, OspF and p39 polypeptide, optionally by chromatography. Additionally, provided herein is an A. phagocytophilum p44 or a B. burgdorferi OspC, OspF and p39 polypeptide produced by the method disclosed above. In some embodiments, the polypeptide may comprise a native glycosylation pattern and/or a native phosphorylation pattern.
An expression vector comprising cDNA encoding a polypeptide or a target molecule is introduced into Escherichia coli, yeast, an insect cell, an animal cell or the like for expression to obtain the polypeptide. Polypeptides used in the present invention can be produced, for example, by expressing a DNA encoding it in a host cell using a method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) or the like. A recombinant vector is produced by inserting a cDNA downstream of a promoter in an appropriate expression vector. The vector is then introduced into a host cell suitable for the expression vector. The host cell can be any cell so long as it can express the gene of interest, and includes bacteria (e.g., Escherichia coli), an animal cell and the like. Expression vector can replicate autonomously in the host cell to be used or vectors which can be integrated into a chromosome comprising an appropriate promoter at such a position that the DNA encoding the polypeptide can be transcribed.
Silent modifications can be made to the nucleic acids that do not alter, substitute or delete the respective amino acid in the recombinant protein. Such modification may be necessary to optimize, for example, the codon usage for a specific recombinant host. The nucleotide sequences can be modified to replace codons that are considered rare or have a low frequency of appropriate t-RNA molecules to a more suitable codon appropriate for the expression host. Such codon tables are known to exist and are readily available to one skilled in the art. In addition, silent modification can be made to the nucleic acid that minimizes secondary structure loops at the level of mRNA that may be deleterious to recombinant protein expression.
In a further aspect, the polypeptides, antibodies and antigenic compositions disclosed above may be used for detecting various infectious organisms including heartworm, E. Canis, A. phagocytophilum, and B. burgdorferi in a subject. In some embodiments, the method may be used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease.
Therefore, provided herein is a method for detecting an antibody that specifically binds to an A. phagocytophilum p44 polypeptide in a sample, which method comprises contacting the P44 polypeptide disclosed above with said sample and detecting a polypeptide-antibody complex formed. In some embodiments, the disease may be granulocytic anaplasmosis.
Further provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method comprises contacting a B. burgdorferi polypeptide selected from the group consisting of OspC, OspF and p39 disclosed above with said sample and detecting a polypeptide-antibody complex formed.
Also provided herein is a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method comprises contacting an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of OspA, OspC, OspF, p39 polypeptide and a fusion peptide of p41 and VLsE disclosed above with said sample and detecting a polypeptide-antibody complex formed. A combination of 2, 3, 4, or 5 polypeptides may be used.
In some embodiments, the method may be used to detect Lyme disease. In addition, the method may be used to distinguish between infection by a Lyme disease pathogen and exposure to a Lyme disease vaccine. Several commercially available Lyme disease vaccines, such as Nobivac™ Lyme (Intervet/Schering-Plough Animal Health, Summit, N.J.), LymeVax® (Fort Dodge Animal Health, New York, N.Y.), and RECOMBITEK® Lyme (Merial Ltd., Duluth, Ga.), provide protection mainly by inducing the production of anti-OspA antibodies. See LaFleur et al. (2009) Clin. Vacc. Immunol. 16:253-259; LaFleur et al. (2010) Clin. Vacc. Immunol. 17:870-874. Therefore, detection of anti-OspA antibodies but not antibodies to other B. burgdorferi polypeptides may indicate that the subject has been vaccinated, while on the other hand, detection of antibodies to other polypeptides in addition to OspA may indicate that the subject has been exposed to a B. burgdorferi antigen naturally. Further, the method may be used to distinguish between exposure to a Nobivac™ Lyme vaccine and exposure to another vaccine, because the Nobivac™ Lyme vaccine induces both anti-OspA and OspC antibodies. See LaFleur et al. (2009) Clin. Vacc. Immunol. 16:253-259. Therefore, detection of both anti-OspA and anti-OspC antibodies but not antibodies to other B. burgdorferi polypeptides may indicate that the subject has been vaccinated with a Nobivac™ Lyme vaccine.
Therefore, the method can be used for classification of Lyme exposure of a mammal, e.g., an animal by calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VLsE using a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample disclosed herein; calculating reference values of the levels of the antibodies; and determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values. The reference values may be calculated using levels of detectable signals of negative controls, and more than one reference values may be calculated for each antibody that specifically binds to a Lyme polypeptide.
The reference values may be established by analyzing results from experimental samples from animals that are infected with or vaccinated against B. burgdorferi. Empirical values may be calculated from the analysis of experimental samples, and used for the calculation of reference values for the antibodies. Initially, artificial values may be set for each reference value and adjusted by an algorithm using experimental data. A minimal value for each reference value may also be established from the analysis of experimental samples and in cases where the reference value calculated is less than the minimal value, the minimal value may be used.
In some embodiments, the reference values for the antibody that specifically binds to OspA may be alpLow, alpMid, alpHigh and/or alpHighest, wherein alpMid may be from about 150% to about 250% of alpLow, alpHigh may be from about 300% to about 400% of alpLow, and/or alpHighest may be from about 500% to about 1,000% of alpLow. In some embodiments, the reference values for the antibody that specifically binds to OspC may be ospcLow and/or ospcHigh; wherein ospcHigh may be from about 150% to about 500% of ospcLow. In some embodiments, the reference values for the antibody that specifically binds to OspF may be ospfLow and/or ospfHigh; wherein ospfHigh may be from about 150% to about 300% of ospfLow. In some embodiments, the reference value for the antibody that specifically binds to p39 may be p39Low. In some embodiments, the reference values for the antibody that specifically binds to the fusion peptide of p41 and VLsE may be slpLow, slpMid and/or slpHigh, wherein slpMid may be from about 150% to about 200% of slpLow, and/or slpHigh may be from about 300% to about 500% of slpLow. The level of antibody that specifically binds to the Anaplasma phagocytophilum P44 polypeptide may be used for detection of ticks in animals being tested. In some embodiments, the P44 polypeptide may comprise the amino acid sequence of SEQ ID NO:7. In some embodiments, the reference value for the antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 may be sub5Low.
In calculating the reference values for the antibodies to the various Lyme polypeptides, results from negative controls may be used in the calculation. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more negative controls may be included in an assay.
In some embodiments, the mammal may be classified as Lyme exposure (LE) if: a) the level of antibody that specifically binds to OspA is lower than alpHigh, and the level of antibody that specifically binds to OspF is greater than or equal to ospfHigh; b) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid, the level of antibody that specifically binds to OspF is lower than ospfHigh, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow or the level of antibody that specifically binds to OspC is greater than or equal to ospcLow, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low or the level of antibody that specifically binds to OspF is greater than or equal to ospfLow; c) the level of antibody that specifically binds to OspA is lower than alpLow, the level of antibody that specifically binds to OspC is lower than ospcLow, the level of antibody that specifically binds to OspF is lower than ospfHigh, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow and lower than slpMid, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low or the level of antibody that specifically binds to OspF is greater than or equal to ospfLow; d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid, the level of antibody that specifically binds to OspC is greater than or equal to ospcLow, the level of antibody that specifically binds to p39 is greater than or equal to p39Low, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow, the level of antibody that specifically binds to OspF is lower than ospfLow, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low; or e) the level of antibody that specifically binds to OspA is lower than alpLow, the level of antibody that specifically binds to OspF is lower than ospfHigh, and the level of antibody that specifically binds to OspC is greater than or equal to ospcLow or the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpMid. In some embodiments, the mammal classified as Lyme exposure may be further classified as Lyme exposure early (LEE) if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure late (LEL).
In some embodiments, the mammal may be classified as Lyme exposure and vaccine (LEV) if: a) the level of antibody that specifically binds to OspA is greater than or equal to alpHigh, and the level of antibody that specifically binds to OspF is greater than or equal to ospfHigh; b) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low, the level of antibody that specifically binds to OspF is lower than ospfHigh, and the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow or the level of antibody that specifically binds to OspC is greater than or equal to ospcLow; c) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to OspC is greater than or equal to ospcHigh, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpHigh, the level of antibody that specifically binds to OspF is lower than ospfHigh, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low; or d) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to OspC is greater than or equal to ospcLow, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low. In some embodiments, the mammal classified as Lyme exposure and vaccine may be further classified as Lyme exposure and vaccine early (LEEV) if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure and vaccine late (LELV).
In some embodiments, the mammal may be classified as Lyme vaccine (LVR) if: a) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, and the level of antibody that specifically binds to OspF is lower than ospfLow; b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to OspC is lower than ospcLow or the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpLow but not both, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low; c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to OspC is lower than ospcLow, and the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpLow; d) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpHigh, the level of antibody that specifically binds to OspC is lower than ospcHigh, and the level of antibody that specifically binds to OspC is greater than or equal to ospcLow or the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow; e) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to OspF is lower than ospfLow, the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low, and the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpHigh or the level of antibody that specifically binds to OspC is lower than ospcHigh; or 0 the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to OspF is lower than ospfHigh, the level of antibody that specifically binds to OspC is lower than ospcLow, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpLow, and the level of antibody that specifically binds to OspF is greater than or equal to ospfLow or the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low.
In some embodiments, the mammal may be classified as indeterminative (IND) if: a) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low, and the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpHigh or the level of antibody that specifically binds to OspC is lower than ospcHigh but not both; b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to OspC is lower than ospcLow or the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is lower than slpLow but not both, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is greater than or equal to sub5Low; c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, the level of antibody that specifically binds to OspF is greater than or equal to ospfLow and lower than ospfHigh, the level of antibody that specifically binds to OspC is greater than or equal to ospcLow, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low; d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid, the level of antibody that specifically binds to OspF is Lower than ospfLow, the level of antibody that specifically binds to OspC is greater than or equal to ospcLow, the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow, the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low, and the level of antibody that specifically binds to p39 is lower than p39Low; or e) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid, the level of antibody that specifically binds to OspF is Lower than ospfLow, the level of antibody that specifically binds to OspC is greater than or equal to ospcLow or the level of antibody that specifically binds to the fusion peptide of p41 and VLsE is greater than or equal to slpLow, and the level of antibody that specifically binds to the amino acid sequence of SEQ ID NO:7 is lower than sub5Low. In some embodiments, the mammal classified as indeterminative may be further classified as possible exposure (PE) if the level of antibody that specifically binds to OspA is lower than alpMid; otherwise Lyme vaccine possible exposure (LVPE).
The following is an exemplary protocol for classifying Lyme exposure in a mammal, e.g., an animal by comparing the levels of various antibodies to the reference values:
For all LE Rules: If ospf<ospfHigh it is LEE, else it is LEL.
LE: alp<alphigh, Ospf>=ospfHigh
LE: alpLow<=alp<alpMid, ospf<ospfHigh, (slpResult>=slpLow OR ospc>=ospcLow), (sub5>=sub5Low OR ospf>=ospfLow)
LE: alp<alpLow, ospc<ospcLow, ospf<ospfHigh, slpLow<=slp<slpMid, (sub5>=sub5Low OR ospf>=ospfLow)
LE: alpLow<=alp<alpMid, ospc>=ospcLow, p39>=p39Low, slp>=slpLow, ospf<ospfLow, sub5<sub5Low
LE: alp<alpLow, ospf<ospfHigh, (ospc>=ospcLow OR slp>=slpMid)
For all LEV rules: if ospf<ospfHigh it is LEEV, else it is LELV
LEV: alp>=alpHigh, ospf>=ospfHigh
LEV: alpMid<=alp<alpHighest, sub5>=sub5Low, ospf<ospfHigh, (slp>=slpLow OR ospc>=ospcLow)
LEV: alpMid<=alp<alpHighest, ospc>=ospcHigh, slp>=slpHigh, ospf<ospfHigh, sub5Result<sub5Low
LEV: alp>=alpHighest, ospfLow<=ospf<ospfHigh, ospc>=ospcLow, slp>=slpLow, sub5>=sub5Low
LVR: alp>=alpHighest, ospf<ospfLow
LVR: alp>=alpHighest, ospfLow<=ospf<ospfHigh, (ospc<ospcLow XOR slp<slpLow), sub5<sub5Low
LVR: alp>=alpHighest, ospfLow<=ospf<ospfHigh, ospc<ospcLow, slp<slpLow
LVR: alpMid<=alp<alpHighest, ospfLow<=ospf<ospfHigh, sub5<sub5Low, slp<slpHigh, ospc<ospcHigh, (ospc>=ospcLow OR slp>=slpLow)
LVR: alpMid<=alp<alpHighest, ospf<ospfLow, sub5<sub5Low, (slp<slpHigh OR ospc<ospcHigh)
LVR: alpMid<=alp<alpHighest, ospf<ospfHigh, ospc<ospcLow, slp<slpLow, (ospf>=ospfLow OR sub5>=sub5Low)
For all IND rules: if alp<alpMid it is PE, else it is LVPE
IND: alpMid<=alp<alpHighest, ospfLow<=ospf<ospfHigh, sub5Result<sub5Low, (slp<slpHigh XOR opsc<ospcHigh)
IND: alp>=alpHighest, ospfLow<=ospf<ospfHigh, (ospc<ospcLow XOR slp<slpLow), sub5>=sub5Low
IND: alp>=alpHighest, ospfLow<=ospf<ospfHigh, ospc>=ospcLow, slp>=slpLow, sub5<sub5Low
IND: alpLow<=alp<alpMid, ospf<ospfLow, ospc>=ospcLow, slp>=slpLow, sub5<sub5Low, p39<p39Low
IND: alpLow<=alp<alpMid, ospf<ospfLow, (ospc>=ospcLow XOR slp>=slpLow), sub5<sub5Low
Keys:
alp level of antibody that specifically binds to OspA
ospc level of antibody that specifically binds to OspC
ospf level of antibody that specifically binds to OspF
p39 level of antibody that specifically binds to p39
slp level of antibody that specifically binds to the fusion peptide of p41 and VLsE
sub5 level of antibody that specifically binds to the multimeric mutant peptide of P44
LEE Lyme exposure early
LEL Lyme exposure late
LVR Lyme vaccine
LELV Lyme exposure late & vaccine
LEEV Lyme exposure early & vaccine
LVRN Lyme vaccine Nobivac™
IND indeterminative
PE possible Lyme exposure
LVPE Lyme vaccine and possible Lyme exposure
XOR one or the other, but not both
Further provided herein is a method for detecting multiple disease antigens and/or antibodies in a sample, which method comprises: a) contacting said sample with a composition for detecting multiple disease antigens and/or antibodies, which composition may comprise at least two, preferably three of the following reagents: an antibody against a Dirofilaria immitis antigen, an E. Canis gp36 polypeptide, an A. phagocytophilum p44 polypeptide, and an antigenic composition comprising a B. burgdorferi polypeptide selected from the group consisting of OspA, OspC, OspF, p39 and a fusion peptide of p41 and VLsE; and b) detecting a polypeptide-antibody complex formed. In some embodiments, the composition may comprise all four of the reagents. In some embodiments, the antibody against a Dirofilaria immitis antigen may be a chicken polyclonal antibody. In some embodiments, the chicken polyclonal antibody may be produced by immunizing chickens with a canine heartworm antigen. In some embodiments, the chicken polyclonal antibody may be a type IgY antibody. In some embodiments, the E. Canis gp36 polypeptide may comprise a polypeptide having an amino acid sequence of SEQ ID NO:26, which may further comprise a tag sequence. In some embodiments, the A. phagocytophilum p44 polypeptide may comprise amino acids 222-236 of SEQ ID NO:1, wherein said polypeptide comprises at least one mutation. In some embodiments, the antigenic composition may at least two B. burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE.
In some embodiments, the sample may be from a subject selected from the group consisting of dog, cat, human and horse. In some embodiments, the method may be used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease. In some embodiments, the sample may be selected from the group consisting of a serum, a plasma and a blood sample. In some embodiments, the sample may be a clinical sample. In some embodiments, the antibody may be a monoclonal or polyclonal antibody or antibody fragment.
The detection of antibodies and/or antigens may be achieved by immunoassays, including any immunoassay known in the art including, but not limited to, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), “sandwich” assay, precipitin reaction, agglutination assay, fluorescent immunoassay, and chemiluminescence-based immunoassay. In some embodiments, the polypeptide-antibody complex may be assessed by a sandwich or competitive assay format, optionally with a binder or antibody. In some embodiments, the binder or antibody may be attached to a surface and functions as a capture antibody. In some embodiments, the capture binder or antibody may be attached to the surface directly or indirectly. In some embodiments, the binder or antibody may be attached to the surface via a biotin-avidin (or streptavidin) linking pair. In some embodiments, at least one of the binders or antibodies may be labeled. In some embodiments, the polypeptide-antibody complex may be assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, plasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay. In some embodiments, the polypeptide-antibody complex may be assessed in a homogeneous or a heterogeneous assay format.
In some embodiments, multiple reagents for detecting infectious organisms may be included in the same assay, such as parallel immunoassay. A parallel immunoassay may include at least 2, 3, 4, 5, 10, 100, 1000 or more reagents, such as antibodies or antigenic polypeptides, in the same assay system.
Numerous technological platforms for performing parallel immunoassays are known. Generally, such methods involve a logical or physical array of either the subject samples, or the protein markers, or both. Common array formats include both liquid and solid phase arrays. For example, assays employing liquid phase arrays, e.g., for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microtiter plates. Microtiter plates with 96, 384 or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general, the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis. Exemplary systems include, e.g., the ORCA™ system from Beckman-Coulter, Inc. (Fullerton, Calif.) and the Zymate systems from Zymark Corporation (Hopkinton, Mass.).
Alternatively, a variety of solid phase arrays can favorably be employed for parallel immunoassays in the context of the invention. Exemplary formats include membrane or filter arrays (e.g., nitrocellulose, nylon), pin arrays, and bead arrays (e.g., in a liquid “slurry”). Typically, probes corresponding to nucleic acid or protein reagents that specifically interact with (e.g., hybridize to or bind to) an expression product corresponding to a member of the candidate library, are immobilized, for example by direct or indirect cross-linking, to the solid support. Essentially any solid support capable of withstanding the reagents and conditions necessary for performing the particular expression assay can be utilized. For example, functionalized glass, silicon, silicon dioxide, modified silicon, any of a variety of polymers, such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof can all serve as the substrate for a solid phase array.
The polypeptides/antibodies may be immobilized to a solid phase support for the detection of antibody binding. As used herein, “solid phase support” is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads and alumina gels. A suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California). In a preferred embodiment for peptide synthesis, solid phase support refers to polydimethylacrylamide resin.
In one embodiment, the array may be a “chip” composed, e.g., of one of the above-specified materials. Polynucleotide probes, e.g., RNA or DNA, such as cDNA, synthetic oligonucleotides, and the like, or binding proteins such as antibodies or antigen-binding fragments or derivatives thereof may be affixed to the chip in a logically ordered manner, i.e., in an array. Detailed discussions of methods for linking nucleic acids and proteins to a chip substrate, are found in, e.g., U.S. Pat. No. 5,143,854, U.S. Pat. No. 5,837,832, U.S. Pat. No. 6,087,112, U.S. Pat. No. 5,215,882, U.S. Pat. No. 5,707,807, U.S. Pat. No. 5,807,522, U.S. Pat. No. 5,958,342, U.S. Pat. No. 5,994,076, U.S. Pat. No. 6,004,755, U.S. Pat. No. 6,048,695, U.S. Pat. No. 6,060,240, U.S. Pat. No. 6,090,556, and U.S. Pat. No. 6,040,138, each of which is hereby incorporated in its entirety.
Microarray signals may be detected by scanning the microarray with a variety of laser or CCD-based scanners, and extracting features with numerous software packages, for example, Imagene (Biodiscovery), Feature Extraction Software (Agilent), Scanalyze (Eisen, M. 1999. SCANALYZE User Manual; Stanford Univ., Stanford, Calif. Ver 2.32.), GenePix (Axon Instruments).
High-throughput protein systems include commercially available systems from Ciphergen Biosystems, Inc. (Fremont, Calif.) such as Protein Chip® arrays and the Schleicher and Schuell protein microspot array (FastQuant Human Chemokine, S&S Bioscences Inc., Keene, N.H., US). In one embodiment, the high-throughput protein assay system may be the Bio-CD system using the SDI™ (Spinning Disc Interferometry) technology by Quadraspec, Inc. (West Lafayette, Ind.). Detailed discussions of the Bio-CD system are found in, e.g., U.S. Pat. No. 6,685,885, U.S. Pat. No. 7,405,831, U.S. Pat. No. 7,552,282, U.S. Pat. No. 7,659,968, U.S. Pat. No. 7,663,092, U.S. Pat. No. 7,787,126, U.S. Pat. No. 7,910,356, U.S. Pat. Pub. No. 2004/0166593, U.S. Pat. Pub. No. 2006/0256676, U.S. Pat. Pub. No. 2007/0023643, U.S. Pat. Pub. No. 2007/0212257, U.S. Pat. Pub. No. 2007/0259366, U.S. Pat. Pub. No. 2008/0175755, U.S. Pat. Pub. No. 2009/0002716, U.S. Pat. Pub. No. 2009/0263913, U.S. Pat. Pub. No. 2010/0145627, and Canadian Pat. Pub. No. 2681722, each of which is hereby incorporated in its entirety.
The parallel immunoassay results obtained as described above can then be used for diagnosis of the specific disorder. The individual proteins/antibodies can be detected or quantified by any of a number of means well known to those of skill in the art. In one aspect, a qualitative change in one or more proteins/antibodies is determined. Qualitative changes include the appearance of a proteins/antibodies spot that is not detectable in samples obtained from normal controls or the disappearance of a proteins/antibodies spot which is detectable in normal controls but not in the sample taken from an affected subject.
In another aspect, a quantitative change in one or more proteins/antibodies may be measured. The concentration of protein/antibody levels may be expressed in absolute terms, for example, optical density as read by image analysis. Alternatively, the concentrations can be expressed as a fraction, relative to normal levels of the same protein/antibody.
In another aspect, provided herein is a computer readable medium containing executable instructions that when executed perform a method of classifying Borrelia burgdorferi infection of a mammal, e.g., an animal, the method comprising: calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VLsE using a method for detecting an antibody that specifically binds to a B. burgdorferi antigen in a sample, which method may comprise contacting the antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of OspA, OspC, OspF, p39 polypeptide and a fusion peptide of p41 and VLsE disclosed above with said sample and detecting a polypeptide-antibody complex formed; calculating reference values of the levels of the antibodies; and determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values.
Further provided herein is a system for classifying Borrelia burgdorferi infection of a mammal, e.g., an animal comprising the computer readable medium disclosed herein and an antigenic composition comprising at least two B. burgdorferi polypeptides, wherein each of said polypeptides may comprise an amino acid sequence selected from the group consisting of: a) an OspA polypeptide, b) an OspC polypeptide, c) an OspF polypeptide, d) a p39 polypeptide, and e) a fusion peptide of p41 and VLsE.
In an additional aspect, provided herein are kits for detecting the various infectious organisms, which kit comprises, in a container, the polypeptides or antigenic compositions. For instance, a polypeptide of the present invention can be included in a kit. A kit can be included in a sealed container. Non-limiting examples of containers include a microtiter plate, a bottle, a metal tube, a laminate tube, a plastic tube, a dispenser, a pressurized container, a barrier container, a package, a compartment, or other types of containers such as injection or blow-molded plastic containers into which the dispersions or compositions or desired bottles, dispensers, or packages are retained. Other examples of containers include glass or plastic vials or bottles. The kit and/or container can include indicia on its surface. The indicia, for example, can be a word, a phrase, an abbreviation, a picture, or a symbol.
The containers can dispense or contain a pre-determined amount of a composition of the present invention. The composition can be dispensed as a liquid, a fluid, or a semi-solid. A kit can also include instructions for using the kit and/or compositions. Instructions can include an explanation of how to use and maintain the compositions.
The following examples are offered to illustrate but not to limit the invention.
Clinical samples of dogs were tested for infection by Borrelia burgdorferi, A. phagocytophilum (AP), and E. canis (EC) using conventional assays such as immunofluorescence assay (IFA) or Western blot analysis, SNAP™ tests (IDEXX Laboratories, Fremont, Calif.) and a new multiplex assay (Accuplex™, VCA Antech Inc., Los Angeles, Calif.). IFA for Lyme disease was conducted using an ELISA assay (Zeus Scientific Inc., Raritan, N.J.).
Accuplex™ is a multiplex assay for detecting various infectious organisms including heartworm, E. Canis, A. phagocytophilum, and B. burgdorferi in a subject using the polypeptides, antibodies and antigenic compositions disclosed herein. Chicken polyclonal antibodies made by immunizing chickens with a canine heartworm antigen were used for heartworm detection. A gp36 polypeptide (SEQ ID NO:26) produced using the pET46 Ek/LIC vector (Novagen) with an inserted coding sequence of SEQ ID NO:27 was used for E. Canis detection. The A. phagocytophilum antigen was a multimeric polypeptide (SEQ ID NO:7) produced using the pET46 Ek/LIC vector with an inserted coding sequence of SEQ ID NO:8. For B. burgdorferi detection, an antigenic composition comprising the OspA, OspC, OspF, p39 polypeptides and a fusion peptide of p41 and VLsE was used. The OspA polypeptide is commercially available from Meridian Life Science, Inc. (Catalog #: R8A131), which contains multiple copies of the B. burgdorferi OspA sequence and a 6-HIS epitope tag. The OspA polypeptide has a total molecular weight of 85 kDa, and reacts with human B. burgdorferi positive serum. The OspC polypeptide having the amino acid sequence of SEQ ID NO:15 was produced using the pET46 Ek/LIC vector with an inserted coding sequence of SEQ ID NO:17. The OspF polypeptide having the amino acid sequence of SEQ ID NO:18 was produced using the pET46 Ek/LIC vector with an inserted coding sequence of SEQ ID NO:20. The p39 polypeptide having the amino acid sequence of SEQ ID NO:21 was produced using the pEV-L8: His8-TEV-LIC vector with an inserted coding sequence of SEQ ID NO:23. The fusion peptide of p41 and VLsE has the amino acid sequence of SEQ ID NO:24 was produced using the pET46 Ek/LIC vector with an inserted coding sequence of SEQ ID NO:25. The polypeptides produced contain tag sequences encoded by the vectors: MAHHHHHHVDDDDK (SEQ ID NO:29) for the pET46 Ek/LIC vector; MHHHHHHHHGVDLGTENLYFQSNA (SEQ ID NO:31) for the pEV-L8: His8-TEV-LIC vector.
Experiments were performed by infecting and/or vaccinating dogs, followed by testing with Accuplex™, SNAP™ and other assays. Results from various testing methods are shown in the tables below.
For classification of B. burgdorferi infections, a value x was calculated using the following formula, wherein multiple negative controls, e.g., six negative controls, were included for each assay:
x=3*STDEV(Negative Controls)+MEDIAN(Negative Controls).
A static value y is used for adjustment when calculating each reference value. A specific y value, which may equal 0, is assigned to each reference value for each antigen based upon data from an experimental study conducted for each test. The following formulas were used to calculate the reference values:
alpLow=0.5x+250;[min1500]
alpMid=x+500;[min2750]
alpHigh=x+2500;[min4500]
alpHighest=x+11500;[min13000]
ospfLow=0.5x+250;[min1500]
ospfHigh=x+500;[min3000]
p39Low=x;[min3750]
slpLow=x;[min2350]
slpMid=x+2500;[min4500]
slpHigh=x+5500;[min7000]
ospcLow=x;[min4500]
ospcHigh=x+15000;[min18000]
sub5Low=x+2000;[min4500]
wherein the underlined values are the y values, and the minimal value for each reference value is included in brackets. In cases where the reference value calculated is less than the minimal value, the minimal value may be used. For example, if x is calculated to be 2000, then the calculated alpLow=1250; alpMid=2500; alpHigh=4500 and alpHighest=13500. Because the calculated alpLow (1250) and alpMid (2500) are less than the minimal values for each reference value (1500 and 2750), the minimal values of 1500 and 2750 will be used for alpLow and alpMid, respectively. For alpHigh, the calculated alpHigh is 4500, which is identical to the minimal value; the alpHigh is set at 4500. And because the calculated alpHighest (13500) is greater than the minimal value (13000), the calculated value of 13500 will be used for alpHighest. Therefore, samples with an OspA value of less than alpLow (1500 in this example) will be OspA negative; samples with an OspA value between alpLow (1500 in this example) and alpMid (2750 in this example) will be OspA low; samples with an OspA value between alpMid (2750 in this example) and alpHigh (4500 in this example) will be OspA mid; samples with an OspA value between alpHigh (4500 in this example) and alpHighest (13500 in this example) will be OspA high; and samples with an OspA value greater than alpHighest (13500 in this example) will be OspA highest, etc. The unit for all the values is fluorescent counts from the Dual Channel Bio-CD detection system by Quadraspec, Inc., using both fluorescence and SDI™ (Spinning Disc Interferometry).
Abbreviations used in the tables:
Accuplex™ interpretation code:
SNAP™ interpretation code:
Keys for IgG titers in IFA tests:
Clinical samples from dogs tested positive for Lyme antigen using Western blot analysis were assayed using the Accuplex™ test and SNAP™ test. The results are summarized in Table 1. The results show that the Accuplex™ test is capable of distinguishing between exposure to Lyme due to natural exposure and vaccination, and between early and late exposure to Lyme.
Table 2 shows test data from clinical samples using Accuplex™ Lyme and AP tests, IFA AP test and SNAP™ test.
A. phagocytophilum clinical samples
Table 3 shows test data from clinical samples using Accuplex™ Lyme, AP and EC tests, IFA EC test and SNAP™ test.
E. canis clinical samples
Table 4 shows test results from experimentally infected dogs using the Accuplex™ Lyme test in comparison to SNAP™ test and ELISA assay (Zeus Scientific, Inc.). The dogs were separated into six groups. Groups 1, 3 and 5 were infected with ticks first, followed by vaccination. Groups 2, 4 and 6 were vaccinated first, followed by ticks infection. The vaccines used are Nobivac™ Lyme (Intervet/Schering-Plough Animal Health, Summit, N.J.), LymeVax® (Fort Dodge Animal Health, New York, N.Y.), and RECOMBITEK® Lyme (Merial Ltd., Duluth, Ga.). Tables 5, 6 and 7 show Lyme Groups 1, 3 & 5 compared with SNAP™ and ELISA tests for mean time to positive (in days from T=0 and before V=0). The average time of detection for all three groups are 26.5 days for Accuplex™, 35.0 days for SNAP™ and 26.8 days for ELISA. The mean time for detection by Accuplex™ of vaccination in Lyme Groups 2, 4 & 6 is: 24.0 (14-36) days for Group 2, 12.7 (6-14) days for Group 4 and 14.0 (14-14) days for Group 6. The average time of detection for all three groups is 16.9 days.
Table 8 shows test results from experimentally infected dogs using the Accuplex™ AP test in comparison to the SNAP™ and IFA tests. A. phagocytophilum (OK Sate University isolate) was administered to all dogs on 22-FEB-2010 (i=0). All dogs were administered 100 mg of doxycycline for 28 days staring on 7-JUN-2010. Table 9 shows the comparison of days for detection among Accuplex™, SNAP™, PCR and IFA tests.
A.
phagocytophilum test results from experimentally infected dogs
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
A. phagocytophilum tests
Table 10 shows test results from experimentally infected dogs using the new Accuplex™ E. canis test in comparison to the SNAP™, PCR and IFA tests. E. canis (OK State University isolate “EBONY”) was administered to all dogs on 11 Jan. 2010. All Dogs were administered 100 mg (PO/BID) of doxycycline for 28 days staring on 8 Mar. 2010. Table 11 shows the comparison of days for detection among different tests.
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
i = 0
Objective:
To evaluate a new automated system for detection of Anaplasma phagocytophilum antibodies in serum of dogs after parenteral inoculation or exposure to wild-caught Ixodes scapularis.
Sample Population:
26 laboratory reared, mixed sex beagles.
Procedures.
Serum and blood was collected temporally from beagles inoculated with culture derived A. phagocytophilum intravenously (5 dogs) or subcutaneously (3 dogs) and 18 dogs that were exposed to wild-caught, adult Ixodes scapularis. An automated fluorescence system based on a silicon wafer was optimized to detect A. phagocytophilum antibodies to a novel mutant peptide and applied to the canine sera. Anaplasma phagocytophilum antibodies were also detected by indirect fluorescent antibody assay and a commercially available kit. Anaplasma phagocytophilum DNA was amplified from blood by polymerase chain reaction (PCR) assay.
Results:
All seven parenterally inoculated dogs that remained in the study and 10 of 18 dogs exposed to I. scapularis were infected by A. phagocytophilum. The time to first positive result for these 10 dogs varied by assay but was only statistically significant amongst groups on week 3 when more samples were PCR positive compared to the antibody assays.
Conclusions:
Results of the three A. phagocytophilum antibody tests were similar which validates the use of the fluorescence-based system. Performance of A. phagocytophilum PCR assays is indicated in dogs with suspected acute anaplasmosis if serum antibody assays are negative.
Introduction
Anaplasma phagocytophilum is a rickettsial organism that is vectored by Ixodes spp. (Dumler et al., Int J Syst Evol Microbiol 51:2145 (2001)). The organism is associated with granulocytic anaplasmosis in a variety of species including humans, horses, dogs, and cats (Chen et al., J Clin Microbiol 32:589 (1994); Foley et al., Vet Rec 160:159 (2007); Lappin et al., J Am Vet Med Assoc (2004) 225:893-896). Distinct strains exist which are associated with host tropisms (Rejmanek et al., J Med. Microbiol. (2012) 61:204-212). Some infected dogs develop clinical illness that is most commonly manifested as fever, polyarthritis, or thrombocytopenia. Detection of antibodies against A. phagocytophilum in serum and amplification of A. phagocytophilum DNA from blood by polymerase chain reaction (PCR) are used most frequently to aid in the diagnosis of canine anaplasmosis (Kirtz et al., J Small Anim Pract 46:300 (2005); Ravnik et al., Vet Microbiol. (2011) 149:172-176; Beall et al., Vector Borne Zoonotic Dis. (2008) 8:455-464).
Serum antibodies against A. phagocytophilum in dog serum can be detected in several types of assays. Many diagnostic laboratories use indirect fluorescent antibody assays (IFA) to detect antibodies against cell culture grown A. phagocytophilum morulae (Prototek Reference Laboratory, Chandler, Ariz.). Western blot immunoassay is used by some laboratories and can be used to determine the immunodominant antigens recognized by individual sera if whole organism preparations are utilized or can be used to determine antibody responses to individual antigens (Chandrashekar et al., Am J Vet Res. (2010) 71:1443-1450; Ge et al., J. Bacteriol. (2007) 189:7819-7828). Based on previous studies, the P44 peptide of A. phagocytophilum is immunodominant and is a common target used to assess for serum antibody responses (Chandrashekar et al., Am J Vet Res. (2010) 71:1443-1450; Ge et al., J. Bacteriol. (2007) 189:7819-7828). One ELISA based protocol for detection of antibodies against A. phagocytophilum is available commercially in the United States (Beall et al., Vector Borne Zoonotic Dis. (2008) 8:455-464; SNAP 4DX, IDEXX Laboratories, Portland, Me.).
Recently, new automated multiplex systems have been developed that are capable of testing for antigens and antibodies against multiple antigens using small volumes of serum (Zhao et al., Appl Opt. (2007) 46:6196-6209). These assays can be very beneficial in service laboratories because the automated system can lessen interassay variability and large numbers of samples can be assayed concurrently. In addition, for some organisms like Borrelia burgdorferi, detection of antibodies against multiple antigens can be used to differentiate vaccinated dogs from those that are naturally infection and acute infections, from chronic infections (Moroff et al., J Vet Diag Invest (In review, 2012)).
The objectives of this study were to validate an automated system (Accuplex™ 4 BioCD) for detection of serum antibodies against a peptide of A. phagocytophilum and to compare the results of the new assay to those of IFA and a commercially available point of care assay as well as to the results of a polymerase chain reaction (PCR) assay that amplifies the DNA of A. phagocytophilum from blood.
Materials and Methods
Animals.
This study was approved by the Institutional Animal Care and Use Committee at Colorado State University (Ixodes exposure) or an independent research laboratory (IV inoculation). The mixed sex beagles (n=26) used in this study were from a laboratory animal facility and ranged in age from 12 to 13 months at the beginning of the experiments. Prior to shipment to the respective research facilities, all dogs were shown to be negative for antibodies against A. phagocytophilum, B. burgdorferi, and Ehrlichia canis as well as for Dirofilaria immitis antigen by use of a commercially available kit (SNAP 4DX, IDEXX Laboratories, Portland, Me.). On arrival, the males were neutered using the facility standard operating procedures. The dogs were housed in groups of two or three dogs and fed ad libitum. Daily animal care was provided by research facility staff members.
Parenteral Inoculation with Cell Cultured A. phagocytophilum.
A field isolate of A. phagocytophilum was grown on HL-60 cells and delivered to Colorado State University by a same day air service stored at ambient temperature (Dr. Susan Little, Oklahoma State University, Stillwater, Okla.). Eight beagles were pre-medicated with 2.2 mg/kg of diphenhydramine administered SQ. The inoculum was divided into eight 2 ml aliquots and administered slowly IV to five dogs. All five dogs had evidence of adverse reactions characterized by panting (five dogs), pale mucous membranes (four dogs), weakness (three dogs), and vomiting and defecation (two dogs) and so the remaining three dogs were inoculated SQ with the inoculum divided into three sites. The adverse events were self-limited in four of the IV dogs over approximately 30 minutes but persisted in one female that was removed from the study. Side-effects were not noted in the dogs inoculated SQ. Samples were collected on Days 0, 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, 147, 154, and 161. Doxycycline was administered at 10 mg/kg, once daily for 28 days starting on Day 105 after inoculation. These dogs were infected to provide sera and blood for assay development as well as to provide temporal information about test results after experimental inoculation.
Anaplasma phagocytophilum Infection by Tick Exposure.
Adult Ixodes scapularis wild-caught in Rhode Island in March 2010 were purchased for use in a parallel study on Borrelia burgdorferi infection (Moroff et al., J Vet Diag Invest (In review, 2012); Dr. Thomas Mather, University of Rhode Island). The prevalence rate of A. phagocytophilum DNA in a representative aliquot of adult ticks from the capture area was approximately 15%. The ticks were maintained at room temperature in humidified chambers until used in the experiments. When placed on 18 of the dogs, 13 female and 12 male ticks were allowed to attach under a tick chamber made of adhesive bandage materials. After 7 days, the ticks were removed with forceps, counted, and stored at −80° C. for future assays. At that time, a tick control product was placed topically (Frontline, Merial LTD, Athens, Ga.). Samples were collected from these 18 dogs weekly for 18 weeks.
Samples.
Blood (6 ml) was collected by jugular venipuncture. After collection, 1.5 ml was placed into EDTA and maintained at 4° C. until assayed. After the remaining blood was allowed to clot, the sample was centrifuged at 1,500×g for 10 minutes and the sera stored in multiple aliquots at −80° C. until assayed.
Assays.
The EDTA blood (cold packs) and sera were shipped by overnight express to a commercial laboratory for performance of a proprietary PCR assay (FastPanel™) that amplifies the of DNA of A. phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, and E. ewingii using the standard operating procedures of the laboratory (Antech Laboratories, Lake Success, N.Y.).
Sera were ultimately assayed for A. phagocytophilum antibodies by IFA us using slides purchased from a commercial laboratory (Prototek Reference Laboratory, Chandler, Ariz.), a commercially available kit following the manufacturer's guidelines (SNAP 4DX, IDEXX Laboratories, Portland, Me.), and the in automated system using a novel mutant peptide derived from A. phagocytophilum as the antigen source as described in the section that follows. An A. phagocytophilum IFA titer of >1:40 was considered positive.
Accuplex™ BioCD System.
This automated system was based on a silicon wafer with a thermal oxide layer (Yamato convection oven DVS-4000, Santa Clara, Calif.). The wafer was treated with both a 3-aminopropyldimethylethoxysilane (APMES) vapor deposition as well as a 1,6-diisocyanatohexane (Di-Iso) liquid deposition. A fluorescent hydrophobic mask was screen printed on the surface to create a 288 well pattern. Using a contact protein printer, 11 different markers were used to print 64 spots in a specific spot pattern in every well. Eight spots were dedicated to each peptide or protein antigen used to capture target antibodies. The assay as currently designed detected infections of Dirofilaria immitis, Borrelia burgdorferi, E. canis, and A. phagocytophilum. After printing of the peptides and antibody, the disc surface was blocked with ethanolamine vapor for 15 minutes at 30° C. to lessen potential for nonspecific binding and was coated in trehalose (2% by volume diluted in deionized water; Sigma-Fluka Analytical, St. Louis, Mo.) for added stability. The finished Accuplex4 disc could hold up to 274 patient samples along with 8 positive controls and 6 negative controls for each of the assays.
Each disc was loaded onto a sample processor which was used for liquid handling and dispensing using a keyed chuck to ensure proper disc loading (SIAS MODEL, Xantus manufactured by Sias, Hombrechtikon Switzerland). The disc was washed with a phosphate buffered saline solution (pH of 7.4) with Tween-20 (PBS-T) for 20 seconds at 400 RPM and then rinsed with deionized water for 20 seconds at the same speed before being centrifuged at 3000 RPM for 15 seconds to dry. Patient serum was loaded into each reaction well (5 μl) and incubated for 30 minutes at 80% humidity. The disc was again washed with PBS-T and deionized water for 20 seconds and centrifuged as described to dry. The fluorescent conjugate was dispensed into each well (6 μl) and incubated for 10 minutes (Protein-A/Alexafluor532, Invitrogen Carlsbad, Calif.). The disc was then washed for the final time with PBS-T and deionized water for 20 seconds and centrifuged as described to dry.
The dual channel reader included both a fluorescent and interferometric detector and also contained the same keyed chuck as the sample processor to ensure proper disc orientation (BioCD reader, Dual Channel Reader, Antech Diagnostics, West Lafayette, Ind.). Once the disc was loaded, it was centrifuged at 4,800 RPM and a 20 mW, 532 nm laser attached to the optical stage was “stepped” across the disc in the x-orientation. As the stage swept across the disc, 2401 data points were recorded through both detectors and sent to the computer workstation.
The interferometric data was used for disc image transformation and well mapping. These data points produced not only the disc image, but an individual image for each well. Using image processing, a spot pattern template was then applied to the fluorescent image where fluorescent counts were taken for each protein spot in all 288 wells. The median value of fluorescent counts was assigned to each individual immunologic reaction. The fluorescent counts for the six negative control wells were used to calculate the cutoffs for each assay. The median was taken from the six negative control wells and added to three standard deviations of the negative control well values along with a constant (Y). The constant was created using increases in fluorescent counts over time post-infection in the IV inoculated dogs. The cutoff format for each immunologic reaction was Median (Negative Controls)+3 STDEV (Negative Controls)+Y. This allowed the cutoffs to adjust for minor variation in discs. These cutoffs were then applied to each reaction, measured in fluorescent counts for an individual patient sample. A suspect sample was considered positive for antibodies against Anaplasma phagocytophilum when the result was greater than the specified threshold.
Accuplex™ 4 BioCD A. phagocytophilum Antibody Assay Optimization Experiments.
The positive and negative control sera used in assay titrations were obtained from the dogs inoculated IV in the study described here. The positive and negative samples were defined by results of PCR for A. phagocytophilum to confirm infection and by IFA for serologic responses. The A. phagocytophilum peptide was a proprietary mutant synthetic peptide derived from A. phagocytophilum P44 that was produced by the commercial laboratory (Antech Laboratories, Lake Success, N.Y.). The optimal concentration was determined by assessing optimal signal:noise, with varying printed mutant peptide concentrations, and buffer compositions. The cut-off point for a positive test result was determined by assay of serum from dogs with known infection status based specifically on differential responses compared to IFA results on serum collected pre-infection (negative IFA) and post-infection (positive IFA).
The intra-assay variation of the assay was calculated by determining the mean and standard deviation for the fluorescent counts for 20 positive control sample wells and calculating the coefficient of variation on one disc. This experiment was performed with the same positive control samples on separate discs on three different days. The inter-assay variation was determined by comparing the coefficient of variations among the three discs.
Statistical Evaluation.
Dogs that became PCR positive for A. phagocytophilum DNA on at least 2 sample dates or that had antibodies against A. phagocytophilum as detected by IFA on at least 2 sample dates were considered to have developed infection by the organism. Results in all 4 assays were recorded as positive or negative. The proportions of dogs that were positive in each assay on each date were analyzed using a generalized linear model with test, week, and the test by week interaction included as fixed effects in the model. Where a significant test effect was detected within a week, all pair-wise comparisons were made. The time to first positive test result was compared among the assays by ANOVA including test as the only fixed effect. Significance was defined as P<0.05.
Results
Accuplex™ 4 BioCD A. phagocytophilum Antibody Assay Optimization Experiments.
In the optimized assay, the intra-assay variation of 20 positive control wells per disc evaluated on separate discs was 15.9%, 15.5%, and 16.3%, respectively. The inter-assay variation of these results among the three discs was 1.5%.
Parenteral Inoculation with Cell Cultured A. phagocytophilum.
All seven dogs inoculated parenterally with cell culture grown A. phagocytophilum met the definition of A. phagocytophilum infection. Clinical signs of disease consistent with anaplasmosis were not recognized in any dog over the duration of the study.
Anaplasma phagocytophilum DNA was first amplified from blood by PCR assay on Day 3 (two dogs) after inoculation (
Anaplasma phagocytophilum Infection by Tick Exposure.
Of the 18 dogs exposed to wild-caught Ixodes spp., 10 dogs met the definition of A. phagocytophilum infection. Clinical signs of disease consistent with anaplasmosis were not recognized in any dog over the duration of the study.
Anaplasma phagocytophilum DNA was first amplified from blood by PCR assay on week 1 (two dogs) after exposure to I. scapularis (
phagocytophilum serological assays and a PCR assay
Least squares mean for PCR (2.5 weeks), IFA (3.7 weeks), SNAP (4.8 weeks), and P44 antibody assay (5.4 weeks) were not significantly different (p=0.0624).
Based on the titration experiments, the Accuplex™ 4 BioCD A. phagocytophilum antibody assay described here was accurate and reproducible for the detection of A. phagocytophilum antibodies in canine sera. As the majority of the assay was automated and rigorous controls were included, thus potential for laboratory error was minimal While the assay required sera to be transported to a central laboratory, antibodies against A. phagocytophilum were robust and were minimally affected by temperature change as documented by use of the same positive and negative control samples repeatedly without changes in results.
In this study, results from three serological assays and a PCR assay were reported for dogs inoculated parenterally with A. phagocytophilum as well as for those infected by exposure to wild-caught I. scapularis. Samples from dogs inoculated parenterally were primarily used to generate sera for assay titrations. The samples from dogs exposed to I. scapularis more closely paralleled results expected from A. phagocytophilum infection in client-owned dogs. While approximately 15% of the I. scapularis in this region of Rhode Island are PCR positive for A. phagocytophilum DNA, only 10 of 18 dogs in this experiment developed A. phagocytophilum infection as defined. The ticks were allowed to feed for up to 7 days and the majority of female ticks attached. These results suggest that some adult beagles can limit infection with A. phagocytophilum. This was most evident in one of the 10 dogs (Table 12; dog 10) that was PCR positive and antibody positive on a few dates after tick attachment but then became PCR negative and serum antibody negative in all tests on all samples collected after week 9.
DNA of A. phagocytophilum could be amplified from blood prior to seroconversion in any of the three serological assays. The results from the dogs described here support the recommendation to perform PCR assays on blood of dogs with suspected A. phagocytophilum infection, particularly if the disease syndrome is acute and serum antibody assay results are negative.
Time to first positive serological test result was in part related to the positive cut-off point selected for each individual assay. The three serological assays performed in the study described here incorporated three different methodologies and had individual positive cut-off points. The cut-off point in the Accuplex™ 4 BioCD A. phagocytophilum was selected to minimize the possibility for false positive reactions being reported based on the inherent interassay variation that occurs with all assays. When the three assays were applied to the sera from the dogs exposed to I. scapularis ticks (Table 12), time to first positive varied between the IFA (Range=Day 14 to Day 56), peptide assay (Range=Day 14-35; one dog never seroconverted), and commercial kit (Range=Day 21-42). While day to first positive result was the same for some dogs in some assays, the commercial kit had the latest first positive test result for five dogs. This differed from a previous report which showed the commercial kit to detect antibodies as soon as 8 days after infection with the NY18 strain (Chandrashekar et al., Am J Vet Res. (2010) 71:1443-1450). The differences in results between the previous study and the one described here may relate to the strains of A. phagocytophilum used or the inoculation dose.
In this study, antibody titers as measured by IFA and the commercially available kit were positive in nine of 10 dogs infected by exposure to I. scapularis ticks up to 12 weeks. In contrast, results of the peptide assay began to fall below the positive cut-off point after week 11 in some of the 9 dogs. These results suggest that the peptide assay results are most strongly correlated to recent infection.
Few data are available evaluating long-term infection of dogs after experimental infections with A. phagocytophilum. In this study, infected dogs were evaluated by PCR assay for 18 weeks (I. scapularis exposure; 10 dogs) or 15 weeks (parenteral inoculation; 7 dogs) prior to doxycycline administration. Several dogs in both groups maintained long term infections based on PCR assay results however, had no apparent clinical signs of illness. These results may reflect the suspected variation in A. phagocytophilum pathogenicity (Foley J et al., Vet Rec 160:159 (2007)). The predominant strain or strains in the area of Rhode Island where these ticks were collected may be relatively non-pathogenic. However, further evaluation of the role played by A. phagocytophilum in chronic illness in dogs should be performed. After parenterally inoculated dogs were administered doxycycline, PCR positive test results were never positive again. However, PCR was only performed on blood and the dogs were not splenectomized or otherwise immune suppressed and so whether infection was cleared by treatment is unknown.
The present invention is further illustrated by the following exemplary embodiments:
1. An Anaplasma phagocytophilum p44 polypeptide comprising amino acids 222-236 of SEQ ID NO:1 (P44-2 disclosed in U.S. Pat. No. 6,436,399 B1), wherein said polypeptide comprises at least one mutation, an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 or an A. phagocytophilum p44 polypeptide comprising amino acids 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1 that comprises at least one mutation.
2. The polypeptide of embodiment 1, wherein the polypeptide comprises 1 to 10 mutations.
3. The polypeptide of embodiment 2, wherein the polypeptide comprises 3 to 7 mutations.
4. The polypeptide of embodiment 1, wherein the mutation is selected from the group consisting of a substitution, an insertion and a deletion.
5. The polypeptide of embodiment 4, wherein the peptide comprises at least 1, 2, 3, 4, 5, 10 or 12 mutations selected from the group consisting of Gly222(Del), His223→Asn, Ser224→Thr, Ser225→Thr, Val227→Ala, Thr228→Ser, Gln229→Asn, Leu233→Val, Leu233→Thr, Phe234→Leu, Ser235→Thr, and Thr236→Ser.
6. The polypeptide of embodiment 1, wherein the polypeptide further comprises a second polypeptide comprising amino acids 237-247 of SEQ ID NO:1.
7. The polypeptide of embodiment 6, wherein the second polypeptide comprises at least one mutation.
8. The polypeptide of embodiment 7, wherein the second polypeptide comprises 1 to 5 mutations.
9. The polypeptide of embodiment 8, wherein the second polypeptide comprises 2 or 3 mutations.
10. The polypeptide of embodiment 7, wherein the mutation is selected from the group consisting of a substitution, an insertion and a deletion.
11. The polypeptide of embodiment 10, wherein the peptide comprises at least 1, 2, 3, 4, 5 or 7 mutations selected from the group consisting of Thr240→Ser, Gln229→Asn, Ile243→Val, Glu245→Asp, Glu245→Asn, Asp246→Lys, and Asp246→Glu.
12. The polypeptide of embodiment 1, wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID Nos:3-6 (SP44-1 to 4).
13. A polypeptide comprising a multimer, a combination, or a chimera of the polypeptides of embodiment 12.
14. The polypeptide of embodiment 13, wherein the polypeptide further comprises a tag sequence.
15. The polypeptide of embodiment 13, wherein the polypeptide further comprises an amino acid linker between the polypeptides of embodiment 12.
16. The polypeptide of embodiment 15, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7 (SP44-134).
17. The polypeptide of embodiment 16, further comprising a tag sequence.
18. An Anaplasma phagocytophilum p44 polypeptide that exhibits at least 75% identity to the amino acid sequence of SEQ ID NO:1, or the amino acids 222-247 of SEQ ID NO:1, wherein said polypeptide is not a wild-type P44 protein, and wherein said polypeptide binds to an antibody that is specific for a wild-type P44 protein.
19. The polypeptide of embodiment 18, wherein the polypeptide exhibits at least 80% identity to the amino acid sequence of SEQ ID NO:1, or the amino acids 222-247 of SEQ ID NO:1.
20. The polypeptide of embodiment 19, wherein the polypeptide exhibits at least 90% identity to the amino acid sequence of SEQ ID NO:1, or the amino acids 222-247 of SEQ ID NO:1.
21. The polypeptide of embodiment 20, wherein the polypeptide exhibits at least 95% identity to the amino acid sequence of SEQ ID NO:1, or the amino acids 222-247 of SEQ ID NO:1.
22. The polypeptide of embodiment 21, wherein the polypeptide exhibits at least 99% identity to the amino acid sequence of SEQ ID NO:1, or the amino acids 222-247 of SEQ ID NO:1.
23. A polynucleotide which encodes an Anaplasma phagocytophilum p44 polypeptide comprising the amino acid sequence of SEQ ID NO:1, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type P44 polynucleotide, or a polynucleotide which encodes an A. phagocytophilum p44 polypeptide having the amino acid sequence of 222-237, 222-238, 222-239, 222-240, 222-241, 222-242, 222-243, 222-244, 222-245, 222-246, or 222-247 of SEQ ID NO:1, or a complimentary strand thereof, and in some embodiments, said polynucleotide is not a wild-type P44 polynucleotide.
24. The polynucleotide of embodiment 23, wherein the polynucleotide exhibits at least 75% identity to the nucleotide sequence of SEQ ID NO:2 (P44-2 disclosed in U.S. Pat. No. 6,436,399 B1), SEQ ID NO:34 or SEQ ID NO:37.
25. The polynucleotide of embodiment 24, wherein the polynucleotide exhibits at least 80% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37.
26. The polynucleotide of embodiment 25, wherein the polynucleotide exhibits at least 90% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37.
27. The polynucleotide of embodiment 26, wherein the polynucleotide exhibits at least 95% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37.
28. The polynucleotide of embodiment 27, wherein the polynucleotide exhibits at least 99% identity to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37.
29. The polynucleotide of embodiment 23, wherein the polynucleotide hybridize to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37 under moderately stringent conditions.
30. The polynucleotide of embodiment 29, wherein the polynucleotide hybridize to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:34 or SEQ ID NO:37 under highly stringent conditions.
31. A polynucleotide which encodes the polypeptide of embodiments 1-22, or a complimentary strand thereof.
32. The polynucleotide of embodiment 31, wherein the polynucleotide is codon-optimized for expression in a non-human organism or a cell.
33. The polynucleotide of embodiment 32, wherein the organism is a virus.
34. The polynucleotide of embodiment 32, wherein the organism is a bacterium.
35. The polynucleotide of embodiment 32, wherein the cell is a yeast cell.
36. The polynucleotide of embodiment 32, wherein the cell is an insect cell.
37. The polynucleotide of embodiment 32, wherein the cell is a mammalian cell.
38. The polynucleotide of embodiment 31, wherein the polynucleotide is DNA or RNA.
39. The polynucleotide of embodiment 31, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:8 (SP44-134).
40. A vector comprising the polynucleotide of embodiment 31.
41. The vector of embodiment 40, wherein the polynucleotide comprises a promoter sequence.
42. The vector of embodiment 40, wherein the polynucleotide further encodes a tag sequence.
43. The vector of embodiment 40, wherein the polynucleotide comprises a poly-A sequence.
44. The vector of embodiment 40, wherein the polynucleotide comprises a translation termination sequence.
45. A non-human organism or a cell transformed with the vector of embodiment 40.
46. The organism of embodiment 45, wherein the organism is a virus.
47. The organism of embodiment 45, wherein the organism is a bacterium.
48. The organism of embodiment 45, wherein the cell is a yeast cell.
49. The organism of embodiment 45, wherein the cell is an insect cell.
50. The organism of embodiment 45, wherein the cell is a mammalian cell.
51. A method for detecting an antibody that specifically binds an Anaplasma phagocytophilum p44 polypeptide in a sample, which method comprises contacting the polypeptide of embodiments 1-22 with said sample and detecting a polypeptide-antibody complex formed.
52. The method of embodiment 51, wherein the sample is from a subject selected from the group consisting of dog, cat, human and horse.
53. The method of embodiment 52, wherein the method is used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease.
54. The method of embodiment 53, wherein the disease is granulocytic anaplasmosis.
55. The method of embodiment 51, wherein the sample is selected from the group consisting of a serum, a plasma and a blood sample.
56. The method of embodiment 51, wherein the sample is a clinical sample.
57. The method of embodiment 51, wherein the antibody is a monoclonal or polyclonal antibody or antibody fragment.
58. The method of embodiment 51, wherein the polypeptide-antibody complex is assessed by a sandwich or competitive assay format, optionally with a binder or antibody.
59. The method of embodiment 58, wherein the binder or antibody is attached to a surface and functions as a capture binder or antibody.
60. The method of embodiment 59, wherein the capture binder or antibody is attached to the surface directly or indirectly.
61. The method of embodiment 60, wherein the capture binder or antibody is attached to the surface via a biotin-avidin (or streptavidin) linking pair.
62. The method of embodiment 58, wherein at least one of the binders or antibodies is labeled.
63. The method of embodiment 51, wherein the polypeptide-antibody complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, lasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
64. The method of embodiment 51, wherein the polypeptide-antibody complex is assessed in a homogeneous or a heterogeneous assay format.
65. A kit for detecting an antibody that specifically binds an Anaplasma phagocytophilum p44 polypeptide, which kit comprises, in a container, the polypeptide of embodiments 1-22.
66. A method of recombinantly making an Anaplasma phagocytophilum p44 polypeptide, which method comprises culturing the organism of embodiment 45, and recovering said polypeptide from said organism.
67. The method of embodiment 66, further comprising isolating the polypeptide, optionally by chromatography.
68. A polypeptide produced by the method of embodiment 66.
69. The polypeptide of embodiment 68, wherein the polypeptide comprises a native glycosylation pattern.
70. The polypeptide of embodiment 68, wherein the polypeptide comprises a native phosphorylation pattern.
71. A polynucleotide which encodes a Borrelia burgdorferi OspC polypeptide comprising the amino acid sequence of SEQ ID NO:15 (OspC), or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspC polynucleotide.
72. The polynucleotide of embodiment 71, wherein the polynucleotide exhibits at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:16.
73. The polynucleotide of embodiment 71, wherein the polynucleotide hybridize to the nucleotide sequence of SEQ ID NO:16 under moderately or highly stringent conditions.
74. The polynucleotide of embodiment 71, wherein the polynucleotide is codon-optimized for expression in a non-human organism or a cell.
75. The polynucleotide of embodiment 74, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
76. The polynucleotide of embodiment 71, wherein the polynucleotide is DNA or RNA.
77. The polynucleotide of embodiment 71, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 (optimized OspC DNA).
78. A vector comprising the polynucleotide of embodiment 71.
79. The vector of embodiment 78, wherein the polynucleotide comprises a promoter sequence.
80. The vector of embodiment 78, wherein the polynucleotide further encodes a tag sequence.
81. The vector of embodiment 78, wherein the polynucleotide comprises a poly-A sequence.
82. The vector of embodiment 78, wherein the polynucleotide comprises a translation termination sequence.
83. A non-human organism or a cell transformed with the vector of embodiment 78.
84. The organism of embodiment 83, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
85. A method of recombinantly making a Borrelia burgdorferi OspC polypeptide, which method comprises culturing the organism of embodiment 83, and recovering said polypeptide from said organism.
86. The method of embodiment 85, further comprising isolating the polypeptide, optionally by chromatography.
87. A Borrelia burgdorferi OspC polypeptide produced by the method of embodiment 85.
88. The polypeptide of embodiment 87, wherein the polypeptide comprises a native glycosylation pattern and/or a native phosphorylation pattern.
89. A method for detecting an antibody that specifically binds to a Borrelia burgdorferi OspC polypeptide in a sample, which method comprises contacting the polypeptide encoded by the polynucleotide of embodiments 71-77 with said sample and detecting a polypeptide-antibody complex formed.
90. A polynucleotide which encodes a Borrelia burgdorferi OspF polypeptide comprising the amino acid sequence of SEQ ID NO:18, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type OspF polynucleotide.
91. The polynucleotide of embodiment 90, wherein the polynucleotide exhibits at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:19.
92. The polynucleotide of embodiment 90, wherein the polynucleotide hybridize to the nucleotide sequence of SEQ ID NO:19 under moderately or highly stringent conditions.
93. The polynucleotide of embodiment 90, wherein the polynucleotide is codon-optimized for expression in a non-human organism or a cell.
94. The polynucleotide of embodiment 93, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
95. The polynucleotide of embodiment 90, wherein the polynucleotide is DNA or RNA.
96. The polynucleotide of embodiment 90, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:20 (optimized OspF DNA).
97. A vector comprising the polynucleotide of embodiment 90.
98. The vector of embodiment 97, wherein the polynucleotide comprises a promoter sequence.
99. The vector of embodiment 97, wherein the polynucleotide further encodes a tag sequence.
100. The vector of embodiment 97, wherein the polynucleotide comprises a poly-A sequence.
101. The vector of embodiment 97, wherein the polynucleotide comprises a translation termination sequence.
102. A non-human organism or a cell transformed with the vector of embodiment 97.
103. The organism of embodiment 102, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
104. A method of recombinantly making a Borrelia burgdorferi OspF polypeptide, which method comprises culturing the organism of embodiment 102, and recovering said polypeptide from said organism.
105. The method of embodiment 104, further comprising isolating the polypeptide, optionally by chromatography.
106. A Borrelia burgdorferi OspF polypeptide produced by the method of embodiment 104.
107. The polypeptide of embodiment 106, wherein the polypeptide comprises a native glycosylation pattern and/or a native phosphorylation pattern.
108. A method for detecting an antibody that specifically binds to a Borrelia burgdorferi OspF in a sample, which method comprises contacting the polypeptide encoded by the polynucleotide of embodiments 90-96 with said sample and detecting a polypeptide-antibody complex formed.
109. A polynucleotide which encodes a Borrelia burgdorferi p39 polypeptide comprising the amino acid sequence of SEQ ID NO:21, or a complimentary strand thereof, wherein said polynucleotide is not a wild-type p39 polynucleotide.
110. The polynucleotide of embodiment 109, wherein the polynucleotide exhibits at least 70%, 75%, 80%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:22.
111. The polynucleotide of embodiment 109, wherein the polynucleotide hybridize to the nucleotide sequence of SEQ ID NO:22 under moderately or highly stringent conditions.
112. The polynucleotide of embodiment 109, wherein the polynucleotide is codon-optimized for expression in a non-human organism or a cell.
113. The polynucleotide of embodiment 112, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
114. The polynucleotide of embodiment 109, wherein the polynucleotide is DNA or RNA.
115. The polynucleotide of embodiment 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:23 (optimized p39 DNA).
116. A vector comprising the polynucleotide of embodiment 109.
117. The vector of embodiment 116, wherein the polynucleotide comprises a promoter sequence.
118. The vector of embodiment 116, wherein the polynucleotide further encodes a tag sequence.
119. The vector of embodiment 116, wherein the polynucleotide comprises a poly-A sequence.
120. The vector of embodiment 116, wherein the polynucleotide comprises a translation termination sequence.
121. A non-human organism or a cell transformed with the vector of embodiment 116.
122. The organism of embodiment 121, wherein the organism or cell is selected from the group consisting of a virus, a bacterium, a yeast cell, an insect cell and a mammalian cell.
123. A method of recombinantly making a Borrelia burgdorferi p39 polypeptide, which method comprises culturing the organism of embodiment 121, and recovering said polypeptide from said organism.
124. The method of embodiment 123, further comprising isolating the polypeptide, optionally by chromatography.
125. A Borrelia burgdorferi p39 polypeptide produced by the method of embodiment 123.
126. The polypeptide of embodiment 125, wherein the polypeptide comprises a native glycosylation pattern and/or a native phosphorylation pattern.
127. A method for detecting an antibody that specifically binds to a Borrelia burgdorferi p39 polypeptide in a sample, which method comprises contacting the polypeptide encoded by the polynucleotide of embodiments 109-115 with said sample and detecting a polypeptide-antibody complex formed.
128. An antigenic composition comprising at least two Borrelia burgdorferi polypeptides, wherein each of said polypeptides comprises an amino acid sequence selected from the group consisting of:
a) an OspA polypeptide,
b) an OspC polypeptide,
c) an OspF polypeptide,
d) a p39 polypeptide, and
e) a fusion peptide of p41 and VlsE,
wherein said antigenic composition does not consist of a) and b).
129. The composition of embodiment 128, which comprises at least 3, 4, or all 5 of said Borrelia burgdorferi polypeptides.
130. The composition of embodiment 128, wherein the OspC polypeptide comprises an amino acid sequence of SEQ ID NO:15.
131. The composition of embodiment 128, wherein the OspF polypeptide comprises an amino acid sequence of SEQ ID NO:18.
132. The composition of embodiment 128, wherein the p39 polypeptide comprises an amino acid sequence of SEQ ID NO:21.
133. The composition of embodiment 128, wherein the fusion peptide of p41 and VlsE comprises an amino acid sequence of SEQ ID NO:24.
134. The composition of embodiment 133, wherein the fusion peptide of p41 and VlsE further comprises a tag sequence.
135. The composition of embodiment 128, wherein the polypeptides form a fusion molecule.
136. A method for detecting an antibody that specifically binds to a Borrelia burgdorferi OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VlsE in a sample, which method comprises
a) contacting said sample with the antigenic composition of embodiment 128; and
b) detecting a polypeptide-antibody complex formed.
137. The method of embodiment 136, wherein the method is used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease.
138. The method of embodiment 137, wherein the disease is Lyme disease.
139. The method of embodiment 138, wherein the method is used to distinguish between infection by a Lyme disease pathogen and exposure to a Lyme disease vaccine.
140. The method of embodiment 138, wherein the method is used to distinguish between exposure to a Nobivac™ Lyme vaccine and exposure to another vaccine.
141. The method of embodiment 136, wherein the antibody is a monoclonal or polyclonal antibody or antibody fragment.
142. The method of embodiment 136, wherein the polypeptide-antibody complex is assessed by a sandwich or competitive assay format, optionally with a binder or antibody.
143. The method of embodiment 142, wherein the binder or antibody is attached to a surface and functions as a capture binder or antibody.
144. The method of embodiment 143, wherein the binder or capture antibody is attached to the surface directly or indirectly.
145. The method of embodiment 144, wherein the binder or capture antibody is attached to the surface via a biotin-avidin (or streptavidin) linking pair.
146. The method of embodiment 142, wherein at least one of the binders or antibodies is labeled.
147. The method of embodiment 136, wherein the complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, lasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
148. The method of embodiment 136, wherein the polypeptide-antibody complex is assessed in a homogeneous or a heterogeneous assay format.
149. A kit for detecting an antibody that specifically binds to a Borrelia burgdorferi OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VlsE, which kit comprises, in a container, the antigenic composition of embodiment 128.
150. A composition for detecting multiple disease antigens and/or antibodies, which composition comprises at least two, preferably three of the following reagents:
a) an antibody against a heartworm (Dirofilaria immitis) antigen,
b) an Ehrlichia Canis gp36 polypeptide,
c) an Anaplasma phagocytophilum p44 polypeptide, and
d) an antigenic composition comprising a Borrelia burgdorferi polypeptide selected from the group consisting of OspA, OspC, OspF, p39 and a fusion peptide of p41 and VlsE.
151. The composition of embodiment 150, which comprises all four of the reagents.
152. The composition of embodiment 150, wherein the reagent a) is a chicken polyclonal antibody.
153. The composition of embodiment 151, wherein the chicken polyclonal antibody is produced by immunizing chickens with a canine heartworm antigen.
154. The composition of embodiment 150, wherein the reagent b) comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:26.
155. The composition of embodiment 154, wherein the polypeptide further comprises a tag sequence.
156. The composition of embodiment 150, wherein the reagent c) comprises the polypeptide of embodiments 1-22.
157. The composition of embodiment 150, wherein the reagent d) comprises the antigenic composition of embodiment 128.
158. A method for detecting multiple disease antigens and/or antibodies in a sample, which method comprises
a) contacting said sample with the composition of embodiment 150; and
b) detecting a polypeptide-antibody complex formed.
159. The method of embodiment 158, wherein the method is used for diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of a disease.
160. The method of embodiment 159, wherein the disease is selected from the group consisting of a heartworm disease, ehrlichiosis, granulocytic anaplasmosis, and Lyme disease.
161. The method of embodiment 158, wherein the sample is selected from the group consisting of a serum, a plasma and a blood sample.
162. The method of embodiment 158, wherein the sample is a clinical sample.
163. The method of embodiment 158, wherein the polypeptide-antibody complex is assessed by a sandwich or competitive assay format, optionally with a binder or antibody.
164. The method of embodiment 163, wherein the binder or antibody is attached to a surface and functions as a capture binder or antibody.
165. The method of embodiment 164, wherein the capture binder or antibody is attached to the surface directly or indirectly.
166. The method of embodiment 165, wherein the capture binder or antibody is attached to the surface via a biotin-avidin (or streptavidin) linking pair.
167. The method of embodiment 163, wherein at least one of the binders or antibodies is labeled.
168. The method of embodiment 158, wherein the polypeptide-antibody complex is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay (IFA), nephelometry, flow cytometry assay, lasmon resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay.
169. The method of embodiment 158, wherein the polypeptide-antibody complex is assessed in a homogeneous or a heterogeneous assay format.
170. A kit for detecting multiple infectious organisms, which kit comprises, in a container, the composition of embodiment 150.
171. A computer readable medium containing executable instructions that when executed perform a method of classifying Borrelia burgdorferi infection of a mammal, e.g., an animal, the method comprising:
calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VlsE using a method according to any one of embodiments 136-148;
calculating reference values of the levels of the antibodies; and
determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values.
172. The computer readable medium of embodiment 171, further comprising calculating a reference value based on one or more negative controls.
173. The computer readable medium of embodiment 171, wherein one or more reference values are calculated for each antibody.
174. The computer readable medium of embodiment 173, wherein the reference values for the antibody that specifically binds to OspA are alpLow, alpMid, alpHigh and/or alpHighest.
175. The computer readable medium of embodiment 173, wherein the reference values for the antibody that specifically binds to OspC are ospcLow and/or ospcHigh.
176. The computer readable medium of embodiment 173, wherein the reference values for the antibody that specifically binds to OspF are ospfLow and/or ospfHigh.
177. The computer readable medium of embodiment 173, wherein the reference value for the antibody that specifically binds to p39 is p39Low.
178. The computer readable medium of embodiment 173, wherein the reference values for the antibody that specifically binds to the fusion peptide of p41 and VlsE are slpLow, slpMid and/or slpHigh.
179. The computer readable medium of embodiment 173, wherein the method further comprises calculating a level and reference value of an antibody that specifically binds to the Anaplasma phagocytophilum P44 polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the reference value for the antibody is sub5Low.
180. The computer readable medium of any one of embodiments 174-179, wherein the mammal is classified as Lyme exposure if:
a) the level of antibody that specifically binds to OspA is lower than alpHigh, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
c) the level of antibody that specifically binds to OspA is lower than alpLow,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
e) the level of antibody that specifically binds to OspA is lower than alpLow,
181. The computer readable medium of embodiment 180, wherein the mammal is classified as Lyme exposure early if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure late.
182. The computer readable medium of any one of embodiments 174-179, wherein the mammal is classified as Lyme exposure and vaccine if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpHigh, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
183. The computer readable medium of embodiment 182, wherein the mammal is classified as Lyme exposure and vaccine early if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure and vaccine late.
184. The computer readable medium of any one of embodiments 174-179, wherein the mammal is classified as Lyme vaccine if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
e) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
f) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
185. The computer readable medium of any one of embodiments 174-179, wherein the mammal is classified as indeterminative if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
e) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
186. The computer readable medium of embodiment 185, wherein the mammal is classified as possible exposure if the level of antibody that specifically binds to OspA is lower than alpMid; otherwise lyme vaccine possible exposure.
187. A method of classifying Borrelia burgdorferi infection of a mammal, e.g., an animal, the method comprising:
calculating levels of antibodies that specifically bind to an OspA, OspC, OspF, p39 polypeptide and/or a fusion peptide of p41 and VlsE using a method according to any one of embodiments 136-148;
calculating reference values of the levels of the antibodies; and
determining the type of Borrelia burgdorferi infection of the mammal by comparing the levels of the antibodies to the reference values.
188. The method of embodiment 187, further comprising calculating a reference value based on negative controls.
189. The method of embodiment 187, wherein one or more reference values are calculated for each antibody.
190. The method of embodiment 189, wherein the reference values for the antibody that specifically binds to OspA are alpLow, alpMid, alpHigh and/or alpHighest.
191. The method of embodiment 189, wherein the reference values for the antibody that specifically binds to OspC are ospcLow and/or ospcHigh.
192. The method of embodiment 189, wherein the reference values for the antibody that specifically binds to OspF are ospfLow and/or ospfHigh.
193. The method of embodiment 189, wherein the reference value for the antibody that specifically binds to p39 is p39Low.
194. The method of embodiment 189, wherein the reference values for the antibody that specifically binds to the fusion peptide of p41 and VlsE are slpLow, slpMid and/or slpHigh.
195. The method of embodiment 189, which further comprises calculating a level and reference value of an antibody that specifically binds to the Anaplasma phagocytophilum P44 polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the reference value for the antibody is sub5Low.
196. The method of any one of embodiments 190-195, wherein the mammal is classified as Lyme exposure if:
a) the level of antibody that specifically binds to OspA is lower than alpHigh, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
c) the level of antibody that specifically binds to OspA is lower than alpLow,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
e) the level of antibody that specifically binds to OspA is lower than alpLow,
197. The method of embodiment 196, wherein the mammal is classified as Lyme exposure early if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure late.
198. The method of any one of embodiments 190-195, wherein the mammal is classified as Lyme exposure and vaccine if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpHigh, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
199. The method of embodiment 198, wherein the mammal is classified as Lyme exposure and vaccine early if the level of antibody that specifically binds to OspF is lower than ospfHigh; otherwise Lyme exposure and vaccine late.
200. The method of any one of embodiments 190-195, wherein the mammal is classified as Lyme vaccine if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest, and
b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
e) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
f) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
201. The method of any one of embodiments 190-195, wherein the mammal is classified as indeterminative if:
a) the level of antibody that specifically binds to OspA is greater than or equal to alpMid and lower than alpHighest,
b) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
c) the level of antibody that specifically binds to OspA is greater than or equal to alpHighest,
d) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
e) the level of antibody that specifically binds to OspA is greater than or equal to alpLow and lower than alpMid,
202. The method of embodiment 201, wherein the mammal is classified as possible exposure if the level of antibody that specifically binds to OspA is lower than alpMid; otherwise Lyme vaccine possible exposure.
203. A system for classifying Borrelia burgdorferi infection of a mammal, e.g., an animal comprising the computer readable medium of embodiment 171 and the antigenic composition of embodiment 128.
Further provided are exemplary Anaplasma phagocytophilum (A. phagocytophilum) tests that are intended to detect A. phagocytophilum infection in canines. Specifically, an exemplary A. phagocytophilum test uses a P20C peptide having the sequence (GHSSGVTQNPKLFSTFVDTVKIAEDK) (SEQ ID NO:35), or a multimer of P20C peptide (a chimeric P20C polypeptide), to detect antibodies to A. phagocytophilum from a sample, e.g., a canine blood sample.
A chimeric P20C polypeptide can comprise any suitable number of P20C peptide, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more of P20C peptide. A chimeric P20C polypeptide can comprise any suitable tag and/or linker sequence(s). In some embodiments, the tag can be a tag from pEV-L8: His8-TEV-LIC vector (from Purdue University, IN) with the amino acid sequence MHHHHHHHHGVDLGTENLYFQSNA (SEQ ID NO: 31). In other embodiments, the tag can be a tag from pET46 Ek/LIC vector (Novagen) with the amino acid sequence MAHHHHHHVDDDDK (SEQ ID NO: 29). The tag can be located at any suitable location(s) within the chimeric P20C polypeptide. For example, the tag can be located at the N-terminus, C-terminus and/or in the middle of the chimeric P20C polypeptide. In some embodiments, an exemplary P20C polypeptide comprises the following amino acid sequence (SEQ ID NO:36):
G
GHSSGVTQNPKLFSTFVDTVKIAEDKGGGHSSGVTQNPKLFSTFVDTV
PG
GHSSGVTQNPKLFSTFVDTVKIAEDKGGGHSSGVTQNPKLFSTFVDT
The chimeric P20C polypeptide can be made by any suitable methods. For example, the chimeric P20C polypeptide can be made recombinantly, e.g., can be made recombinantly in E. coli, using the following DNA sequence (SEQ ID NO:37):
In some embodiments, the chimeric P20C polypeptide may also comprise at its N-terminus, a tag from pEV-L8: His8-TEV-LIC vector (from Purdue University, IN). The tag from pEV-L8: His8-TEV-LIC vector has the amino acid sequence MHHHHHHHHGVDLGTENLYFQSNA (SEQ ID NO:31). In case the tag from pEV-L8: His8-TEV-LIC vector is cleaved, the chimeric P20C polypeptide will have the remaining 3 (SNA) amino acids at the N-terminus. The tag from pEV-L8: His8-TEV-LIC vector can be encoded by any suitable polynucleotide sequence, e.g., the DNA sequence, atgcaccatcatcatcatcatcatcatggtgttgatctgggtaccgagaacctgtacttccaatccaatgcc (SEQ ID NO:30).
The chimeric P20C polypeptide can be used in any suitable assay format. In some embodiments, the chimeric P20C polypeptide is immobilized on a substrate (e.g., a solid surface such as a silicon disk, a microtiterplate or a nitrocellulose membrane). In use, a sample, e.g., a canine blood sample, is applied to the substrate with immobilized chimeric P20C polypeptide on it. If the blood sample has canine antibodies to A. phagocytophilum antigen containing P20C epitope, the antibodies will bind to the immobilized chimeric P20C polypeptide. Subsequently, a signal moiety, e.g., a protein A or G conjugated to a detectable label, is applied and bound to the canine anti-A. phagocytophilum antibodies. The detection of the bound label indicates that the canine blood sample is positive for canine antibodies to A. phagocytophilum antigen.
The present application claims the priority benefit of U.S. provisional application Ser. No. 61/555,399, filed Nov. 3, 2011 and claims the priority benefit of U.S. provisional application Ser. No. 61/650,386, filed May 22, 2012. The contents of these applications are incorporated by reference herein in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 61555399 | Nov 2011 | US | |
| 61650386 | May 2012 | US |
