Human granulocytic ehrlichiosis (HGE) is a recently described illness caused by a bacterial species that is phylogenetically similar to, or conspecific with, an ehrlichial genogroup comprising the veterinary pathogens Ehrlichia equi and E. phagocytophilia (Chen et al., J. Clin. Micro., 32, 589 (1994); Dumler et al., J. Clin. Micro., 33, 1098 (1995)). The only vectors demonstrated to transmit the HGE agent are Ixodes sp. ticks, although anecdotal evidence suggests that the American dog tick (Dermacentor variabilis) may also be implicated in HGE transmission. HGE typically presents as an acute febrile illness with headache, myalgias, leukopenia, thrombocytopenia, and elevated levels of alkaline phosphatase, lactate dehydrogenase, and aminotransferases (Bakken et al., JAMA, 272, 212 (1994); Bakken et al., JAMA, 275, 199 (1996); Pancholi et al., J. Infect, Dis, 172, 1007 (1995); Telford et al., Proc. Natl. Acad. Sci USA, 93, 6209 (1996)). Severe cases, when left untreated, may be fatal.
For the direct recovery of the HGE agent from clinical specimens, mouse inoculation, molecular detection, or cultivation of the organism in HL60 cells have been used. However, the duration of bacteremia with the HGE agent is not known, so the reliability of these tests beyond the acute stage of the illness has not been established. Antibody tests based on seroconversion to E. equi antigen have been used to identify infected persons after the initial stage of the illness (Bakken et al., JAMA, 272, 212 (1994); Bakken et al., JAMA, 275, 199 (1996)). However, the indirect fluorescent antibody (IFA) assay based on E. equi antigen is labor intensive, not widely available, and the interpretation of results is often subject to substantial individual variation (Bakken et al., JAMA, 275, 199 (1996); Dumler et al., supra; Dumler et al., Dermat. Clinics, 12, 25 (1994)).
Thus, what is needed is an improved method to detect the presence of an infectious agent associated with HGE.
The present invention is based upon the direct recovery (or isolation) of a gene encoding an immunogenic polypeptide associated with granulocytic ehrlichiosis in humans from patient tissues. Direct recovery of genes encoding immunodominant antigens from infected tissues, i.e., without prior growth in culture, is particularly useful to prepare immunodiagnostic reagents for emerging, uncultured, or difficult to culture, pathogens, e.g., the agents associated with human granulocytic ehrlichiosis (HGE), Tropheryma whippelii, the bacillary agent associated with Whipple""s disease, and other organisms related to mycobacteria which are yet to be cultivated in vitro. Moreover, direct recovery of DNA comprising open reading frames can also facilitate development of customized immunodiagnostic reagents, even when recovery of the infectious agent is not possible or when extensive agent variability (i.e., xe2x80x9cquasispeciesxe2x80x9d) precludes development of universal immunodiagnostic reagents.
Thus, the invention provides an isolated and purified DNA molecule comprising a preselected DNA sequence encoding an immunogenic polypeptide, a biologically active subunit, or a biologically active variant thereof, the presence of which is associated with granulocytic ehrlichiosis in a mammal, e.g., in humans. Preferably, the preselected DNA sequence encodes an immunogenic polypeptide which is specifically associated with the agent which causes human granulocytic ehrlichiosis (HGE). A preferred embodiment of the invention provides a preselected DNA sequence which encodes an immunogenic polypeptide having an amino acid sequence comprising SEQ ID NO:2, e.g., the preselected DNA sequence comprises SEQ ID NO:1. The invention further provides an isolated and purified DNA molecule which is complementary to SEQ ID NO:1.
The invention also provides an expression cassette comprising a preselected DNA sequence operably linked to a promoter functional in a host cell wherein said DNA sequence encodes an immunogenic polypeptide, a biologically active variant or subunit thereof, wherein said polypeptide is associated with granulocytic ehrlichiosis in a mammal. The expression of the preselected DNA sequence in host cells yields a polypeptide which is an ehrlichial antigen. Preferably, the expression cassette of the invention comprises a preselected DNA sequence encoding an immunogenic fusion polypeptide, a portion of which comprises an ehrlichial-specific polypeptide. The remainder of the DNA sequence preferably encodes a polypeptide that enhances the immunogenicity of the fusion polypeptide in vivo.
As used herein, xe2x80x9can immunogenic polypeptide associated with granulocytic ehrlichiosisxe2x80x9d is preferably a polypeptide having the amino acid Li sequence comprising SEQ ID NO:2, as well as variants of SEQ ID NO:2 which have at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity or homology to SEQ ID NO:2, or a biologically active subunit thereof. Biologically active subunits of the immunogenic polypeptide of the invention, and biologically active variants of the immunogenic polypeptide of the invention and subunits thereof, falling within the scope of the invention have at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the activity of the polypeptide comprising SEQ ID NO:2. The activity of an immunogenic polypeptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the polypeptide to be bound by antibodies specific for the infectious agent associated with granulocytic ehrlichiosis (see Example II), or the ability of the polypeptide to elicit a sequence-specific immunologic response when the polypeptide is administered to an organism, e.g., a mammal such as rabbit, goat, sheep, rat or mouse. Preferably, the immunologic response is a humoral response, i.e, antibody response, directed to a particular epitope on the polypeptide. More preferably, the presence of antibodies specific for that epitope correlates with the granulocytic ehrlichiosis infection status of the organism.
Further provided is an isolated and purified immunogenic polypeptide, a biologically active subunit or variant thereof, which is associated with granulocytic ehrlichiosis in a mammal. The polypeptide is useful to detect the presence of antibodies in mammals, e.g., deer, mice, rats, dogs and humans, which are specific for an infectious agent associated with granulocytic ehrlichiosis. Preferably, the polypeptide has an amino acid sequence comprising SEQ ID NO:2. The isolated and purified polypeptides of the invention are useful to prepare an immunogenic composition, such as a vaccine comprising the polypeptide, in combination with a pharmaceutically acceptable carrier, wherein the administration of the immunogenic composition or vaccine to a mammal induces the production of antibodies to the polypeptide. In particular, an immunogenic composition or vaccine which comprises a polypeptide having an epitope which is specific for the agent associated with human granulocytic ehrlichiosis is preferred.
The invention provides a method of expressing a nucleic acid molecule encoding an immunogenic polypeptide, the presence of which is associated with granulocytic ehrlichiosis, in a cultured host cell stably transformed with a chimeric vector comprising said nucleic acid molecule operably linked to control sequences recognized by the host cell transformed with the vector, and recovering the polypeptide from the host cell.
The invention fturther provides a diagnostic method comprising contacting an amount of DNA obtained from a physiological sample which comprises cells from a mammal at risk of, or afflicted with, granulocytic ehrlichiosis, with an amount of at least two complementary oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified DNA. At least one oligonucleotide binds specifically to a DNA sequence encoding a polypeptide derived or isolated from an infectious agent associated with granulocytic ehrlichiosis. The presence of the amplified DNA is then detected or determined. The presence of the amplified DNA is indicative of a mammal at risk of, or afflicted with, granulocytic ehrlichiosis. The results described below show that an amplified DNA was detected using DNA obtained from blood of two humans afflicted with granulocytic ehrlichiosis. Sequence and phylogenetic analysis of the polypeptide encoded by the amplified DNA shows that the polypeptide is homologous to the E. coli heat shock protein-60 (HSP60) and related to, but distinct from, a homologous protein found in E. chaffeensis (Sumner et al., Infection and Immunity, 61, 3546 (1993)) and from other rickettsia-like organisms known to infect humans.
Also provided is a method for detecting DNA encoding an immunogenic polypeptide associated with granulocytic ehrlichiosis in a mammal. The method comprises isolating or deriving an amount of DNA from a mammalian physiological sample which comprises cells suspected of containing DNA encoding the immunogenic polypeptide. The DNA is contacted with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified DNA. At least one oligonucleotide is specific for the DNA encoding the immunogenic polypeptide. The presence of the amplified DNA is then determined or detected.
As used herein, the term an oligonucleotide xe2x80x9cspecific for the DNA encoding a polypeptide from an infectious agent associated with granulocytic ehrlichiosisxe2x80x9d or xe2x80x9cspecific for DNA encoding an immunogenic polypeptide associated with granulocytic ehrlichiosisxe2x80x9d means a DNA sequence that has at least about 80%, more preferably at least about 90%, and more preferably at least about 95%, sequence identity or homology to SEQ ID NO:1 An oligonucleotide or primer of the invention has at least about 7-50, preferably at least about 10-40, and more preferably at least about 15-35, nucleotides. Preferably, the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3xe2x80x2 of the oligonucleotide primer which have at least about 80%, more preferably at least about 85%, and more preferably at least about 90%, identity to SEQ ID NO:1. Thus, the oligonucleotides of the invention may also include sequences which are unrelated to nucleic acid sequences of an infectious agent associated with granulocytic ehrlichiosis, e.g., they may encode restriction endonuclease recognition sequences. Preferred oligonucleotides of the invention include an oligonucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.
Also provided is a diagnostic kit for detecting DNA which encodes at least a portion of a polypeptide specific to an infectious agent associated with granulocytic ehrlichiosis in a physiological sample suspected of containing said DNA. The kit comprises packaging containing, separately packaged, (a) a known amount of a first oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to SEQ ID NO:1, and (b) a known amount of a second oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to a nucleotide sequence which is complementary to SEQ ID NO:1.
Thus, the invention also provides an oligonucleotide which consists of at least about 7-50 nucleotides and which has at least about 80% identity to, or has complementarity to a nucleotide sequence having, SEQ ID NO:1.
Also provided is a method for detecting or determining the presence of antibodies in a mammalian physiological sample, which antibodies are specific for an infectious agent that is associated with granulocytic ehrlichiosis. The method comprises contacting an amount of purified polypeptide with the sample which is suspected of comprising antibodies specific for the infectious agent, for a sufficient time to form binary complexes between at least a portion of the antibodies and a portion of the purified polypeptide. The polypeptide is one which is expressed by, and is encoded by the nucleic acid of, the infectious agent. The presence or amount of the complexes is then determined or detected. The results reported hereinbelow demonstrated that a portion of purified polypeptide, expressed in vitro from a DNA molecule of the invention, is bound by antibodies from mice experimentally infected with the agent associated with human granulocytic ehrlichiosis. Thus, the detection of antibody responses to this and other ehrlichial antigens can be used in ELISA-based immunoassays for the serodiagnosis of granulocytic ehrlichial infection in animals and in humans.
Moreover, the results show that the expression of a gene encoding an antigenic polypeptide, wherein the gene is obtained by direct recovery from a clinical sample without the benefit of prior biological amplification via cultivation in vitro, is useful to detect a humoral immune response to an infectious agent, i.e., the agent associated with human granulocytic ehrlichiosis.
Further provided is a method for detecting granulocytic ehrlichiosis in a mammal. The method comprises contacting an amount of purified polypeptide with a sample suspected of containing antibodies to said polypeptide, for a sufficient time to form binary complexes between at least a portion of the antibodies and a portion of the purified polypeptide. The polypeptide is one which is encoded by the nucleic acid from an infectious agent associated with granulocytic ehrlichiosis. The presence or amount of the binary complexes is then determined or detected. The presence of said complexes is indicative of a mammal at risk of, or afflicted with, granulocytic ehrlichiosis.
Also provided is a diagnostic kit for detecting or determining antibodies that specifically react with an infectious agent which is associated with granulocytic ehrlichiosis. The kit comprises packaging, containing, separately packaged, a solid phase capable of binding a polypeptide and a known amount of a purified polypeptide, the presence of which is associated with granulocytic ehrlichiosis. Preferably, the polypeptide has an amino acid sequence comprising SEQ ID NO:2.