WO1999052370A1

WO1999052370A1 - Nucleic acids encoding outer membrane protein of human granulocytic ehrlichiosis agent

Info

Publication number: WO1999052370A1
Application number: PCT/US1999/007759
Authority: WO
Inventors: Yasuko Rikhisa; Ning Zhi; Norio Ohashi
Original assignee: The Ohio State Research Foundation
Priority date: 1998-04-09
Filing date: 1999-04-08
Publication date: 1999-10-21
Also published as: CA2326717A1; EP1069827A4; AU3483599A; EP1069827A1

Abstract

The invention provides tools for serodiagnosing HGE. The tools are structurally related proteins, 'Group 44 proteins', and antibodies to such proteins. The Group 44 proteins comprise a single central hypervariable region, a first conserved region and a second conserved region which flank the central hypervariable region. The Group 44 proteins comprise P44, P44-2, P44-12, P44-15, P44-18, P44-19, and variants of such proteins. The invention also provides isolated polynucleotides or nucleic acids, referred to as 'Group 44 polynucleotides', which encode the Group 44 proteins and fragments thereof. Each oligopeptide comprises a sequence specific to one Group 44 protein. The present invention also provides antibodies immunospecific for and which bind to members of the P44 family of proteins. The present invention provides kits containing reagent for diagnosing HGE.

Description

NUCLEIC ACIDS ENCODING OUTER MEMBRANE PROTEIN OF HUMAN GRANULOCYΗC EHRLICHIOSIS AGENT

5 BACKGROUND

This work was supported by grant RO1 AI33123 and grant RO1 A140934 from the National Institutes of Health. The Government has certain rights in this invention.

Human granulocytic ehrlichiosis (HGE), an emerging human infectious

10 disease, is increasingly being recognized in the United States. Serological and PCR studies suggest that HGE infection also exists in Europe. HGE is caused by infection with an obligatory intracellular bacterium, HGE agent. Comparison of 16S rRNA gene sequences and ultrastructure indicates that the HGE agent is closely related to Ehrlichia phagocytophila, the agent of tick-borne fever, and E. equi. the agent of

15 equine ehrlichiosis. The HGE agent is transmitted by the Ixodes sp. tick, and the white-footed mouse is considered to be the major reservoir of the HGE agent in the United States.

HGE infection is characterized by the presence of ehrlichial inclusions called morulae in human or animal peripheral blood granulocytes. The symptoms of HGE

20 include chills, headache, myalgia, hematological abnormalities, including leukopenia and thrombocytopenia. HGE frequently requires prolonged hospitalization. When treatment is delayed due to misdiagnosis, HGE can be fatal.

Although several laboratories have used IFA testing, acute-phase blood smear, nested PCR, and culture isolation for diagnosis of HGE, each of these diagnostic tests

25 has disadvantages. IFA testing using the HGE agent or E. egw/^'-infected cells has been most widely used for serodiagnosis of ehrlichiosis. IFA requires a tissue culture system for preparation of HGE-infected cell antigen slides, fluorescent microscope, and trained persons especially for evaluation of the serum reactivity to the antigen on the slide.

30 The nested PCR, while better suited to early diagnosis of HGE than IFA testing, requires thermocycler and trained personel, and the reagents are relatively expensive. The sensitivity of culture isolation in HGE diagnosis seems to be relatively lower than that of IFA test and the nested PCR. Moreover, diagnosis by culture isolation requires serologic evidence, PCR, or 16S rRNA gene sequence for confirmation of the identity Accordingly, a convenient and sensitive method with high specificity for the diagnosis of HGE infection is still desirable.

SUMMARY OF THE INVENTION present invention relates to improved diagnostic tools for serodiagnosing

HGE. The diagnostic tools are structurally related proteins, collectively referred to hereinafter as the "Group 44 proteins", and to antibodies to such proteins. As compared to each other, the Group 44 proteins comprise a single central hypervariable region of approximately 94 amino acid residues, a first conserved region and a second conserved region which flank the central hypervariable region. The first conserved region comprises 52 amino acids and is linked directly or through a linker of from 1 to 10 amino acids to the amino terminus, i.e., the N-terminus, of the hypervariable region. The second conserved region comprises about 56 amino acids and is linked either directly or through a linker of from 1 to 10 amino acids to the carboxy terminus, i.e., the C-terminus, of the hypervariable region. The hypervariable region of each Group 44 protein has a higher hydrophilicity, a higher antigenic index, and a higher surface probability than either the first conserved region or the second conserved region of the respective protein. The hypervariable region is basic and has an isoelectric point, i.e., a pi, of from about 7.1 to about 9.2 and a molecular mass, i.e., an Mr, of from about 8.5 kDa to about 11 kDa. The hypervariable region has a first semiconserved region of 8 amino acids near the amino terminus thereof and a second semiconserved region of 11 amino acids at the carboxy terminus thereof. The hypervariable region also contains a third semiconserved region of 6 amino acids. It is believed that the third semiconserved region, which is between and separated from the first semiconserved region and the second semiconserved region is perhaps involved in adhesion of the HGE agent to other cells.

The Group 44 proteins comprise the P44 protein, the P44-2 protein, the P44- 12 protein, the P44-15 protein, the P44-18 protein, and the P44-19 protein and variants of such proteins. The P44 protein and variants thereof specifically bind to antibodies found in the sera from horses infected with the HGE agent and to antibodies in the sera from humans infected with the HGE agent and horse anti- Ehrlichia equi serum. The P44 protein and variants thereof do not bind to antibodies in sera from patients infected with E.chaffeensis or Borrelia burgdorferi. In one embodiment, the P44 protein comprises the amino acid sequence, SEQ ID NO: 2, shown in Figure 1 A.

The P44-2 protein has a molecular mass of about 45 kDa. In one embodiment the P44-2 protein comprises the amino acid sequence, SEQ ID NO: 4, shown in Figure 2. The P44-12 protein has a molecular mass of about 41.2 kDa. In one embodiment the P44-12 protein comprises the amino acid sequence. SEQ ID NO: 6, shown in Figure 3. The P44-15 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 10.8 kDa. In one embodiment, the P44-15 protein comprises the amino acid sequence, SEQ ID NO: 8, shown in Figure 4. The P44-18 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 26.4 kDa. In one embodiment, the P44-18 protein comprises the amino acid sequence. SEQ ID NO: 10 shown in Figure 5. The P44-19 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 8.6 kDa and an isoelectric point of about 8.8. The P44-19 protein comprises the amino acid sequence, SEQ ID NO: 12, shown in Figure 6.

The Group 44 proteins are immunogenic and, thus, are useful for preparing antibodies. The isolated proteins of the P44 family, either individually or as a panel of proteins, are useful for detecting antibodies to the HGE agent in the blood of patients with clinical signs of HGE .The isolated, individual Group 44 proteins are also useful in an immunogenic composition for ameliorating HGE. The isolated, individual Group 44 proteins are also useful in a vaccine for protecting against infection with the HGE agent.

The present invention also provides isolated polynucleotides or nucleic acids, referred to collectively hereinafter as the "Group 44 polynucleotides". which encode the Group 44 proteins and fragments thereof. The P44 polynucleotides encode the P44 protein. One embodiment of the P44 polynucleotides comprises the nucleotide sequence, SEQ ID NO:l, shown in Figure IB. The P44-2 polynucleotides encode the P44-2 protein. One embodiment of the P44-2 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 3. shown in Figure 2. The P44-12 polynucleotides encode the P44-12. One embodiment of the P44-12 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 5, shown in Figure 3. The P44-15 polynucleotides encode the P44-15 protein. One embodiment of the P44-15 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 7, shown in Figure 4. The P44-18 polynucleotides encode the P44-18 protein. One embodiment of the P44-18 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 9, shown in Figure 5. The P44-19 polynucleotides encode the P 44-19 protein. One embodiment of the P44-19 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 11, shown in Figure 6. The Group 44 polynucleotides are useful for preparing the Group 44 proteins and variants thereof. Group 44 polynucleotides which encode fragments of the Group 44 proteins are useful as primers and probes.

The present invention provides synthetic oligopeptides of 14-16 amino acids in length. Each of the oligopeptides comprise a sequence which is specific to one Group 44 protein. Such peptides are useful for preparing antibodies which are immunospeciflc for one or more Group 44 proteins.

The present invention also relates to antibodies which are immunospeciflc for and bind to members of the P44 family of proteins. Such antibodies are useful for immunolabeling isolates of the HGE agent and for detecting the presence of HGE in body fluids, tissues, and particularly, monocytes and macrophages. The present invention also relates to kits containing reagents for diagnosing HGE.

BRIEF DESCRIPTION OF THE FIGURES

FIGS 1 A and IB show the amino acid sequence, SEQ ID NO: 2, of the P44 protein and a nucleotide sequence, SEQ ID NO: 1 which encodes the P44 protein. The deduced amino acid sequence of P44 is shown beneath the DNA sequence. N terminal amino acid sequence of native isolated P44 is underlined. Arrowheads indicate 5' annealing positions of the primer pair (24 mers for forward and 26 mers for reverse) designed for PCR amplification of partial P44 gene which was subcloned into pET30A. an expression vector.

FIGS. 2 A and 2B show the amino acid sequence, SEQ ID NO:4, of the P44-2 protein and a nucleotide sequence, SEQ ID NO:3. which encodes the P44-2 protein.

FIGS. 3 A and 3B show the amino acid sequence. SEQ ID NO:6, of the P44-12 protein and a nucleotide sequence, SEQ ID NO:5. which encodes the P44-12 protein. FIGS. 4A and 4B show the amino acid sequence. SEQ ID NO:8, of the P44-15 protein and a nucleotide sequence, SEQ ID NO:7, which encodes the P44-15 protein. FIGS. 5 A and 5B show the amino acid sequence, SEQ ID NO: 10, of the P44- 18 protein and a nucleotide sequence, SEQ ID NO:9, which encodes the P44-18 protein. FIGS. 6A and 6B show the amino acid sequence, SEQ ID NO: 12, of the P44- 19 protein and a nucleotide sequence, SEQ ID NO:l 1, which encodes the P44-19 protein.

FIG 7. depicts the hydrophilicity profiles of the Group 44 proteins. FIG 8. depicts the antigenic index profiles of the Group 44 proteins.

FIG 9. shows the surface probability profiles of the Group 44 proteins. FIG 10. is an alignment of amino acid sequences deduced from cDNA clones and the corresponding /^-homologous genes of the HGE agent. Aligned positions of identical amino acids with the P44 protein are shown with dot. Gaps indicate by dashed lines were introduced for an optimal alignment of all proteins. A boxed area in the middle indicates the amino acid sequences deduced from nucleotide sequences ofcDNAs. Hypervariable regions are shown in boldface. A bar indicates the N- terminal amino acid sequence of the native P44 protein and an arrowhead shows the cleavage site of the putative signal peptide. The amino acid sequences underlined in the hypervariable regions of P44-2 and P44-18 indicate the sequences which were used to prepare synthetic oligopeptides, Pep2 and Pep 18, respectively. The arrows point out the positions of the primers used in RT-PCR. The numbers on the right side indicate the positions of amino acid residues in P44-homologous proteins from the N terminus to C terminus. FIG. 11 is a restriction map of 6.9-kb genomic DNA fragment including p44 gene of the HGE agent. The closed boxes with arrow indicate four open reading frames (ORF) which are identified in this fragment. The arrows indicate the orientation of these ORFs. Shaded boxes (Rl and R2) show two regions identical in the ORF1 which encoded 59- and 65-amino acids, respectively. The solid bar at bottom indicates the region which was cloned into the pET30a expression vector. FIG.12 shows the primer positions in the p44 gene and the truncated ORF homologous to p44 in the recombinant plasmid pHGE1221. The primers p3708 and p4257 were designed for RT-PCR to amplify the cDNA of /^-homologous genes. The primers hvf and hvr were designed to prepare the cDNA-specific probes by PCR for Southern and Northern blot analysis. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a group of structurally related proteins, the Group 44 proteins, which are encoded by a group of homologous genes which are contained within the genome of the HGE causative agent. The Group 44 proteins have an amino acid sequence identity, as determined using the MEGALIGN project in the DNASTAR program (DNA STAR, Inc., Madison, Wisconsin), ranging from 59% to 83.4%. The Group 44 proteins comprise a single central hypervariable region of about 94 amino acid residues, a first conserved region of about 52 amino acids which is linked either directly or through a linker of from 1 to about 10 amino acids to the amino or N-terminus of the hypervariable region, and a second conserved region of about 56 amino acids which is linked either directly or through a linker of from 1 to about 10 amino acids to the carboxy or C-terminus of the hypervariable region. The Group 44 proteins also comprise an N-terminal end sequence which is linked to the N-terminal amino acid of the first conserved region and a C terminal end sequence which is linked to the C-terminal amino acid of the second conserved region. The Group 44 proteins comprise the P44 protein and variants thereof, the P44-2 protein and variants thereof, the P44-15 protein and variants thereof, the P44-18 protein and variants thereof, and the P44-19 protein and variants thereof. The hypervariable regions of the Group 44 proteins have a higher hydrophilicity, a higher antigenic index, and a higher surface probability than either the first conserved region or the second conserved regions. The hypervariable regions of the Group 44 proteins have an average hydrophilicity index from about 0.45 to about 0.95, as determined using the Kyte-Doolitle hydrophilicity profile which is described in Kyte, Jack and R. F.Doolitle (1982). J. Mol. Biol. 157:105-132. The hypervariable regions of the Group 44 proteins have an average antigenic index of from about 0.65 to about 1.5, as determined using the Jameson- Wo If antigenic index profile, which is described in Jameson, B.A. and H. Wolf (1988) CABIOS. 4:181- 186. The hypervariable regions of the Group 44 proteins have an average surface probability index of from about 1.0 to about 1.55 as determined using the Emini surface probability profile, which is described in Emini, E. A. et al (1985) J. Virology. 55:836-839. The hypervariable regions of the Group 44 proteins have an Mr of from about 8.6 kDa to about 10.9 kDa and an isoelectric point (pi) of from about 7.1 to about 9.2. The hypervariable regions of the Group 44 proteins comprise a first semiconserved region at their N-terminus and a second semiconserved region at their C-terminus. The first semiconserved region comprises the sequence lie, XI, X2, Lys, X3, Cys wherein XI is Asp or Gly , X2 is Gly or Lys , and X3 is Val or He. The second semiconserved region comprises the sequence Asn, Ala, XI, X2, X3, Ala, X4, X5 , Leu, X6, and X7 wherein XI is Asn, Thr, Lys, or Glu, X2 is Ala or Lys, X3 is Met or Val; X4 is Lys, Gly, or Thr; X5 is Asp or Glu; X6 is Val or Thr, and X7 is Gin or Lys . The hypervariable regions of the Group 44 proteins also comprise a third semiconserved region which is located between the first semiconserved region and the second semiconserved region. The third semiconserved region comprises the sequence Lys-Xl-Trp-Pro-Thr-X2; wherein XI is Asn or His and X2 is Gly, Thr, or Ser. It is believed that the third semiconserved region permits adhesion of the HGE agent to other cells. The characteristics of the hypervariable regions of individual Group 44 proteins are shown in Table 1 below:

Table 1: Characteristics of hypervariable regions of Group 44 Proteins

Group 44 Total Mr Mr of PI. of Kyte-hydro⁰ James-antige" Emini-

Proteins hypervariable hypervariable surface^c

Hv region region

P44-hv 41,293 10,327 9.16 0.45 0.65 1.0

P44-2hv 44,969 10,454 7.17 0.53 1.29 1.03

P44-12hv 41,197 10,378 8.17 0.82 1.38 1.04

P44-15hv 29,287 10,831 8.77 0.82 1.37 1.25

P44-18hv 26,400 10,186 8.65 0.94 1.49 1.53

P44-19hv 8,645 8.8 0.67 1.07 1.05 a. Kyte-Doolittle hydrophilicity. Kyte, Jack And R.F. Doolittle (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132. b. Jameson-Wolf antigenic index. Jameson, B.A. and H. Wolf (1988). The antigenic index: a novel algorithm for predicting antigen determinants. CABIOS. 4: 181-186. c. Emini surface probability. Emini, E.A. and J. Hughes, D. Perlow and J. Boger (1985). Induction of hepatits A virus-neutralizing and antibody by a virus specific synthetic peptide.

Virology. 55: 836-839.

The present invention also encompasses variants of the proteins of the Group 44 proteins whose amino acid sequences are shown in SEQ ID Nos 2, 4, 6, 8 , 10, and 12. A "variant" as used herein, refers to a protein whose amino acid sequence is similar to the amino acid sequence of a particular Group 44 protein, hereinafter referred to as the reference amino acid sequence, but does not have 100% identity with the respective reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 90% identical to the reference sequence, preferably, at least 95%o identical, more preferably at least 96% identical, most preferably at least 97%) identical to the reference sequence. Variant sequences which are at least 90% identical have no more than 10 alterations, i.e any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using MEGALIGN project in the DNA STAR program.

While it is possible to have nonconservative amino acid substitutions, it is preferred that the substitutions be conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydoxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.

Preferably, the amino acid sequence of the first conserved region is at least 95%o identical, more preferably at least 97%> identical, most prefera-bly at least 98%> identical to the amino acid sequence of the first conserved region of P44, P44-2, P44- 15, P44-18, or P44-19. Preferably, the amino acid sequence of the second conserved region is at least 95%> identical, more preferably at least 97% identical, most preferably at least 98% identical to the amino acid sequence of the second conserved region of P44, P44-2, P44-15, P44-18, or P44-19.

The alterations are designed not to abolish the immunoreactivity of the variant with antibodies that bind to the reference protein. Guidance in determining which amino acid residues may be substituted, inserted or deleted without abolishing such immunoreactivity of the variant protein are found using computer programs well known in the art, for example, DNASTAR software. Preferably, the alterations are not located in the variant protein regions which correspond to the first conserved region or the second conserved region of the respective Group 44 protein

The present invention also encompasses fusion proteins in which a tag or one or more amino acids, preferably from about 2 to 15 amino acids, more preferably from about 34 to about 62 amino acids are added to the amino or carboxy terminus of the amino acid sequence of a Group 44 protein or a variant of such protein. Typically, such additions are made to stabilize the resulting fusion protein or to simplify purification of an expressed recombinant form of the corresponding P44 protein or variant of such protein. Such tags are known in the art. Representative examples of such tags include sequences which encode a series of histidine residues, the Herpes simplex glycoprotein D, or glutathione S-transferase.

The present invention also encompasses Group 44 proteins in which one or more amino acids, preferably no more than 10 amino acids, in the respective Group 44 protein are altered by posttranslation processes or synthetic methods. Examples of such modifications include, but are not limited to, acetylation, amidation, ADP- ribosylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a lipid, cross-linking gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, sulfation, and transfer-RNA mediated additions of amino acids to proteins such as arginylation and ubiquitination.

One Group 44 protein is the P44 protein, which weighs approximately 41 kDa and has an isoelectric point of 5.11. The P44 protein and variants thereof specifically bind to antibodies found in the sera from horses infected with the HGE agent and to antibodies in the sera from humans infected with the HGE agent and horse anti- Ehrlichia equi serum. The P44 protein does not bind to antibodies in sera from patients infected with E.chaffeensis or Borrelia burgdorferi. The P44 protein comprises the amino acid sequence, SEQ ID NO: 2, shown in Figure 1 . The hypervariable region of the P44 protein extends from amino acid 176 to amino acid 277 of the sequence, SEQ ID NO:2, shown in Figure 1. The hypervariable region of the P44 protein and variants thereof have an Mr of from about 8.6 to about 10.8 kDa. The first conserved region of the P44 protein extends from 124 amino acid to amino acid 184 of SEQ ID NO: 2. The second conserved region oFf the P44 protein extends from amino acid 114 to amino acid 175 of SEQ ID NO:2. The first semiconserved region of the hypervariable region of the P44 protein has the amino acid sequence IDGKVC; the second semiconserved region of the hypervariable region of the P44 protein NANAMAKDLVK, SEQ ID NO: ; the third semiconserved region of the hypervariable region of the P44 protein has the amino acid sequence KNWPTG. The P44-2 protein has an Mr of about 45 kDa and an isoelectric point of 5.20.

The P44-2 protein comprises the amino acid sequence, SEQ ID NO: 4 shown in Figure 2. The hypervariable region of the P44-2 protein extends from amino acid 176 to amino acid 277 and has the characteristics shown in Table 1. The first conserved region of the P44-2 protein extends from amino acid 114 to amino acid 175 of SEQ ID NO: 4. The second conserved region of the P44-2 protein extends from amino acid 278 to amino acid 344 of SEQ ID NO: 4. The first semiconserved region of the hypervariable region of the P44 protein has the amino acid sequence IDGKVC; the second semiconserved region of the hypervariable region of the P44-2 protein has the amino acid sequence NAKAVATDLVQ, SEQ ID NO: ; the third semiconserved

10 region of the hypervariable region of the P44-2 protein has the amino acid sequence KNWPTG-

The P44-12 protein has an Mr of about 41.2 kDa and an isoelectric point of 5.19. The P44-12 protein comprises the amino acid sequence, SEQ ID NO:6, shown in Figure 3. The hypervariable region of the P44-12 protein extends from amino acid 176 to amino acid 279 and has the characteristics shown in Table 1. The first conserved region of the P44-12 protein extends from amino acid 114 to amino acid 175 of SEQ ID NO: 6. The second conserved region of the P44-12 protein extends from amino acid 280 to amino acid 345 of SEQ. ID. NO. 6. The first semiconserved region of the hypervariable region of the P44-12 protein has the amino acid sequence IDKKVC; the second semiconserved region of the hypervariable region of the P44-

12 protein has the amino acid sequence NAEAVAKDLVQ, SEQ ID NO: ; the third semiconserved region of the hypervariable region of the P44-12 protein has the amino acid sequence KNWPTS. The P44- 15 protein comprises a hypervariable region having an Mr of about

10.8 kDa and an isoelectric point of about 8.8. The P44-15 protein comprises the amino acid sequence, SEQ ID NO:8, shown in Figure 4 . The hypervariable region of the P44-15 protein extends from amino acid 82 to amino acid 184 and has the characteristics shown in Table 1 below. The first conserved region of the P44-15 protein extends from amino acid 30 to amino acid 83 of SEQ ID NO: 8. The second conserved region of the P44-15 protein extends from amino acid 185 to amino acid 250 of SEQ. ID. 8. The first semiconserved region of the hypervariable region of the P44-15 protein has the amino acid sequence IDGKVC; the second semiconserved region of the hypervariable region of the P44-15 protein has the amino acid sequence NAKAVAKDLVQ, SEQ ID NO: ; the third semiconserved region of the hypervariable region of the P44-2 protein has the amino acid sequence KNWPTS.

The P44-18 protein comprises a hypervariable region having an Mr of about 10.2 kDa and an isoelectric point of about 8.7. The P44-8 protein comprises the amino acid sequence, SEQ ID NO: 10, shown in Figure 5. The hypervariable region of the P44-18 protein extends from amino acid 73 to amino acid 170 of the sequence shown in SEQ ID NO: 10. The first conserved region of the P44-18 protein extends from amino acid 21 to amino acid 72 of SEQ ID NO: 10. The second conserved region of the P44-18 protein extends from amino acid 171 to amino acid 237 of the SEQ. ID. NO. 10. The first semiconserved region of the hypervariable region of the

11 P44-18 protein has the amino acid sequence IGKKVC; the second semiconserved region of the hypervariable region of the P44-18 protein has the amino acid sequence

NAKAVAGDLTK, SEQ ID NO: ; the third semiconserved region of the hypervariable region of the P44-18 protein has the amino acid sequence KNWPTS. The P44- 19 protein comprises a hypervariable region having an Mr of about

8.6 kDa and an isoelectric point of about 8.8. The P44-19 protein comprises the amino acid sequence, SEQ ID NO: 12. shown in Figure 6 . The hypervariable region of the P44-19 protein extends from amino acid 39 to amino acid 133. The first conserved region of the P44-19 protein extends from amino acid 1 to amino acid 38 of SEQ ID NO: 12. The second conserved region of the P44-19 protein extends from amino acid 134 to amino acid 164. The first semiconserved region of the hypervariable region of the P44-15 protein has the amino acid sequence IDGKIC; the second semiconserved region of the hypervariable region of the P44-19 protein has the amino acid sequence NATKVAGELTK, SEQ ID NO: ; the third semiconserved region of the hypervariable region of the P44-19 protein has the amino acid sequence KHWPTT. The members of the P44 family of proteins are immunogenic and, thus, are useful for preparing antibodies. Such antibodies are useful for immunolabeling isolates of the HGE agent and for detecting the presence of the HGE agent in body fluids, tissues, and particularly in neutrophils. The isolated proteins of the P44 family, particularly P44 are useful for detecting antibodies to the HGE agent in the blood of patients with clinical signs of HGE. The isolated members of the P44 family are also useful in a vaccine for protecting against infection with the HGE agent.

In another aspect, the present invention provides an isolated peptide which comprises a fragment of the P44 protein, hereinafter referred to as rP44 polypeptide. rP44 polypeptide weighs approximately 23 kDa and comprises the amino-terminal half of the P44 protein. In one embodiment, rP44 comprises the amino acid sequence extending from amino acid 30 through amino acid 248 of the amino acid sequence shown in Fig. 1. In another embodiment the rP44 comprises the amino acid sequence extending from amino acid 38 through amino acid 248 of the amino acid sequence shown in Fig. 1, and, thus, lacks the amino acids of the signal sequence of the P44 protein. The rP44 peptide is a useful diagnostic tools for detecting the presence of antibodies in the bodily fluids, particularly sera, of individuals infected with the causative agent of HGE.

12 The present invention provides oligopeptides of from about 14-16 amino acids in length and having a sequence which is specific to one or more of the P44 family proteins. Such peptides are useful for preparing chimeric peptides that are used to prepare antibodies that are immunospeciflc for one or more, of the P44 family proteins. In one embodiment, the synthetic oligopeptide comprises the amino acid sequence LSNGSAEAAHKYLSK, SEQ ID NO: , which is specific for the P44 protein. In another embodiment, the synthetic oligopeptide comprises the amino acid sequence GHSSGVTQNPKLFST, SEQ ID NO:_, which is specific for the P44-2 protein. In another embodiment, the synthetic oligopeptide comprises an amino acid sequence GKKSGDNGSLADYTD, SEQ ID NO: , which is specific for the P44-12 protein. In another embodiment, the synthetic oligopeptide comprises an amino acid sequence PLYSDETHTKGASEGR, SEQ ID NO:— which is specific for the P44-15 protein. In another embodiment, the synthetic oligopeptide comprises the amino acid sequence KNQKSSDTDTGVEKA, SEQ ID NO: , which is specific for the P44-18 protein. In another embodiment, the synthetic oligopeptide comprises the amino acid sequence TGSNKYGTGTNSGELT, SEQ ID NO: ,which is specific for the P44-

19 protein. .

The present invention also provides isolated polynucleotides or nucleic acids which encode the proteins of the P-44 family or variants of the P44 family proteins. The polynucleotide is a DNA or RNA molecule, preferably a DNA molecule, and comprises a sequence which codes for a Group 44 protein or a variant thereof. One polynucleotide, referred to hereinafter as the p44 polynucleotide" encodes the P44 protein. In one embodiment, the p44 polynucleotide comprises the nucleotide sequence , SEQ ID NO. 1, shown in Figure 1. Another polynucleotide, the p44-2 polynucleotide encodes the P44-2 protein. In one embodiment the p44-2 polynucleotide comprises the nucleotide sequence, SEQ ID NO: 3 shown in Figure 2. Another polynucleotide, the p 44-12 polynucleotide encodes the P44-12 protein. In one embodiment the p44-12 polynucleotide comprises the nucleotide sequence, SEQ ID NO: 5, shown in Figure 3. Another polynucleotide, the p 44-15 polynucleotide encodes the P44-15 protein. In one embodiment the p44-l 5 polynucleotide comprises the nucleotide sequence, SEQ ID NO: 7, shown in Figure 4. Another polynucleotide, the p44-18 polynucleotide encodes the P44-18 protein. In one embodiment the p44-18 polynucleotide comprises the nucleotide sequence, SEQ ID NO: 9, shown in Figure 5. Another polynucleotide, the p44-19 polynucleotide encodes the P44-19 protein. In

13 one embodiment the p44-19 polynucleotide comprises the nucleotide sequence, SEQ ID NO: 11 , shown in Figure 6.

Polynucleotides that encode the Group 44 proteins are useful tools that can be used in recombinant techniques for producing the Group 44 proteins. Polynucleotides encoding the Group 44 proteins are also useful for designing hybridization probes for isolating and identifying cDNA clones and genomic clones encoding the Group 44 proteins or allelic forms thereof. Such hybridization techniques are known to those of skill in the art. Sequence of polynucleotides that encode the Group 44 proteins are also useful for designing primers for polymerase chain reaction (PCR), a technique useful for obtaining large quantities of cDNA molecules that encode the Group 44 proteins.

Also encompassed by the present invention, are single stranded polynucleotides, hereinafter referred to as antisense polynucleotides, having sequences which are complementary to the DNA and RNA sequences which encode the P44 protein, the P44-2 protein, the P44-15 protein, the P44-18 protein, and the P44-19 protein. The term complementary as used herein refers to the natural binding of the polynucleotides under permissive salt and temperature conditions by base pairing,

The present invention also encompasses oligonucleotides that are used as primers in polymerase chain reaction (PCR) technologies to amplify transcripts of the genes which encode the P44 family proteins or portions of such transcripts. Preferably, the primers comprise 18-30 nucleotides, more preferably 19-25 nucleotides. Preferably, the primers have a G+C content of 40% or greater. Such oligonucleotides are at least 98%> complementary with a portion of the DNA strand, i.e., the sense strand, which encodes the respective P44 family protein or a portion of its corresponding antisense strand. Preferably, the primer has at least 99% complementarity, more preferably 100%) complementarity, with such sense strand or its corresponding antisense strand. Primers which are which have 100%o complementarity with the antisense strand of a double-stranded DNA molecule which encodes a P44 family protein have a sequence which is identical to a sequence contained within the sense strand. The identity of primers which are 15 nucleotides in length and have full complementarity with a portion of the antisense strand of a double-stranded DNA molecule which encodes the P44 protein is determined using the nucleotide sequence, SEQ ID NO:l , shown in FIG 1A and described by the

14 general formula a-b, where a is any integer between 1 to 1239, where b is equal to a+14, and where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 1.

The present invention also encompasses oligonucleotides that are useful as hybridization probes for detecting transcripts of the genes which encode the P44 family proteins or for mapping of the genes which encode the P44 family proteins Preferably, such oligonucleotides comprise at least 210 nucleotides, more preferably at least 230, most preferably from about 210 to 280 nucleotides. Such hybridization probes have a sequence which is at least 90%> complementary with a sequence contained within the sense strand of a DNA molecule which encodes a P44 family protein or with a sequence contained within its corresponding antisense strand. Such hybridization probes bind to the sense strand under stringent conditions. The term "stringent conditions" as used herein is the binding which occurs within a range from about Tm 5°C (5°C below the melting temperature Tm of the probe) to about 20°C to 25°C below Tm. The probes are used in Northern assays to detect transcripts of p44 homologous genes and in Southern assays to detect p44 homologous genes. The identity of probes which are 200 nucleotides in length and have full complementarity with a portion of the antisense strand of a double-stranded DNA molecule which encodes the P44 protein is determined using the nucleotide sequence, SEQ ID NO: 2, shown in FIG 1 A and described by the general formula a-b, where a is any integer between 1 to 1239, b is equal to a +200, and where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:l.

The present invention also encompasses isolated polynucleotides which are alleles of the genes which encode the Group 44 proteins. As used herein, an allele or allelic sequence is an alternative form of the gene which may result from one or more mutations in the sequences which encode the Group 44 proteins. Such mutations typically arise from natural addition, deletion of substitution of nucleotides in the open reading frame sequences Any gene may have none, one, or several allelic forms. Such alleles are identified using conventional techniques, such as for example screening libraries with probes having sequences identical to or complementary with one or more Group 44 polynucleotides.

The present invention also encompasses altered polynucleotides which encode Group 44 proteins or Group 44 protein variants. Such alterations include deletions, additions, or substitutions. Such alterations may produce a silent change and result in

15 a Group 44 protein having the same amino acid sequence as the Group 44 protein encoded by the unaltered polynucleotide. Such alterations may produce a nucleotide sequence possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eucaryotic host may be incorporated into the nucleotide sequences shown in Figures 1-6 to increase the rate of expression of the proteins encoded by such sequences. Such alterations may also introduce new restriction sites into the sequence or result in the production of a Group 44 protein variant. Typically, such alterations are accomplished using site-directed mutagenesis.

In another aspect, the present invention relates to antibodies which are specific for and bind to at least one Group 44 protein or a peptide specific for one or more Group 44 proteins. Such antibodies are useful research tools for identifying cells, particularly granulocytes, infected with the causative agent of HGE and for purifying the major outer membrane protein of the causative agent of HGE from partially purified preparations by affinity chromatography. Such antibodies are also useful for identifying bacterial colonies, particularly colonies of genetically-engineered bacteria, that are expressing the major outer membrane protein of the causative agent of HGE.

The present invention also relates to kits containing reagents for diagnosing HGE.. The kit comprises one or more Group 44 proteins or antigenic fragments thereof. Preferably, the kit comprises a panel of Group 44 proteins or peptide fragments thereof. For ease of detection, it is preferred that the Group 44 proteins or peptides be attached to a substrate such as a column, plastic dish, matrix, or membrane, preferably nitrocellulose. The kit may further comprise a biomolecule, preferably a secondary antibody, for detecting interactions between the isolated Group 44 protein or peptide and antibodies in a patient sample. Preferably, the biomolecule is coupled to a detectable tag such as an enzyme, chromophore, fluorophore, or radio- isotope. The kit is used by contacting a patient sample with the Group 44 proteins or peptides under conditions that permit formation of antigen-antibody complexes. Then the biomolecule is added and the presence or absence of any resulting antigen- antibody complexes is detected by assaying for a change in the sample, for example, by observing the formation of a precipitate in the sample, the presence of radioactivity on the substrate, or a color change in the sample or on the substrate.

The present invention also provides a method for detecting antibodies to the HGE agent in a sample of a bodily fluid from a patient. The method comprises providing one or more Group 44 proteins or antigenic fragments thereof, contacting

16 the Group 44 protein or antigenic fragment thereof with a bodily sample taken from the patient; and assaying for the formation of a complex between Group 44 protein or antigenic fragment thereof and antibodies in the bodily sample. The sample may be a tissue or a biological fluid, including urine, whole blood, or exudate, preferably serum. The sample may be untreated, subjected to precipitation, fractionation, separation, or purification before combining with the Group 44 protein. Interactions between antibodies in the sample and the Group 44 protein are detected by radiometric, colorimetric, or fluorometric means, size-separation, or precipitation. Preferably, detection is by addition of a secondary antibody that is coupled to a detectable tag, such as for example, an enzyme, fluorophore, or chromophore.

Formation of the antigen-antibody complex is indicative of the presence of anti-HGE antibodies, either IgM or IgG, in the patient. Thus, the method is used to determine whether a patient is infected with the HGE agent. The method can also be used to distinguish patients infected with HGE from patients infected with Ehrlichia chaffeensis or Borrelia burgdorferi as there is no cross-reactivity between the isolated Group 44 proteins, particularly the P44 protein or the rP44 polypeptide, and antibodies produced in individuals infected with either Ehrlichia chaffeensis or Borrelia burgdorferi.

Preparing the Group 44 Proteins and Peptides

The Group 44 proteins, the rP44 polypeptide, and the synthetic P44 oligopeptides may be produced by conventional peptide synthesizers. The Group 44 proteins and rP44 polypeptide may also be produced using cell-free translation systems and RNA molecules derived from DNA constructs that encode the Group 44 proteins and the rP44 polypeptide. Alternatively, the Group 44 proteins and the rP44 polypeptide are made by transfecting host cells with expression vectors that comprise a DNA sequence that encodes the respective Group 44 protein or the rP44 polypeptide and then inducing expression of the protein or polypeptide in the host cells. For recombinant production, recombinant constructs comprising one or more of the sequences which encode the respective Group 44 protein or rP44 peptide are introduced into host cells by conventional methods such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape lading, ballistic introduction or infection.

17 The Group 44 proteins and rP44 polypeptide may be expressed in suitable host cells, such as for example, mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters using conventional techniques. Following transformation of the suitable host strain and growth of the host strain to an appropriate cell density, the cells are harvested by centrifugation. disrupted by physical or chemical means, and the resulting crude extract retained for further purification of the P44 protein or rP44 polypeptide.

Conventional procedures for isolating recombinant proteins from transformed host cells, such as isolation by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, and high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate recombinant Group 44 proteins or rP44 polypeptide.

Synthetic oligopeptides specific for a particular Group 44 protein are identified by examining and comparing the hypervariable regions of the known Group 44 proteins. Preparation of Antibodies

The Group 44 proteins, the rP44 polypeptide, and the P44 synthetic oligopeptides are used as immunogens to produce antibodies immunospeciflc for one or more Group 44 proteins. The term "immunospeciflc" means the antibodies have substantially greater affinity for one or more Group 44 proteins than for other proteins. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, and Fab fragments.

It is preferred that the peptides, fragments, or oligopeptides that are used to induce antibodies to the P44 family proteins have an amino acid sequence of at least five amino acids, and more preferably, at least 10 amino acids that are identical to a portion of the amino acid sequence of a Group 44 protein. Preferably, the oligopeptide has a sequence which is identical to a portion of the hypervariable region of a Group 44 protein. Such peptides are conventionally fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.

Polyclonal antibodies are generated using conventional techniques by administering the Group 44 protein or a portion thereof, i.e., the rP44 polypeptide, or the P44 chimeric molecule to a host animal. Depending on the host species, various

18 adjuvants may be used to increase immunological response. Among adjuvants used in humnas, BCG (bacilli Calmette-Guerin, and Corynebacterium parvum are especially preferable. Conventional protocols are also used to collect blood from the immunized animals and to isolate the serum and or the IgG fraction from the blood. For preparation of monoclonal antibodies, conventional hybridoma techniques are used. Such antibodies are produced by continuous cell lines in culture. Suitable techniques for preparing monoclonal antibodies include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV hybridoma technique. Various immunoassays may be used for screening to identify antibodies having the desired specificity. These include protocols which involve competitive binding or immunoradiometric assays and typically involve the measurement of complex formation between the respective Group 44 protein and the anibody.

Polynucleotides that encode Group 44 Proteins or variants thereof

Polynucleotides comprising sequences encoding a Group 44 protein or a variant thereof may be synthesized in whole or in part using chemical methods. Polynucleotides which encode a Group 44 protein, particularly alleles of the genes which encode a Group 44 protein, may be obtained by screening a genomic library of an HGE isolate with a probe comprising sequences identical or complementary to the sequences shown in Figures 1-6 or with antibodies immunospecific for a Group 44 protein to identify clones containing such polynucleotide.

The probes are used in Southern blot or colony hybridization assays under high stringency conditions. Alternatively, polynucleotides encoding Group 44 proteins may be made using polymerase chain reaction (PCR) technology and primers which bind specifically to sequences which are known to encode a Group 44 protein.

The following examples are for purposes of illustration only and are not intended to limit the scope of the claims which are appended hereto.

EXAMPLES EXAMPLE 1 : D44 POLYNUCLEOTIDE A. Cloning

HGE agent No. 2, 3, 6, 11 and 13 isolates isolated from patients in New York State were cultivated in HL-60 cells. The organisms were purified by the Sephacryl S- 1000 chromatography method as described by Rikihisa et al in Analysis of Ehrlichia

19 canis and canine granulocytic Ehrlichia infection. J. Clin. Microbiol. 30: 143-148 (1992). Genomic DNA of the HGE agent was isolated from purified ehrlichial organisms by SDS lysis, pronase digestion, phenol-chloroform extraction, and ethanol precipitation, according to the procedure of Ohashi et al (1998). Immunodominant major outer membrane proteins of Ehrlicha chaffeensis are encoded by a polymorphic multigene family. Infect Immun. 66: 132-139. Purified genomic DNA was completely digested with 20 units of EcoRI at 37°C for 4 h, and then litigated into the λZAPII vector. All procedures were carried out by using λZAPII/EcoRI/CIAP cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's instruction. The gene library was constructed by infecting E. coli ALl-blue MRFl strain with the recombinant phage. Clones and strain with the recombinant phage. Clones expressing ehrlichial proteins were identified by using horse anti-HGE serum which has been preabsorbed with E. coli lysate. Positive recombinant pBluescript phagemids were excised from the λZAPII phages in the presence of helper fl and were used to transform E. coli SOL R cells (Stratagene). All of the positive clones were analyzed by Western blotting using the horse anti-tfE gent serum. Phagemids purification, restriction enzyme digestion, and gel electrophoresis were carried out as described by Sambrook et al in Molecular cloning: a laboratory manuals. 2nd ed. Cold Spring Harbor Laboratory Press. Plainview, NY (1989). DNA sequencing was determined by dideoxyl-termination method with an

Applied Biosystems 373 DNA sequencer. DNA sequencing was performed by primer- walking method using synthetic oligonucleotides as primers. Translation of the nucleotide sequence and alignment of the amino acid sequence were made by using DNASIS computer software (Hitachi Software Engineering Co. Ltd., Yokohama, Japan). A homology search was done with data base of GenBank (National Center for Biotechnology Information, Bethesda, MD) by using the software local alignment search tool (Altschul, S. F et al, Basic local alignment search tool. J. Mol. Biol. 215: 403-410. 1990) in the BLAST network service (NCBI). GenBank.

One of the positive clone which expressed a 44-kDa antigenic protein was named pHGE1221. The insert size of pHGE1221 was approximately 6.9 kb. The map of pHGE1221 contained five ORFs and the second ORF of 1239 bp encoding a 413- amino acid protein with a molecular mass of 43,739 Da including signal peptide. This protein was designated the P44 proteinThe nucleotide sequence, SEQ ID NO:l, of

20 ORF2 and the amino acid sequence, SEQ ID NO:2, encoded by ORF2 is shown in FIG. 1. The ORF1 contained 759 nucleotide base pairs encoding 253-amino acids without start codon. Two regions in the ORF1 were found to share the exact same sequences within ORF2. These conserved regions were named Rl and R2, which encoded 59- and 65-amino acids, respectively. B. p44 Polynucleotide Probe

A 1.2-kb p44 gene fragment generated by PCR from the clone pHGE1221 was labeled with [ P] dATP by the random primer method using a kit (Boehringer Mannheim, Indianapolis, IN) and the labeled fragment and used as a DNA probe to detect p44 homologous genes. Genomic DNA (200 ng) extracted from the purified HGE agent HZ strain was completely digested with 20 units of restriction endonucleases at 37°C for 4 h, electrophoresed, and transferred to Hybond-N+ nylon membrane (Amersham, Arlington Heights, IL) by a standard method . Hybridization was 16 h. The nylon sheet was washed in Ol.xSSC )lxSSC containing 0.15M sodium chloride and 0.015M sodium citrate) with 1% SDS at 55° C and hybridized probes were exposed to Hyperfllm (Amsersham) at -80° C. Genomic Southern blot analysis revealed more than 10 bands bound to thep44 gene probe, suggesting the existence of additional p44- homologous genes in the HGE agent genome.

EXAMPLE 2: Polynucleotides Encoding P44-2 Protein. P44-12 Protein. P44-15 Protein. P44-18 Protein, and P44-19 Protein. A. RT-PCR and Cloning of cDNAs

A pair of oligonucleotides used for RT-PCR (p3708 and p4257 as shown in Figure 12 and Table 2 below) was designed based on the conserved regions between DNA sequences of the p44 gene and a truncated /^-homologous gene downstream from tia.ep44. Total RNA was extracted from HL-60 cells infected with the HGE agent by using TRIzol reagent (GIBCO-BRL, Gaithersburg, Md.). The isolated RNA (3 μg) was heated at 70°C for 10 min and reverse transcribed in a 20-μl reaction mixture (0.5 mM deoxynuleoside triphophate mixture (dNTP), 200 U of Superscript II reverse transcriptase (GIBCO-BRL), 2 pmole of p4257 primer, and 3 mM MgCl₂) at 42°C for 50 min. PCR was performed in a 100-μl reaction mixture containing 2 μl of the cDNA product, 10 pmole of each of p3726 and p4257 primers, 0.2 mM dNTP mixture, 5 U of Taq DNA polymerase, and 1.5 mM MgCl , with 3 min of denaturation at 94°C followed by 30 cycles consisting of 1 min of denaturation at

21 94°C, 1 min of annealing at 52°C, and 2 min of extension at 72°C. To rule out contamination of DNA in the RNA preparation, RT-PCR without reverse transcriptase was carried out (negative control). The amplified RT-PCR products were cloned in a pCRII vector by using the TA Cloning Kit (Invitrogen Co., San Diego, Calif). Twenty five cDNA clones, which were randomly selected from the transformants, were sequenced by dideoxy chain termination method with an Applied Biosystems 373 DNA sequencer.

After cloning the amplicons, only five different nucleotide sequences were found in the 25 randomly selected cDNA. The five sequences of the cDNAs were represented as p44-2 cDNA, p44- 18cDN A, p44- 12 cDNA, p44- 15 cDN A, p44- 18 cDNA and p44-19 cDNA. A comparison of the deduced amino acid sequences among then proteins and polypeptides encoded by the five different cDNAs and the p44 gene revealed that a central region of approximately 94-amino acid residues corresponding to the 175th- to 269th- amino acid sequence of the P44 protein was hypervariable, and flanked regions of approximately 30 residues each was highly conserved (Figure 11). Within the hypervariable region, the highest amino acid sequence similarity was 32.8% between the P44-2 and P44-18 proteins, and the lowest similarity was 19.9% between P44-15 and P44 proteins. In comparison with flanking regions, the hypervariable region had higher hydrophilicity and antigenic index. The N-terminal amino acid sequence (HDDVSALETG) of the native 44-kDa protein was found in P44, P44-2 and P44- 12 proteins. A comparison of deduced amino acid sequences of these proteins revealed anidentical amino acid sequence consisting of 15 residues at their N terminus. It is believed that this sequence is a signal peptide. The alignment also showed that the N- and C-terminal portions are highly conserved among three Group 4 proteins (P44-2, P44-12 and P44), except existence of additional 34-amino acid residues at C terminus of P44-2 (Fig. 11). At N and C termini of both P44-15 and P44-18 proteins there is a short stretch of an amino acid sequence consisting of 8 to 28 residues without any homology to other Group 44 proteins including P44, P44-2 and P44-12 (Fig. 11). B. P44-2, p44-12, p44-14, p44-18, and p44- 19 probes

DNA probes specific to each of the cDNAs for Southern blotting were designed based on a comparison of deduced amino acid sequences among these cDNAs. The central hypervariable regions in each cDNA and in the p44 gene were amplified by PCR with primer pairs as shown in Table 2 below. The amplicons were

22 cloned into a pCRII vector. The DNA insert excised from each recombinant plasmid was labeled with [α-³²P]dATP by the random primer method with a kit (Amersham, Arlington Heights, 111.) and used as a probe to detect genes in the genome of the HGE agent. DNA Hybridization was performed in a Rapid Hybridization Buffer

(Amersham). The membrane was exposed to a Hyperfilm (Amersham). A single DNA band was detected in all restriction digestions tested. Since restriction enzymes used do not cut within ap44 gene and all cDNA clones, this suggests that each gene corresponding to the four probes is a single copy in the HGE agent genome. The probes specific to the cDNAs of P44-2 and P44-15 generated two or three bands, showing that two to three gene copies with sequences identical or highly homologous to those of the cDNAs of P44-2 or P44-15 exist in the HGE agent genome.

TheXbαl DNA fragments of the HGE agent, which were detected by genomic Southern blot analysis, were inserted into a pBluescript II KS (+) vector, and the recombinant plasmids were introduced into Escherichia coli DH5 . By using the colony hybridization method (29) with the specific DNA probes same as those used for Southern blot analysis, three positive clones were isolated and the DNA inserts were sequenced. The clones were designated pHGE3.0, pHGE3.4, and pHGE3.9 containing ehrlichial DNA fragments of 3.0, 3.4, and 3.9 kb, respectively. Sequencing of the 3.9- and 3.0-kb fragments revealed two complete ORFs of

1,275 bp and 1,173 bp encoding 425- and 391-amino acid proteins with Mr of 44,969 and 41,179, respectively. As expected, these ORFs (p44-2 andp44-12) contained the sequences identical to the cDNAs of P44-2 and P44-12, respectively (Fig. 2, 4The 3.4-kb DNA fragment contained an ORF of 834 bp encoding 278-amino acid protein with Mr of 29,387. This ORF (p44-15) included a sequence identical to P44-15 cDNA. T Qp44-15 did not have a universal start codon and lacked DNA sequence corresponding to 82-amino acid residues at the N terminus of the P44 protein. Consensus sequences of σ⁷⁰ promoter (AT-rich region about 10 base pairs upstream of the transcription start site; the -10 sequence) and Shine-Dalgarno sequence were found in the regions upstream from the start codon of p44-2 andp44-12. The nucleotide sequence(s) of the cDNAs and genes have been submitted to GenBank™/EBI Data Bank and assigned accession number(s) AF 135254, AF135255, AF135256, AF135257, AF135258, AF135259, AF132260, AF132261,

23 AF 132262, and AF 132263. The nucleotide sequences of the cDNAs (SEQ ID Nos 3, 5, 7, 9, and 11) and the amino acid sequences, SEQ ID Nos 4, 6, 8, 10, and 12, of the proteins and polypeptides, encoded by the cDNas are shown in Figures 2-6.

Table 2. Oligodeocynucleotides used in RT-PCR and PCR

Target Genes Primers Nucleotide Sequences Regions³ PCR Usage

(5'-3') (nt.) products (pb)

P3708 GCTAAGGAGTTAGCTTATGAT 41 1-432

P4257 AAGAAGATCATAACAAGCAT 936-960 549 Cloning of DNA

(hvf and hvr^b

P44-2 2hvf TACTGGTAGCCATGCTGACC 542-563

2hvr AATCACCCGTCTTCAGTGATG 769-789 231 Southern/Northern blot

P44-18 18hvf GGAGATTTCTAATTCCGGTAT 542-563

18hvf AGTCATTATTCGATTTAGACG 769-789 234 Southern/Northern blot

P44-12 12hvf TGATAAGAAGGTTTGTGATGG 542-563

12hvr AGAGCACATTAACGTTGTCAC 769-789 270 Southern blot

P44-15 15hvf GAAGGTTTGTAAGACGAAGGC 542-563

15hvr AGTTCGTGACAGGTTTTGGAG 769-789 240 Southern blot

P44-19 19hvf CATTGATGGGAAGATTTGTAAG 542-564

19hvr AGGTGAGCTTTGTTAGTTCTC 769-789 234 Southern blot

P44 44hvf GAAGGTTTGTAGTGGAAAGCA 542-563

44hvr ATGCTCCAACTACAATGCTAT 769-789 247 Southern/Northern blot

3' end of p44- 12 pnfl2 CAAGTTTGACTGGAACACTCC 218-239

(p44-12n)^c pnrl2 AACAATATCTTTACCAGAGG 478-498 280 Southern blot

5' end oϊp44-12 pcfl2 CTAAAGACCTAGTACAGGAGC 880-901

(p44-12C)^c per 12 GAGAGAGCTGATAACTCAACC 1072-1093 210 Southern blot a. Numbers correspond to the nucleotides in the p44 gene which was cloned in reference 34 (GenBank accession number: AF059181) and the primer positions are shown in Fig. 1. b. Primer pairs are used to amplify hypervariable regions of each of cDNA's. c. Probes p44-12N and p44-12C are specific to 5'- and 3'- end conserved regions oϊp44 homologous genes.

Example 3 P44 Protein and rP44 Polypeptide. Amino acid sequence analysis of the P44 protein indicated that it is a typical transmembrane protein which contains alternative hydrophilic and hydrophobic motifs with a signal peptide of 37 amino acid residues. Searching of the GenBank database revealed amino acid sequence similarity between the HGE agent P44 and Anaplasma marginale, a bovine intraerythocytic bacteria, major surface protein 2 (66%) similarity; 44% identity).

The entire P44 protein was expressed by cloning whole p44 gene into pET30a vector. However, the expression level is low.

To effectively overexpress antigenic epitopes of recombinant P44, the deduced amino acid sequences based on the sequences of a plasmid pHGE1221,

24 which was positive in immunoscreening and contained an ORF encoding the proposed P44, were analyzed with DNASTAR computer software (DNASTAR Inc., 1228 South Park Street, Madison.). Several motifs with high antigenic index and probability of surface exposure were found in NH2-terminal portion. Therefore, the primers were designed to amplify the DNA sequence encoding for a 219-amino acid polypeptide from NH₂-terminus including 8 amino acid residues of signal peptide, and were prepared by BioServe (Laurel, MD). The 5' oligonucleotide primer consists of the DNA sequence coding for the NH₂-terminal region of the HGE agent P44 and the Ncol restriction sites (underlined) [5'-CGCCATGGCTGGGAGTGATGTCA-3'] and the 3' oligonucleotide primers consists of the DNA sequence coding for the amino acids from No. 243 to 247 with the addition of a stop codon (TAG, boldface) and a EcoRI restriction site (underlined) [5'-GCGAATTCTACGCACTACCATTACTCA- 3') (Fig. 1). PCR amplification was carried out with a Perkin-Elmer Cetus DNA Thermal Cycler (Model 480) by using standard procedures. The 657-bp amplified product containing approximately half of the P44 gene was digested with Ncol and EcoRI, and ligated into dephosphorylated Ncol- and EcoRI -digested pET30a expression vector (Novagen, Inc., Madison, WI). The recombinant plasmid was designated as pEP44. E. coli NovaBlue strain (Novagen, Inc.) was transformed with recombinant pET30a. A plasmid preparation of pEP44 from transformed NovaBlue was then used to transform E. coli BL21(DE3)pLysS.. The purification of rP44 protein was performed by using the His-Bind Buffer Kit (Novagen, Inc.) according to the manufacturer's instruction. The expressed partial recombinant P44 antigenic polypeptide (rP44) purified by affinity chromatography was 35 kDa in SDS-PAGE. It was a fusion polypeptide in which a 44-amino acid sequence including the His tag peptide derived from pET30a expression vector was located at the NH -terminus.

Example 3 : Detecting HGE Infection with the rP44 Polypeptide A. Western immunoblot analysis

The affinity-purified rP44 polypeptide and purified HGE organisms were used for Western immunoblot analysis of patient sera. All HGE patient sera No. 2, 3, 4, 6, 11, 13 , 21 and 22 were collected in the patients from Westchester County Medical Center in New York State. The diagnosis was confirmed by using PCR, IFA testing and culture islation. Patient sera No. 21-1, -2, -3, -4 and -5 were collected at different stages of illness (7/25/95-first acute stage, 8/24/95 -convalescent

25 stage, 10/5/95, 6/13/96, and 7/17/97-second acute stage) from a patient suspected of having re-infection with the HGE agent.

Purified HGE agent and rP44 polypeptide separated by 10 % PAGE were transferred to a nitrocellulose sheet, then immersed in TBS buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4]) containing 0.05% Tween 20 (T-TBS) and 5% milk at 4° C overnight to saturate protein-binding site. The nitrocellulose membrane was incubated with the primary mouse, human or horse sera at a 1 : 1,000 dilution and then with peroxidase-conjugated affinity purified anti-human, anti-horse or anti-mouse immunoglobulin G (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) at a 1 : 1,000 or 1 :2,000 dilution. The peroxidase-positive bands were detected by immersing the sheet in a developing solution (70 mM sodium acetate, pH 6.2) containing 0.005%) diaminobenzidine tetrahydrochloride (Nacalai Tesque, Inc., Kyoto, Japan) and 0.03%> H₂O₂ at room temperature for 5 min. The enzyme reaction was terminated by washing the sheet in 0.1 M H₂SO₄. Horse anti-HGE serum and eleven sera from the HGE patients specifically recognized the rP44 polypeptide. All these patients were confirmed to have HGE by PCR and/or culture isolation previously. Five sera collected over 2 year period at different stages of illness from a patient (No. 21) reacted with rP44. This patient was suspected of having re-infection with HGE agent, since IFA titer at 8 days before 21-5 serum collection date was 1 :40. The result indicates that regardless of stages of infection or reinfection P44 is the major antigen recognized by the patient sera. The horse anti-E. equi serum strongly reacted with both native 44-kDa protein in whole cell organisms and rP44 polypeptide. Human anti-£. chaffeensis and rabbit anti-5. burgdorferi did not recognize rP44. This indicates that rP44 polypeptide can be used as a testing antigen to differentiate infection with HGE agent from infection with E. chaffeensis or B. burgdorferi.

B. Dot immunoblot assay

The affinity-purified rP44 polypeptide in TBS was blotted onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and then immersed T- TBS containing 5%> milk at room temperature for 30 min, air dried, and stored at -20° C until required. Based on the results of quantitative analysis, 0.5 ug per dot of rP44 was used in the immunoassay to assay the clinical specimens. For immunoassay, sera

26 to be tested were diluted at 1:1,000 in T-TBS containing 5% milk and incubated with the antigen dots for 1 h at room temperature After being washed three times with T- TBS, the nitrocellulose sheets were incubated with peroxidase-conjugated affinity- purified anti-human IgG (Kirkegaard & Perry) at 1 :2,000 dilution. The peroxidase- positive bands were detected by immersing the sheet in a developing solution as described in the Western immunoblot section. The color density was measured using background correction of ImageQuant program Molecular Dynamics, Sunnyvale, CA).

To determine the optimal amount of antigen of per dot and dilution of patient sera for dot blot immunoassay, nitrocellulose membrane strips, each having eight dots containing a different amount of purified rP44 antigen, were incubated with five different sera with IFA titers in a range from 1 : 2,560 to <1 : 20. There was a positive correlation between color densities of dot reactions and IFA titers when > 50 ng of recombinant antigen was used per dot. No reaction was detected using negative control sera (IFA titer <1 : 20). Since the difference of color density among sera with different IFA titers was quite distinct, and the color density progressively increased (especially at antigen amounts of 0.25 ug to 1 ug per dot), 0.5 ug per dot was used in the following assays. This amount of protein can distinguish both high and low titers. A total of 25 clinical patient sera with different IFA titers (from 1 :2,560 to < 1 : 20) were examined by dot blot immunoassay using 0.5 ug per dot of affinity-purified rP44. As shown in Fig. 6A, the color density of the each dot is highly correlated with the IFA titer. In five tested sera with an IFA titer < 1 : 20, the color density of one dot can be clearly distinguished from other negative sera by the naked eye. This sera was collected at acute phase from the patient No. 3 who was nested PCR and culture isolation positive for the HGE agent. This patient developed a convalescent IFA titer of 1 : 640 . The remaining four < 1 : 20 sera were derived from patients who were negative by convalescent serum IFA, PCR, or isolation. These results indicate that the dot immunoassay using rP44 protein provides a simpler serodiagnosis of HGE infection than the IFA test.

Example 4: Oligopeptides specific to the P44 protein, the P44-2 Protein, the P44- 15 protein, the P44- 18 protein, and the P44-19 Protein

Synthetic oligopeptides specific to each Group 44 Protein were prepared. Such oligopeptides were identified, by using DNASTAR program (DNASTAR Inc.,

27 Madison, Wis.) and selected from the hypervariable region of the P44 protein, the P44-2 protein, the P44-15 protein and the P44-18 protein, and the P44-19 protein. Two oligopeptides with sequences of GHSSGVTQNPKLFST and KNQKSSDTDTGVEKA were synthesized (Alpha Diagnostic, San Antonio, Tex.) and named Pep2 and Pepl 8, respectively. Pep2 and Pepl 8 were conjugated to keyhole limpet hemocyanin (KLH) and and the resulting KLH-conjugated oligopeptide Pep2 and Pep 18 were used as antigens in a dot blot assay. The assay was carried out as described above in example 3. Convalescent sera tested were from two patients (no. 7, and no. 11) with clinical signs of HGE was confirmed by IFA and PCR. The results showed that oligopeptides are useful for detecting antibodies to the HGE causative agent in serum from patients afflicted with HGE.

Example 5: anti-rP44 polyclonal antiserum.

Hyperimmune anti-rP44 polyclonal antiserum was generated by intraperitoneal immunization of BALB/c male mice (6 weeks old) with rP44 affinity- purified as described above. Primary immunization of each animal was with 15 ug of purified rP44 in Freund's complete adjuvant. Two boosts of 10 ug each of rP44 in Freund's incomplete adjuvant followed at day 14 and 28. Hyperimmune serum was obtained 14 days after last boost The mouse antiserum specific to rP44 was used in a western immunoblot analysis. The antiserum strongly reacted with 44- and 42-kDa proteins in all isolates tested, and recognized a 27-kDa protein in No. 2 and 6 isolates, and a 15-kDa protein in No.2, 3, 6 and USG isolates.

Example 6 Antisera to KLH-conjugated oligopeptide Pep2 and Pep 18

Antisera against the KLH-conjugated oligopeptide Pep2 and Pep 18 were generated by immunization of a rabbit and a mouse with keyhole limpet hemacyanin (KLH) (PIERCE, Rockford, Ill.)-conjugated synthetic oligopeptide Pep2 and Pepl 8, respectively.

The resulting sera was used in a Western immunoblot analysis of the HGE agent lysate as described in example 3. The mouse anti-recombinant rP44 polypeptide anti- serum was used as a positive control. For double IFA staining, a lissamine rhodamine (LR)-labeled goat anti-rabbit IgG and fluorescein isothiocyanate

28 (FITC) -labeled goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) were used as secondary antibodies. The rabbit anti-Pep2 and the mouse anti-Pep 18 sera reacted with a single band of 44 kDa and 43 kDa, respectively in the HGE agent lysate, respectively . In host cells the HGE agent resides in membrane-bound inclusions that appear as clusters of organisms like mulberries, therefore, are called morulae. The morulae are considered as microcolonies derived from a single organism. Six morulae in an HL-60 cell which reacted with the mouse anti-rP44 serum were also recognized by the rabbit anti-Pep2 serum. All organisms were double labeled in 100 infected cells scored in three independent labeling experiments. This means ap44-2 gene is probably expressed in all of the HGE agent organisms cultivated in HL-60 cells. With a mouse anti-Pep 18 serum even at low (1:5) dilution, the immunofluorescence labeling of organisms or morulae was extremely weak.

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Claims

CLAIMSWhat is claimed is:

1. An isolated outer membrane protein of the human granulocytic ehrlichiosis causative agent, said protein comprising: a) a hypervariable region of about 94 amino acids, wherein said hypervariable region has an isolelectric point of from about 7.1 to about 9.2 and an Mr of from about 8.5kDa to about 11 kDa; b) a first conserved region of about 52 amino acids linked directly or through a linker of from 1 to about 10 amino acids to the N-terminus of the hypervariable region; c) a second conserved region of about 56 amino acids linked directly or through a linker of from 1 to about 10 amino acids to the C teminus of said hypervariable region; wherein said hypervariable region has a hydrophilicity index of from about 0.45 to 0.95, an antigenic index of from about 0.65 to 1.5 , and a surface probability index of from about 1.0 to 1.55.

2. The isolated protein of claim 1 wherein said isolated protein is immunoreactive with antiserum from patients with human granulocytic ehrlichiosis.

3. The isolated protein of claim 1 wherein said hypervariable region comprises: a) a first semiconserved region near the amino terminus thereof, said first semiconserved region comprising the sequence Xl-X2-Lys-X3-Cys; wherein XI is Asp or Gly , X2 is Gly or Lys , and X3 is Val or He; b) a second semiconserved region at the carboxy terminus thereof, said second second semiconserved region comprising the sequence Asn-Ala-Xl-X2-X3-Ala-X4- X5-Leu-X6-X7; wherein XI is Asn, Thr, Lys, or Glu, X2 is Ala or Lys, X3 is Met or Val; X4 is Lys, Gly, or Thr; X5 is Asp or Glu; X6 is Val or Thr, and X7 is Gin or Lys; and

30 c) a third semiconserved region between the first and second semiconserved region, said third semiconserved region comprising the sequence Lys-Xl-Trp-Pro- Thr-X2; wherein XI is Asn or His and X2 is Gly, Thr, or Ser.

4. The isolated protein of claim 1 wherein said first conserved region has an amino acid sequence which is at least 95% identical to the amino acid sequence of the first conserved region of a protein selected from the group consisting of: the P44 protein, the P44-2 protein, the P44-12 protein, the P44-15 protein, the P44-18 protein, and the P44-19 protein, and wherein said second conserved region has an amino acid sequence which is at least 95%) identical to the amino acid sequence of the second conserved region of a protein selected from the group consisting of: the P44 protein, the P44-2 protein, the P44-12 protein, the P44-15 protein, the P44- 18 protein, and the P44-19 protein.

5. An isolated outer membrane protein of the causative agent to human granulocytic ehrlichiosis, wherein said protein is selected from the group consisting of a P44 protein or a variant thereof; a P44-2 protein or a variant thereof; a P44-12 protein or a variant thereof; a P44-15 protein or a variant thereof, a P44-18 protein or a variant thereof; and a P44-19 protein or a variant thereof.

6. The isolated protein of claim 5 wherein said protein comprises an amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group consisting of the amino acid sequence SEQ ID NO:2, shown in Figure 1; the amino acid sequnece, SEQ ID NO: 4, shown in Figure 2; the amino acid sequence, SEQ ID NO:6, shown in Figure 3; the amino acid sequence, SEQ ID NO:8, shown in Figure 4; the amino acid sequence, SEQ ID NO: 10 shown in Figure 5; and the amino acid sequence, SEQ ID NO: 12, shown in Figure 6.

7. The isolated protein of claim 6 wherein said protein is a mature protein and the amino acid sequence lacks the signal sequence.

8. An isolated polynucleotide encoding the protein of claim 1.

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9. The isolated polynucleotide of claim 8 wherein said polynucleotide comprises a sequence which encodes a protein selected from the group consisting of a P44 protein or a variant thereof; a P44-2 protein or a variant thereof; a P44-12 protein or a variant thereof; a P44-15 protein or a variant thereof, a P44-18 protein or a variant thereof; and a P44-19 protein or a variant thereof.

10. The isolated polynucleotide of claim 9 wherein said polynucleotide encodes an amino acid sequence which is at least 90%> identical to an amino acid sequence selected from the group consisting of the amino acid sequence SEQ ID NO:2, shown in Figure 1; the amino acid sequence, SEQ ID NO: 4, shown in Figure 2, the amino acid sequence, SEQ ID NO:6, shown in Figure 3; the amino acid sequence; SEQ ID NO:8, shown in Figure 4; the amino acid sequence, SEQ ID NO: 10, shown in Figure 5; and the amino acid sequence, SEQ ID NO: 12, shown in Figure 6.

11. The isolated polynucleotide of claim 10 wherein said polynucleotide comprises a sequence which is at least 90%> identical to a sequence selected from the group consisting of the nucleotide sequence, SEQ ID NO:l, as shown in Figure 1; the nucleotide sequence, SEQ ID NO:3, as shown in Figure 2, the nucleotide sequence, SEQ ID NO:5, as shown in Figure 3; the nucleotide sequence, SEQ ID NO:7, as shown in Figure 4; the nucleotide sequence, SEQ ID NO:9, as shown in Figure 5; and the nucleotide sequence, SEQ ID NO:l 1, as shown in Figure 6.

12. A polypeptide of from 12 to 250 amino acids for producing antibodies immunospecific for one or more Group 44 proteins, said polypeptide comprising a sequence which is at least 98%> identical to a portion of an amino sequence selected from the group consisting of the amino acid sequence, SEQ ID NO:2, shown in Figure 1 ; the amino acid sequence, SEQ ID NO: 4, shown in Figure 2; the amino acid sequence, SEQ ID NO:6, shown in Figure 3; the amino acid sequence; SEQ ID NO:8, shown in Figure 4; the amino acid sequence, SEQ ID NO:10 shown in Figure 5; and the amino acid sequence, SEQ ID NO: 12, shown in Figure 6.

13. The polypeptide of claim 12 wherein said polypeptide comprises a sequence which is identical to a sequence in the hypervariable region of a protein selected from

32 the group consisting of the P44 protein, the P44-2 protein, the P44-12 protein, the P44- 15 protein, the P44- 18 protein, and the P44- 19 protein.

14. The polypeptide of claim 13, wherein said polypeptide comprises the sequence of amino acid 30 through amino acid 248 of the P44 protein or the sequence of amino acid 38 through amino acid 248 of the P44 protein.

15. The polypeptide of claim 13 wherein said polypeptide comprises a sequence selected from the group consisting of: LSNGSAEAAHKYLSK, SEQ ID NO:,

GHSSGVTQNPKLFST, SEQ ID NO:,

GKKSGDNGSLADYTD, SEQ ID NO:;

PLYSDETHTKGASEGR, SEQ ID NO:;

KNQKSSDTDTGVEKA, SEQ ID NO:, and TGSNKYGTGTNSGELT, SEQ ID NO: .

16. An antibody which binds to a protein selected from the group consisting of the P44 protein, the P44-2 protein, the P44-12 protein, the P44-15 protein, the P44-18 protein, and the P44-19 protein.

17. A method for detecting antibodies to the HGE agent in a bodily sample of a patient, said method comprising: a) contacting said bodily sample with a Group 44 protein or an antigenic fragment thereof; and b) assaying for the formation of an antigen-antibody complex between antibodies in said sample and said Group 44 protein or said antigenic fragment.

18. A kit for diagnosing human granulocytic ehrlichiosis in a patient, said kit comprising one or more Group 44 proteins or antigenic fragments thereof.

19. The kit of claim 18 wherein said kit further comprises a biomolecule, for detecting interactions between the isolated Group 44 protein or antigenic fragment thereof.

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20. The kit of claim 18 wherein said kit comprises a panel of Group.44 proteins or antigenic fragments thereof.

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