AU8154801A - Compositions and methods for the prevention and diagnosis of human granulocytic enrlichiosis - Google Patents

Description

1

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Actual Inventors: Address for Service: Yale University Erol FIKRIG Stephen W BARTHOLD Jacob IJDO Wei SUN CULLEN CO., Patent Trade Mark Attorneys, 239 George Street, Brisbane, QId. 4000, Australia.

0 0 0 Invention Title: Compositions and Methods for the Prevention and Diagnosis of Human Granulocytic Ehrlichiosis Australian Patent Application No. 47416/97 Details of Associated Applications: filed 24 March 1999 The following statement is a full description of this invention, including the best method of performing it known to us: la COMPOSITIONS AND METHODS FOR THE PREVENTION AND DIAGNOSIS

OF

HUMAN GANIULOCYTIC

EHRLICHIOSIS

This application claims priority under U.S.C. 120 from pending United States provisional application Serial Number 60/027,180, filed October 1, 1996.

This invention was made with government support under Grant numbers AI 26815, AI 37993 and AI 39002 awarded by the National Institutes of Health.

The government has certain rights in the invention.

*t CTECHNICAL FIELD OF THE INVENTION *This invention relates to compositions and methods useful for studying the pathogenicity of and for the prevention, treatment and diagnosis of human granulocytic ehrlichiosis

(HGE).

More particularly, this invention relates to polypeptides and DNA sequences which encode them, from 20 the agent of HGE, referred to herein as "aoHGE" Such polypeptides and DNA sequences are useful to detect the presence of aoHGE in humans, to diagnose human granulocytic ehrlichiosis and related disorders caused by aoHGE infection, and to elicit an immune response 2 which is effective to prevent or lessen the severity, for some period of time, of aoHGE infection. Also within the scope of this invention are antibodies directed against aoHGE polypeptides, compositions including vaccines comprising the antibodies.

This invention also relates to vaccines comprising aoHGE, one or more of the aoHGE polypeptides or antibodies of this invention. Also within the scope of this invention are diagnostic kits comprising the aoHGE polypeptides, DNA sequences encoding them or antibodies of this invention.

This invention also relates to methods for selecting protective aoHGE polypeptides and antibodies.

Methods for using the aforementioned polypeptides,

DNA

sequences and antibodies are also within the scope of this invention.

BACKGROUND OF THE INVENTION Human ehrlichioses are emerging zoonotic infections caused by gram-negative obligate 20 intracellular bacteria of the genus Ehrlichia

[J.S.

Dumler et al., "Ehrlichial diseases of humans: emerging tick-borne infections," Clin. Infect. Dis., 20, pp.

1102-10 (1995); J. Dumler and D. Walker, "Emergence of the Ehrlichioses as Human Health Problems," Emerging 25 Infectious Diseases, 2, pp. 18-29 (1996)]. Two distinct human ehrlichioses occur in the United States: a human monocytic ehrlichiosis, caused by Ehrlichia chaffeensis, which specifically infects monocytes, and human granulocytic ehrlichiosis, which infects granulocytes.

3 The causative agent of human granulocytic ehrlichiosis is a recently identified bacteria of the genus Ehrlichia which has not yet been named. It is sometimes referred to as E. microti or as "the agent of HGE" Telford et al., "Perpetuation of the Agent of Human Granulocytic Ehrlichiosis In a Deer Tick- Rodent Cycle," Proc. Natl. Acad. Sci. USA, 93, pp.

6209-6214 (1996)]. The Ehrlichia which causes human granulocytic ehrlichiosis will be referred to herein as "aoHGE." Although many veterinary ehrlichioses have been described over the last several decades, human ehrlichioses have only recently been characterized.

E.

chaffeensis was discovered in 1990 and HGE was first described in 1994 Chen et al., "Identification of a Granulocytotropic Ehrlichia Species As the Etiologic Agent of Human Disease," J. Clin. Microbiol., 32, pp.

589-595 (1994)]. Since 1994, more than 200 cases of human granulocytic ehrlichiosis have been documented, 20 predominantly in upper midwestern and northeastern states, but also in the northwest Walker et al., "Emergence of the Ehrlichioses as Human Health SProblems," Emerging Infectious Diseases, 2, pp. 18-29 (1996)].

Human granulocytic ehrlichiosis is associated with a wide range of clinical symptoms. The illness is most commonly char -terized by influenza-like symptoms .(including fever, myalgia and headache), leukopenia, anemia, thrombocytopenia and elevated serum transaminase levels Dumler et al., "Ehrlichial Diseases of Humans: Emerging Tick-borne Infections," Clin. Infect. Dis., 20, pp. 1102-1110 (1995)]. The 4 spectrum of symptoms ranges from undiagnosed, subclinical infection to severe disease including gastrointestinal and pulmonary hemorrhage and death.

Furthermore, in some cases of human granulocytic ehrlichiosis, opportunistic infections have been demonstrated, suggesting altered neutrophil function as well as possible defects in acquired immune responses Walker et al., Emerging Infectious Diseases, supra]. The detection of ehrlichial DNA by PCR twentyeight days after the onset of symptoms in a patient who was not treated with antibiotics suggests that the bacteria may persist within the host Walker et al., Emerging Infectious Diseases, supra].

Human ehrlichioses are tick-borne infections.

Although many vertebrates are potentially infected with aoHGE, the white-footed mouse (Peromyscus leucopus) has been identified as the major animal reservoir for aoHGE Telford, III et al., "Perpetuation of the Agent .of Human Granulocytic Ehrlichiosis In a Deer Tick- 20 Rodent Cycle," Proc. Natl. Acad. Sci. USA, 93, pp.

6209-6214 (1996) The tick vector has been shown to be Ixodes scapularis (also referred to as Ixodes dammini) in the Ixodes ricinus complex IS. et al., Proc. Natl. Acad. Sci. USA, 93, supra]. Ticks acquire aoHGE by feeding on an infected host. Humans are infected by the bite of infected ticks. Not unexpectedly, the disease is prevalent in regions of the country where Lyme disease and babesiosis, diseases :also associated with I. scapularis, are common [L.A.

Magnarelli et al., "Coexistence of antibodies To Tickborne Pathogens of Babesiosis, Ehrlichiosis and Lyme Borreliosis in Human Sera, J. Clin. Microbiol., 33, pp.

5 3054-3057 (1995)]. In endemic areas of Connecticut and in Westchester County in New York, up to 50% of ticks collected may carry aoHGE and approximately 20% may simultaneously be infected with B. burgdorferi (the agent of Lyme disease) Fish, unpublished data).

Diagnosis of human granulocytic ehrlichiosis is difficult. Currently, the most definitive method for diagnosing acute infection is identification of the organism, and the characteristic morulae, within the cytoplasm of granulocytes in a peripheral blood smear.

However, because less than 1% of the granulocytes may be infected and because the patient usually exhibits leukopenia, detection of the intracytoplasmic inclusions is unreliable and results in a substantial number of false negative diagnoses.

Direct cultivation of aoHGE in HL-60 cells and the development of granulocytic morulae in the peripheral blood of mice inoculated with patient serum has recently been reported et al., supra.

S: 20 However, these methods are expensive and laborintensive and have not yet been used for diagnosis.

PCR analysis based on aoHGE specific 16S ribosomal

DNA

sequences shows some promise but also may produce false-positive and false-negative results. Nor does PCR indicate the existence of viable organisms.

Immunofluorescent serology using as antigen cells infected with E. equi, to which aoHGE is closely related, has been moderately successful for diagnosing S: aoHGE. However, serologic testing is not yet 30 standardized, and results may vary between laboratories and commercial kits, causing false negative and, more commonly, false positive results.

6 In addition, the disease often goes unrecognized, as the ticks are small and easy to miss.

Unlike Lyme disease, aoHGE infection does not produce a characteristic rash. Accordingly, there is an urgent need to identify aoHGE proteins for diagnostic use.

No reliable laboratory model for human granulocytic ehrlichiosis has been developed. in order to study the pathogenesis of human granulocytic ehrlichiosis and to develop agents and methods.to prevent and diagnose the disease, a cost-effective animal model which mimics the human disease and the immune response to selected aoHGE antigens is required.

Human granulocytic ehrlichiosis is a potentially fatal disease if antibiotic therapy is not initiated in a timely fashion. As prevention of tick infestation is imperfect, and human granulocytic ehrlichiosis may be .missed or misdiagnosed when.it does appear, there is a great need for vaccines against the disease. No aoHGE polypeptides for use in vaccines have 20 been identified to date. Thus, there exists an urgent 0. need for the determination of the antigens of aoHGE and related proteins which are able to elicit a protective immune response.

In addition, human granulocytic ehrlichiosis is becoming a recognized human health problem in endemic areas and the incidence of the disease is expected to rise over the next several years. Human granulocytic ehrlichiosis is transmitted by ticks that carry a number of different pathogens including Babesia 30 microti, the agent of babesiosis, and Borrelia burgdorferi, the agent of Lyme disease. A greater understanding of human granulocytic ehrlichiosis may 7 provide insight into clinical symptoms that result in misdiagnosis of other tick-borne infections, most notably Lyme disease.

DISCLOSURE OF THE INVENTION The present invention solves the problems referred to above by providing means to study, diagnose, prevent and treat aoHGE infection and human granulocytic ehrlichiosis and related disorders caused by aoHGE infection. More particularly, this invention provides aoHGE polypeptides, DNA sequences that encode the polypeptides, antibodies directed against the polypeptides and compositions and methods comprising the aoHGE polypeptides, DNA sequences and antibodies.

This invention further provides a single or multicomponent vaccine comprising aoHGE or one or more aoHGE polypeptides or antibodies of this invention.

This invention provides DNA sequences that code for the aoHGE polypeptides of this invention, recombinant DNA molecules that are characterized by 20 those DNA sequences, unicellular hosts transformed with those DNA sequen.;3 and molecules, and methods of using those sequences, molecules and hosts to produce the aoHGE polypeptides and multicomponent vaccines of this invention. DNA sequences of this invention are also 25 advantageously used in methods and means for the diagnosis of aoHGE infection and human granulocytic ehrlichiosis.

Also within the scope of this invention are S" diagnostic means and methods characterized by aoHGE polypeptides, DNA sequences encoding them or antibodies directed against these polypeptides. These means and 8 methods are useful for the detection of human granulocytic ehrlichiosis and aoHGE infection. They are also useful in following the course of treatment against such infection. In patients previously inoculated with the vaccines of this invention, the detection means and methods disclosed herein are also useful for determining if booster inoculations are appropriate.

This invention further provides an immunocompetent, non-human, mammalian model for human granulocytic ehrlichiosis for use in studying the pathology of the disease and in screening for aoHGE polypeptides and antibodies that are capable of protecting a treated subject against aoHGE infection or human granulocytic ehrlichiosis and related disorders caused by aoHGE infection.

Finally, this invention also.provides methods for the identification and isolation of additional aoHGE polypeptides, as well as compositions and methods comprising such polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-D depicts the DNA and amino acid sequences of the E6 polypeptide of aoHGE isolate NCH-1 (SEQ ID NOS: 1 and 2) 25 Figure 2A-D depicts the DNA and amino acid sequences of the E7 polypeptide of aoHGE isolate NCH-1 (SEQ ID NOS: 3 and 4).

Figure 3 depicts the amino acid sequence of the 44-1 polypeptide from the 44 kDa protein of aoHGE isolate NCH-1 (SEQ ID NO: 9 Figure 4 depicts the amino acid sequence of the 44-2 polypeptide from the 44 kDa protein of aoHGE isolate NCH-1 (SEQ ID NO: 6).

Figure 5A-B depicts the DNA sequence of the 44 kDa protein of aoHGE isolate NCH-1 (SEQ ID NO: Figure 6 depicts the amino acid sequence of the 44 kDa protein of aoHGE isolate NCH-1 (SEQ ID NO: and indicates the position of the 44-1 and 44-2 polypeptides.

Figure 7 depicts the amino acid sequence of the 80-1 polypeptide from the 80 kDa protein of aoHGE isolate NCH-1 (SEQ ID NO: x indicates the amino acid positions in which a characteristic chromatogram was not obtained.

Figure 8 shows the ELISA and IFA antibody titers in sera from aoHGE infected mice to antigen at 10, 17 and 24 days after tick-borne infection. Titers are expressed as the last positive 2-fold reciprocal dilution of serum, 4 mice/interval.

20 Figure 9 shows immunoblot results of serum samples from 18 aoHGE patients. Titers 1:80 and above were considered positive. ND: not done. a: acute serum. c: convalescent serum. For patient 18, there were two convalescent sera, one at 3 weeks and one at 6 weeks after tick bite.

Figure 10 depicts the 5' and 3' primers used to amplify the e6 gene (SEQ ID NOS: 8 and The underlined portion of the 3' primer indicates the inserted XhoI site. The underlined portion of the 30 primer indicates the inserted EcoRI site.

Figure 11A-C depicts the DNA sequence of the eM4 polypeptide of aoHGE isolate NCH-1 (SEQ ID NO: 12) 10 Figure 12A-B depicts the amino acid sequence of the eM4 polypeptide of aoHGE isolate NCH-1 (SEQ ID NO: Figure 13A-B depicts the DNA sequence designated E5-3A (SEQ ID NO: which was isolated from a genomic aoHGE isolate NCH-1 library using oligonucleotide probes derived from the 44-kDa DNA sequence (SEQ ID NO: Figure 14A-B depicts the DNA sequence designated E5-3B (SEQ ID NO: which was isolated from a genomic aoHGE isolate NCH-1 library using oligonucleotide probes derived from the 44-kDa DNA sequence (SEQ ID NO: Figure 15A-B depicts the DNA sequence designated E5-5A (SEQ ID NO: which was isolated from a genomic aoHGE isolate NCH-1 library using oligonucleotide probes derived from the 44-kDa DNA sequence (SEQ ID NO: Figure 16 depicts the DNA sequence designated 20 E5-5B (SEQ ID NO: which was isolated from a genomic aoHGE isolate NCH-1 library using oligonucleotide probes derived from the 44-kDa DNA sequence (SEQ ID NO: Figure 17A-C depicts the DNA sequence designated E5-6 (SEQ ID NO: which was isolated from a genomic aoHGE isolate NCH-1 library using oligonucleotide probes derived from the 44-kDa DNA sequence (SEQ ID NO: Figure 18 is a matrix plot depicting a region .i 30 of homology between approximately nucleotides 200-400 and 600-1000 the ES-3B DNA sequence and approximately 11 nucleotides 400-600 and 900-1200, respectively, of the 44-kDa DNA sequence.

Figure 19 is a matrix plot depicting a region of homology between approximately nucleotides 300-650 of the E5-5B DNA .sequence and approximately nucleotides 900-1200 of the 44-kDa DNA sequence.

Figure 20 is a matrix plot depicting a region of homology between approximately nucleotides 1000-1400 and 1700-1900 of the E5-5B DNA sequence and approximately nucleotides 400-600 and 900-1300 of the 44-kDa DNA sequence.

DETATTLED DESCRPTION OF THE INVENTION This invention relates to aoHGE polypeptides and DNA sequences encoding them, antibodies directed against those polypeptides, compositions comprising the polypeptides, DNA sequences or antibodies, and methods for identifying additional aoHGE polypeptides and antibodies and methods for the detection, treatment and prevention of human granulocytic ehrlichiosis and S 2. 20 related disorders -aused by aoHGE infection.

More specifically, in one embodiment, this invention provides a 40-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a 44-kDa aoHGE polypeptide and fragments 44-1 and 44-2 thereof, .nd compositions and methods comprising the polypeptide and fragments.

in another embodiment, this invention provides a 65-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

11 12 In another embodiment, this invention provides a 80-kDa aoHGE polypeptide and the 80-1 fragment thereof, and compositions and methods comprising the polypeptide and fragment.

In another embodiment, this invention provides a 94-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a 105-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a 110-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a 115-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a 125-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.

20 In another embodiment, this invention provides an E6 polypeptide and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides an E7 polypeptide encoded and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides an eM4 polypeptide encoded and compositions and methods comprising the polypeptide.

In another embodiment, this invention 30 provides an E5-3A, E5-3B, E5-5A, E5-5B and E5-6 DNA ssequences, and compositions and methods comprising them.

13 The preferred compositions and methods of each of the aforementioned embodiments are characterized by immunogenic aoHGE polypeptides. As used herein, an "immunogenic aoHGE polypeptide" is any aoHGE polypeptide that, when administered to an animal, is capable of eliciting a corresponding antibody. In particular, immunogenic aoHGE polypeptides are intended to include additional aoHGE polypeptides which may be identified according to the methods disclosed herein.

The most preferred compositions and methods of each of the aforementioned embodiments are characterized by aoHGE polypeptides which elicit in treated animals, the formation of an immune response which is effective to prevent or lessen the severity, for some period of time, of aoHGE infection.

In another preferred embodiment, this invention provides a vaccine comprising aoHGE, one or more aoHGE polypeptides of this invention or one or more antibodies directed against aoHGE or a polypeptide 20 of this invention.

All of the aoHGE polypeptides provided by this invention, and the DNA :equences encoding them, are substantially free of an Ehrlichia bacterium or fragments thereof, and thus may be used in a variety of applications without the risk of unintentional infection or contamination with undesired Ehrlichia components. Accordingly, the aoHGE polypeptides of this invention are particularly advantageous in compositions and methods for the diagnosis and 30 prevention of aoHGE infection.

As used herein, a polypeptide that is "substantially free of an Ehrlichia bacterium or 14 fragments thereof" is a polypeptide that, when introduced into an animal susceptible to aoHGE infection, fails to produce any Ehrlichia bacteria detectable by microscopic examination of a blood or tissue smear, by PCR amplification using aoHGE specific primers, by in situ hybridization with aoHGE specific probes or by any other method for detecting aoHGE infection. Alternatively, it is a polypeptide that is detectable as a single band on an immunoblot probed o0 with polyclonal anti-aoHGE anti-serum.

In another preferred embodiment, this invention provides immunodominant aoHGE polypeptides.

As used herein, an "immunodominant aoHGE polypeptide" denotes an aoHGE polypeptide, or derivative thereof, that is recognized by antibodies elicited by infection with aoHGE, but which is substantially less reactive with antibodies elicited by infection with other bacteria. As used herein, an "immunodominant region" of an aoHGE polypeptide denotes a region of an aoHGE 20 polypeptide, or derivatives thereof, that is recognized by antibodies elicited by aoHGE infection but that is substantially less reactive than the full-length aoHGE protein when reacted with antibodies elicited by infection with other bacteria.

As used herein, "substantially less reactive" means, that when reacted in an ELISA or on an immunoblot with patient serum which contains antibodies elicited by infection with bacteria other than aoHGE, the level of reactivity would be at least 10-fold lower 30 than the level of reactivity with serum from patients infected with aoHGE. More preferably, the immunodominant polypeptides would be bound at a level 15 at least 50-fold lower than the level of binding that occurs with antibodies in sera from patients infected with aoHGE. Most preferably, there would be no detectable binding.

In yet another embodiment, this invention provides antibodies directed against the aoHGE polypeptides of this invention, and pharmaceutically effective compositions and methods comprising those antibodies. The antibodies of this embodiment are those that are reactive with the aoHGE polypeptides of this invention, and are effective to diagnose, treat or protect against aoHGE infection and human granulocytic ehrlichiosis. Such antibodies may be used in a variety of applications, including to detect the presence of aoHGE, to screen for expression of novel aoHGE polypeptides, to purify novel aoHGE polypeptides, to block or bind to the aoHGE polypeptides, to direct molecules to the surface of aoHGE or aoHGE infected cells and to prevent or lessen the severity, for some period of time, of aoHGE infection.

In still another embodiment, this invention relates to diagnostic means and methods characterized by the aoHGE polyPeptides, DNA sequences or antibodies of the invention.

25 This invention further provides an immunocompetent nonhuman, mammalian model for human HGE. The laborator-, mouse model, described herein, is characterized by clinical features that closely mimic HGE in humans. Thus, the mouse model is useful for 30 selecting the preferred aoHGE polypeptides and S" antibodies of this invention that are effective to 16 protect against aoHGE infection and human granulocytic ehrlichiosis.

A further embodiment of this invention is a novel diagnostic assay for detecting the presence of aoHGE in a biological sample. The assay provided herein tests the ability of the biological sample to produce aoHGE infection in infant laboratory mice. In a preferred embodiment, the infant mice are 5 days old or less. In a more preferred embodiment, the mice are 3 days old or less. In the most preferred embodiment, the mice are 1 day of age.

In order to further define.this invention, the following terms and definitions are herein provided.

As used herein, an "aoHGE polypeptide" is a polypeptide encoded by a DNA sequence of aoHGE. For example, aoHGE polypeptides include the 40, 44, 65, *94, 110, 115, or 125-kDa polypeptide expressed by aoHGE, as described in Example I, infra, an E6, E7 or 20 eM4 polypeptide or fragments or derivatives thereof.

As used herein, an "aoHGE polypeptide" includes polypeptides encoded by a DNA sequence of any organism that causes HGE.

As used herein, a "40-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: a 40-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an 30 animal infected with aoHGE, and serotypic variants thereof; 17 fragments comprising at least 8 amino acids taken as a block from the 40-kDa aoHGE polypeptide of derivatives of a 40-kDa aoHGE polypeptide of or said derivatives being at least identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 40-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and the 40-kDa aoHGE polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 40-kDa aoHGE polypeptide of or or 20 As used herein, a "44-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: the 44-kDa aoHGE protein having the amino acid 25 sequence of SEQ ID NO: and serotypic variants thereof; the 44-1 polypeptide of SEQ ID NO: the 44-2 polypeptide of SEQ ID NO: 6; a polypeptide encoded by a DNA sequence which 30 hybridizes to a DNA sequence encoding the polypeptide of under stringent conditions; 18 fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of a derivative of a polypeptide of said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a polypeptide of aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a polypeptide of and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a polypeptide of As used herein, a "65-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is 20 selected from the group consisting of: a 65-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the 65-kDa aoHGE polypeptide of a derivative of a 65-kDa aoHGE polypeptide of or said derivative being at least 30 identical in amino acid sequence to the corresponding polypeptide of or 19 aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 65-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and the 65-kDa aoHGE polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 65-kDa aoHGE polypeptide of or or As used herein, an "80-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: an 80-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, and serotypic variants 20 thereof; the 80-1 polypeptide of SEQ ID NO: 7; fragiuentr comprising at least 8 amino acids taken as a block from the 80-kDa aoHGE polypeptide of or a derivative of a polypeptide of said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a 30 mammalian host with aoHGE, which antibodies are immunologically reactive with a polypeptide of 20 aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a polypeptide of and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a polypeptide of As used herein, a "94-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: a 94-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the 94-kDa aoHGE polypeptide of a derivative of a 94-kDa aoHGE polypeptide of or said derivative being at least 20 identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 94-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 94-kDa aoHGE polypeptide of or (b) o 30 or and 21 aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 94-kDa aoHGE polypeptide of or or As used herein, a "105-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: a 105-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the 105-kDa aoHGE polypeptide of a derivative of a 105-kDa aoHGE polypeptide of or said derivative being at least identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive witn a 105-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 105-kDa aoHGE polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with 30 a 105-kDa aoHGE polypeptide of or or As used herein, a "110-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of 22 Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: a 110-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the 100-kDa aoHGE polypeptide of a derivative of a 10-kDa aoHGE polypeptide of or said derivative being at least identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 110-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of 20 eliciting antibodies that are immunologically reactive with aoHGE and a 110-kDa aoHGE polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 110-kDa aoHGE polypeptide of or or As used herein, a "115-kDa aoHGE polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: 30 a 115-kDa aoHGE protein appearing as a single band on a Western blot after reacting with sera from an 23 animal infected with aoHGE, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the 115-kDa aoHGE polypeptide of a derivative of a 115-kDa aoHGE polypeptide of or said derivative being at least identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 115-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 115-kDa polypeptide of or or and aoHGE polypeptides that are immunologically 20 reactive with antibodies elicited by immunization with a 115-kDa aoHGE polypeptide of or or (c) As used herein, a "125-kDa aoHGE polypeptide" denotes a polypee-ide which is substantially free of Ehrlichia bacterium or fragments thereof and which is 25 selected from the group consisting of: a 125-kDa aoHGE protein appearing as a single band on a Western 'ots after reacting with sera from an animal infected with aoHGE, and serotypic variants thereof; 30 fragments comprising at least 8 amino acids taken as a block from the 125-kDa aoHGE polypeptide of 24 a derivative of a 125-kDa aoHGE polypeptide of or said derivative being at least identical in amino acid sequence to the corresponding polypeptide of or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with a 125-kDa aoHGE polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 125-kDa aoHGE polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 125-kDa aoHGE polypeptide of or or As used herein, an "E6 polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected 20 from the group consisting of:

G

an E6 protein of having the amino acid sequence set forth in SEQ ID NO: 2 and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the E6 polypeptide of a derivative of an E6 polypeptide of or said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of or 30 aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are 25 immunologically reactive with an E6 polypeptide of (a) or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and an E6 polypeptide of or or and aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with an E6 polypeptide of or or As used herein, an "E7 polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: an E7 protein having the amino acid sequence set forth in SEQ ID NO: 4 and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the E7 protein of a derivative of an E7 polypeptide of or 20 said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of S (a)or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a 25 mammalian host with aoHGE, which antibodies are im!munologically reactive with an E7 polypeptide of (a) f) aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive 30 with aoHGE and an E7 polypeptide of and e 26 aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with an E7 polypeptide of As used herein, an "eM4 polypeptide" denotes a polypeptide which is substantially free of Ehrlichia bacterium or fragments thereof and which is selected from the group consisting of: the eM4 polypeptide having an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 12, and serotypic variants thereof; fragments comprising at least 8 amino acids taken as a block from the polypeptide of a derivative of a polypeptide of or said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are 20 immunologically reactive with a polypeptide of or or aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a polypeptide of or or and 25 aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a polypeptide of or or As used herein, a "serotypic variant" of an aoHGE polypeptide of this invention, also referred to 30 herein as a "variant", is any naturally occurring aoHGE polypeptide which may be encoded, in whole or in part, by a DNA sequence which hybridizes, at 20-27"C below 27 Tm, to any portion of the DNA sequence encoding the aoHGE polypeptide disclosed herein.

One of skill in the art will understand that serotypic variants of aoHGE polypeptides include those polypeptides encoded by DNA sequences of which any portion may be amplified by using the polymerase chain reaction and oligonucleotide primers derived from any portion of the DNA sequence encoding the aoHGE polypeptide.

As used herein, a "protective aoHGE polypeptide" is any aoHGE polypeptide that, when administered to an animal, is capable of eliciting an immune response that is effective to prevent or lessen the severity, for some period of time, of aoHGE infection or HGE. Preventing or lessening the severity of infection may be evidenced by a change in the physiological manifestations of aoHGE infection, including fever, myalgia, arthralgia, anemia, leukocytopenia, thrombocytopenia, neutropenia, elevated 20 hepatic enzyme levels, gastro-intestinal or pulmonary hemorrhaging and other disorders caused by aoHGE infection. I. may be evidenced by a decrease in or absence of aoHGE in the treated animal. And, it may be evidenced by a decrease in the level of aoHGE in 25 infected ticks which have fed on treated animals.

One of skill in the art will understand that probes and oligonucleotide primers derived from the DNA encoding an aoHGE polypeptide may be used to isolate and clone further variants of aoHGE proteins from other 30 aoHGE isolates and perhaps from other rickettsia as well, which are useful in the methods and compositions of this invention.

28 As used herein, a "derivative" an aoHGE polypeptide is a polypeptide in which one or more physical, chemical, or biological properties has been altered. Such modifications include, but are not limited to: amino acid substitutions, modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide with other organic and non-organic molecules; and other modifications, any of which may result in changes in primary, secondary or tertiary structure.

As used herein, a "protective epitope" is an epitope which is recognized by a protective antibody, and/or an epitope which, when used to immunize an animal, elicits an immune response sufficient to prevent or lessen the severity for some period of time, of aoHGE infection or HGE. Again, preventing or lessening the severity of infection may 20 be evidenced by a change in the physiological manifestations of aoHGE infection including fever, myalgia, arthralgia, anemia, leukocytopenia, thrombocytopenia, neutropenia, elevated hepatic enzyme levels, gastro-intestinal or pulmonary hemorrhaging, 25 and other related disorders. It may be evidenced by a decrease in the level of aoHGE in the treated animal.

And, it may also be evidenced by a decrease in the level of aoHGE in infected ticks feeding on treated.

animals. A protective epitope may comprise a T cell 3o epitope, a B cell epitope, or combinations thereof.

As used herein, a "protective antibody" is an antibody that confers protection, for some period of 29 time, against aoHGE infection or any one of the physiological disorders associated with aoHGE infection or HGE.

As used herein, a "T cell epitope" is an epitope which, when presented to T cells by antigen presenting cells, results in a T cell response such as clonal expansion or expression of lymphokines or other immunostimulatory molecules. A T cell epitope may also be an epitope recognized by cytotoxic T cells that may io affect intracellular aoHGE infection. A strong T cell epitope is a T cell epitope which elicits a strong T cell response.

As used herein, a "B cell epitope" is the simplest spatial conformation of an antigen which reacts with a specific antibody.

As used herein, a "therapeutically effective amount" of a polypeptide or of an antibody is the amount that, when administered to an animal, elicits an immune response that is effective to prevent or lessen 20 the severity, for some period of time, of aoHGE infection.

As used herein, "an anti-aoHGE polypeptide antibody," also referred to as "an antibody of this invention," is an antibody directed against an aoHGE 25 polypeptide of this invention. For example, an antibody of this invention may be directed against a 44-kDa, 65-kDa, 80-kDa, 94-KDa, 110-kDa, 115kDa, 125-kDa polypeptide expressed by aoHGE, as described in Example I, infra, an E6, E7 or eM4 30 polypeptide, or a fragment, derivative or serotypic variant of the aforementioned polypeptides. An antiaoHGE polypeptide antibody of this invention includes 30 antibodies directed against polypeptides expressed by aoHGE, or fragments or derivatives thereof, that are immunologically cross-reactive with any one of the aforementioned polypeptides. Finally, an anti-aoHGE polypeptide antibody of this invention includes antibodies directed against other aoHGE polypeptides identified according to methods taught herein.

As used herein, an "anti-aoHGE polypeptide antibody" is an immunoglobulin molecule, or portion thereof, that is immunologically reactive with an aoHGE polypeptide of the present invention and that was either elicited by immunization with aoHGE or an aoHGE polypeptide of this invention or was isolated or identified by its reactivity with an aoHGE polypeptide of this invention.

An anti-aoHGE polypeptide antibody may be an intact immunoglobulin molecule or a portion of an immunoglobulin molecule that contains an intact antigen binding site, including those portions known in the art as Fab, Fab' and F(ab')2. It should be understood that an anti-aoHGE polypeptide antibody may also be a protective antibody.

The aoHGE polypeptides disclosed herein are immunologically reactive with antisera generated by 25 infection of a mammalian host with aoHGE. Accordingly, they are useful in methods and compositions to diagnose human granulocytic ehrlichiosis, and in therapAutic compositions to stimulate immunological clearance of aoHGE during ongoing infection.

30 In addition, because at least some, if not all of the aoHGE polypeptides disclosed herein are protective surface proteins of aoHGE, they are 31 particularly useful in single and multicomponent vaccines against human granulocytic ehrlichiosis. In this regard, multicomponent vaccines are preferred because such vaccines may be formulated to more closely resemble the immunogens presented by replicationcompetent aoHGE, and because such vaccines are more likely to confer broad-spectrum protection than a vaccine comprising only a single aoHGE polypeptide.

Multicomponent vaccines according to this invention may also contain polypeptides which characterize other vaccines useful for immunization against diseases other than human granulocytic ehrlichiosis such as, for example, Lyme disease, human monocytic ehrlichiosis, babesiosis, diphtheria, polio, hepatitis, and measles. Such multicomponent vaccines are typically incorporated into a single composition.

The preferred compositions and methods of this invention comprise aoHGE polypeptides having enhanced immunogenicity. Such polypeptides may result S.i 20 when the native forms of the polypeptides or fragments thereof are modified or subjected to treatments to enhance their immunogenic character in the intended recipient.

Numerous techniques are available and well 25 known to those of skill in the art which may be used, without undue experimentation, to substantially increase the immu, genicity of the aoHGE polypeptides herein disclosed. For example, aoHGE polypeptides of this invention may be modified by coupling to 30 dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS. Particularly if the y p e p t i d e s a r e s m a hemicall synthesized polypeptides are small, chemically synthesized 32 polypeptides, it may be desirable to couple them to an immunogenic carrier. The coupling, of course, must not interfere with the ability of either the polypeptide or the carrier to function appropriately. For a review of some general considerations in coupling strategies, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988).

Useful immunogenic carriers are well known in the art. Examples of such carriers are keyhole limpet hemocyanin (KLH); albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.

Modification of the amino acid sequence of the aoHGE polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity or alter their biochemical properties. For example, the polypeptides 20 or fragments thereof may be expressed with or without the signal and other sequences that may direct addition of lipid moieties.

As will be apparent from the disclosure to follow, the polypeptides may also be prepared with the 25 objective of increasing stability or rendering the molecules more amenable to purification and S. preparation. One such technique is to express the polypeptides as fusion proteins comprising other aoHGE or non-aoHGE sequences.

30 In accordance with this invention, derivatives of the aoHGE polypeptides may be prepared by a variety of methods, including by in vitro 33 manipulation of the DNA encoding the native polypeptides and subsequent expression of the modified DNA, by chemical synthesis of derivatized

DNA

sequences, or by chemical or biological manipulation of expressed amino acid sequences.

For example, derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid. Those of skill in the art will understand that conservative substitution is preferred, 3-methylhistidine may be substituted for histidine, 4 -hydroxyproline may be substituted for proline, 5 -hydroxylysine may be substituted for lysine, and the like.

Causing amino acid substitutions which are less conservative may also result in desired derivatives, by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution 20 of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge 25 for a residue having a net negative charge.

When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the 30 desired characteristics. In particular, the immunogenicity, immunodominance and/or protectiveness 34 of a derivative of this invention can be readily determined using methods disclosed in the Examples.

In a preferred embodiment of this invention, the aoHGE polypeptides disclosed herein are prepared as part of a larger fusion protein. For example, an aoHGE polypeptide of this invention may be fused at its Nterminus or C-terminus to a different immunogenic aoHGE polypeptide, to a non-aoHGE polypeptide or to combinations thereof, to produce fusion proteins o0 comprising the aoHGE polypeptide.

In a preferred embodiment of this invention, fusion proteins comprising aoHGE polypeptides are constructed comprising B cell and/or T cell epitopes from multiple serotypic variants of aoHGE, each variant differing from another with respect to the locations or sequences of the epitopes within the polypeptide. In a more preferred embodiment, fusion proteins are constructed which comprise one or more of the aoHGE :i polypeptides fused to other aoHGE polypeptides. Such 20 fusion proteins are particularly effective in the prevention, treatment and diagnosis of human granulocytic ehrlichiosis as caused by a wide spectrum of aoHGE isolates.

In another preferred embodiment of this 25 invention, the aoHGE polypeptides are fused to moieties, such as immunoglobulin domains, which may increase the stability and prolong the in vivo plasma half-life of the polypeptide. Such fusions may be prepared without undue experimentation according to 30 methods well known to those of skill in the art, for example, in accordance with the teachings of United States patent 4,946,778, or United States patent 35 5,116,964. The exact site of the fusion is not critical as long as the polypeptide retains the desired biological activity. Such determinations may be made according to the teachings herein or by other methods known to those of skill in the art.

It is preferred that the fusion proteins comprising the aoHGE polypeptides be produced at the DNA level, by constructing a nucleic acid molecule encoding the fusion protein, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods.

The aoHGE polypeptides may also be part of larger multimeric molecules which may be produced recombinantly or may be synthesized chemically. Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including 20 lipids and carbohydrates.

Preferably, the multimeric proteins will consist of multipse T or B cell epitopes or combinations thereof repeated within the same molecule, either randomly, or with spacers (amino acid or 25 otherwise) between them.

In a preferred embodiment of this invention, aoHGE is incorporated into a vaccine. As disclosed in Examples and animals immunized with such a vaccine produce antibodies that confer protection 30 against aoHGE infection.

In another preferred embodiment of this invention, an aoHGE polypeptide of this invention which 36 is also a protective aoHGE polypeptide is incorporated into a single component vaccine. In a more preferred embodiment of this invention, aoHGE polypeptides of this invention which are also protective aoHGE polypeptides are incorporated into a multicomponent vaccine comprising other protective aoHGE polypeptides.

In addition to A multicomponent vaccine may also contain protective polypeptides useful for immunization against other diseases such as, for example, Lyme disease, human monocytic ehrlichiosis, babesiosis, diphtheria, polio, hepatitis, and measles. Such a vaccine, by virtue of its ability to elicit antibodies to a variety of protective aoHGE polypeptides, will be effective to protect against human granulocytic ehrlichiosis as caused by a broad spectrum of different aoHGE isolates, even those that may not express one or more of the aoHGE proteins.

The multicomponent vaccine may contain the aoHGE polypeptides as part of a multimeric molecule in 20 which the various components are covalently associated.

Alternatively, it may contain multiple individual components. For example, a multicomponent vaccine may be prepared comprising two or more of the aoHGE polypeptides, wherein each polypeptide is expressed and 25 purified from independent cell cultures and the polypeptides are combined prior to or during formulation.

Alternatively, a multicomponent vaccine may be prepared from heterodimers or tetramers wherein the S: 30 polypeptides have been fused to immunoglobulin chains or portions thereof. Such a vaccine could comprise, for example, a 44-kDa aoHGE polypeptide fused to an 37 immunoglobulin heavy chain and an E6 aoHGE polypeptide fused to an immunoglobulin light chain, and could be produced by transforming a host cell with DNA encoding the heavy chain fusion and DNA encoding the light chain fusion. One of skill in the art will understand that the host cell selected should be capable of assembling the two chains appropriately. Alternatively, the heavy and light chain fusions could be produced from separate cell lines and allowed to associate after purification 0 The desirability of including a particular component and the relative proportions of each component may be determined by using the assay systems disclosed herein, or by using other systems known to those in the art. Most preferably, the multicomponent vaccine will comprise numerous T cell and B cell epitopes of protective aoHGE polypeptides.

This invention also contemplates that the aoHGE polypeptides of this invention, either alone or combined, may be administered to an animal via a 20 liposome delivery system in order to enhance their stability and/or immunogenicity. Delivery of the aoHGE polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated animal. Such cells, 25 upon ingesting the liposome, would digest the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules *see *0 a required to elicit a strong immune response.

a The liposome system may be any variety of 30 unilamellar vesicles, multilamellar vesicles, or stable 6 plurilamellar vesicles, and may be prepared and administered according to methods well known to those 38 of skill in the art, for example in accordance with the teachings of United States patents 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, it may be desirable to express the aoHGE polypeptides of this invention, as well as other selected aoHGE polypeptides, as lipoproteins, in order to enhance their binding to liposomes.

Any of the aoHGE polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

According to this invention, we describe a method which comprises the steps of treating an animal with a therapeutically effective amount of an aoHGE polypeptide, or a fusion protein or a multimeric protein comprising an aoHGE polypeptide, in a manner 20 sufficient to prevent or lessen the severity, for some period of time, of aoHGE infection. The polypeptides that are preferred for use in such methods are those that contain protective epitopes. Such protective epitopes may be B cell epitopes, T cell epitopes, or combinations thereof.

According to another embodiment of this invention, we describe a method which comprises the steps of treating an animal with a multicomponent vaccine comprising a therapeutically effective amount 30 of an aoHGE polypeptide, or a fusion protein or multimeric protein comprising such polypeptide in a manner sufficient to prevent or lessen the severity, 39 for some period of time, of aoHGE infection. Again, the polypeptides, fusion proteins and multimeric proteins that are preferred for use in such methods are those that contain protective epitopes, which may be B cell epitopes, T cell epitopes, or combinations thereof.

The most preferred polypeptides, fusion proteins and multimeric proteins for use in these compositions and methods are those containing both strong T cell and B cell epitopes. Without being bound by theory, we believe that this is the best way to stimulate high titer antibodies that are effective to neutralize aoHGE infection. Such preferred polypeptides will be internalized by B cells expressing surface immunoglobulin that recognizes the B cell epitope(s). The B cells will then process the antigen and present it to T cells. The T cells will recognize the T cell epitope(s) and respond by proliferating and producing lymphokines which in turn cause B cells to 20 differentiate into antibody producing plasma cells.

Thus, in this system, a closed autocataiytic circuit exists which will result in the amplification of both B and T cell responses, leading ultimately to production Sof a strong immune response which includes high titer antibodies against the aoHGE polypeptide.

One of skill in the art will also understand that it may be ao:antageous to administer the aoHGE polypeptides of this invention in a form that will favor the production of T-helper cells type 1 (THl) 30 which help activate macrophages, and/or T-helper cells type 2 which help B cells to generate antibody responses. Aside from administering epitopes which are 40 strong T cell or B cell epitopes, the induction of T 1 or T, 2 cells may also be favored by the mode of administration of the polypeptide. For example, aoHGE polypeptides may be administered in certain doses or with particular adjuvants and immunomodulators, for example with interferon-gamma or interleuken-12 (TH1 response) or interleukin-4 or interleuken-10 (T,2 response).

To prepare the preferred polypeptides of this invention, in one embodiment, overlapping fragments of the aoHGE polypeptides of this invention are constructed as described herein. The polypeptides that contain -B cell epitopes may be identified in a variety of ways for example by their ability to remove protective antibodies from polyclonal antiserum directed against the polypeptide or elicit an immune response which is effective to prevent or lessen the severity of aoHGE infection.

Alternatively, the polypeptides may be used to produce monoclonal antibodies which are screened for their ability to confer protection against aoHGE infection when used to immunize naive animals. Once a given monoclonal antibody is found to confer protection, the particular epitope that is recognized by that antibody may then be identified.

As recognition of T cell epitopes is MHC restricted, the polypeptides that contain T cell epitopes may be identified in vitro by testing them for their ability to stimulate proliferation and/or 30 cytokine production by T cell clones generated from humans of various HLA types, from the lymph nodes, spleens, or peripheral blood lymphocytes of C3H or 41 other laboratory mice, or from domestic animals.

Compositions comprising multiple T cell epitopes recognized by individuals with different Class

II

antigens are useful for prevention and treatment of human granulocytic ehrlichiosis in a broad spectrum of patients.

In a preferred embodiment of the present invention, an aoHGE polypeptide containing a B cell epitope is fused to one or more other immunogenic aoHGE polypeptides containing strong T cell epitopes. The fusion protein that carries both strong T cell and B cell epitopes is able to participate in elicitation of a high titer antibody response effective to neutralize infection with aoHGE.

Strong T cell epitopes may also be provided by non-aoHGE molecules. For example, strong T cell epitopes have been observed in hepatitis B virus core antigen (HBcAg). Furthermore, it has been shown that linkage of one of these segments to segments of the surface antigen of Hepatitis B virus, which are poorly recognized by T cells, results in a major amplification of the anti-HBV surface antigen response, Milich et al., "Antibody Production To The Nucleocapsid And Envelope Of The Hepatitis B Virus Primed By A Single Synthetic T Cell Site", Nature, 329, pp. 547-49 (1987)].

Therefore, in yet another preferred S* embodiment, B cell epitopes of the aoHGE polypeptides are fused to segments of HBcAG or to other antigens which contain strong T cell epitopes, to produce a fusion protein that can elicit a high titer antibody response against aoHGE. In addition, it may be 42 particularly advantageous to link an aoHGE polypeptide of this invention to a strong immunogen that is also widely recognized, for example tetanus toxoid.

It will be readily appreciated by one of ordinary skill in the art that the aoHGE polypeptides of this invention, as well as fusion proteins and multimeric proteins containing them, may be prepared by recombinant means, chemical means, or combinations thereof.

For example, the polypeptides may be generated by recombinant means using the DNA sequences of aoHGE isolate NCH-1 as set forth in the sequence listings contained herein. DNA encoding serotypic variants of the polypeptides may likewise be cloned, using PCR and oligonucleotide primers derived from the sequences herein disclosed.

In this regard, it may be particularly desirable to isolate the genes encoding aoHGE polypeptides from isolates that differ antigenically 20 from strain NCH-1, aoHGE isolates against which aoHGE NCH-1 polypeptides are ineffective to protect, in order to obtain a broad spectrum of different epitopes which would be useful in the methods and compositions of this invention.

Oligonucleotide primers and other nucleic acid probes derived from the genes encoding the aoHGE polypeptides of this invention may also be used to isolate and clone other related proteins from aoHGE an.

related rickettsia which may contain regions of DNA sequence homologous to the DNA sequences of this Sinvention. In addition, the DNA sequences of this 43 invention may also be used in PCR reactions to detect the presence of aoHGE in a suspected infected sample.

If the aoHGE polypeptides of this invention are produced recombinantly, they may be expressed in unicellular hosts. As is well known to one of skill in the art, in order to obtain high expression levels of foreign DNA sequences in a host, the sequences are generally operatively linked to transcriptional and translational expression control sequences that are functional in the chosen host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further comprises a selection marker.

The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is secreted from the eukaryotic host.

20 An amino terminal methionine may or may not be present on the expressed polypeptides of this invention. x f Ll ie terminal methionine is not cleaved by the expression host, it may, if desired, be

S

chemically removed by standard techniques.

25 A wide variety of expression host/vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors for eukaryotic hosts, include, for example, vectors .comprising expression control sequences from 30 bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retroviruses including lentiviruses. Useful expression vectors for bacterial 44 hosts include bacterial plasmids, such as those from E. coli, including pBluescript, pGEX-2T, pUC vectors, col El, pCRl, pBR322, pMB9 and their derivatives, pETwider host range plasmids, such as RP4, phage DNAs, the numerous derivatives of phage lambda, e.g.

and AGT11, and other phages. Useful expression vectors for yeast cells include the 2u plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the Laa 20 system, the r.E system, the IAQ or IBC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast amating system and other constitutive and inducible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

30 In a preferred embodiment, DNA sequences encoding the aoHGE polypeptides of this invention are cloned in the expression vector lambda ZAP II 45 (Stratagene, La Jolla, CA), in which expression from the lac promoter may be induced by IPTG.

In another preferred embodiment, DNA encoding the aoHGE polypeptides of this invention is inserted in frame into an expression vector that allows high level expression of the polypeptide as a glutathione

S-

transferase fusion protein. Such a fusion protein thus contains amino acids encoded by the vector sequences as well as amino acids of the aoHGE polypeptide.

A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COSl-, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells.

20 It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because 30 the vector must be replicated in it. The vector's copy number, the ability to control that copy number, the ability to control integration, if any, and the 46 expression of any other proteins encoded by the vector, such as antibiotic or other selection markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the promoter sequence, its controllability, and its compatibility with the DNA sequence of this invention, particularly with regard to potential secondary io structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention.

Within these parameters, one of skill in the art may select various vector/expression control 20 sequence/host combinations that will express the DNA sequences of this invention on fermentation or in other large scale cultures.

The molecules comprising the aoHGE polypeptides encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with 30 inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like. One 47 of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.

In addition, the aoHGE polypeptides may be generated by any of several chemical techniques. For example, they may be prepared using the solid-phase synthetic technique originally described by R. B.

Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc, 83 pp. 2149-54 (1963), or they may be prepared by synthesis in solution. A summary of peptide synthesis techniques may be found in.E. Gross H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984).

Typically, these synthetic methods comprise the sequenzial addition of one or more amino acid residues to a growing peptide chain. Often peptide 20 coupling agents are used to facilitate this reaction.

*For a recitation of peptide coupling agents suitable for the uses described herein see M. Bodansky, supra.

Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different protecting group is utilized for amino acids containing a reactive side .goup, lysine. A variety of S* protecting groups known in the field of peptide synthesis and recognized by conventional abbreviations therein, may be found in T. Greene, Protective Groups In Organic Synthesis, Academic Press (1981) 48 According to another embodiment of this invention, antibodies directed against the aoHGE polypeptides are-generated. Such antibodies are immunoglobulin molecules or portions thereof that are immunologically reactive with an aoHGE polypeptide of the present invention. It should be understood that the antibodies of this invention include antibodies immunologically reactive with fusion proteins and multimeric proteins comprising an aoHGE polypeptide.

Antibodies directed against an aoHGE polypeptide may be generated by a variety of, means including infection of a mammalian host with aoHGE, or by immunization of a mammalian host with an aoHGE polypeptide of the present invention. Such antibodies may be polyclonal or monoclonal, it is preferred that they are monoclonal. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art. For a review of such methods, see Antibodies, A Laboratory Manual, supra, and D.E.

20 Yelton, et al., Ann. Rev. of Biochem., 50, pp. 657-80 (1981). Determination of immunoreactivity with an aoHGE polypeptide of this invention may be made by any of several methods well known in the art, including by immunoblot assay and ELISA.

An antibody of this invention may also be a hybrid molecule formed from immunoglobulin sequences from different species mouse and human or from portions of immunoglobulin light and heavy chain sequences from the same species. It may be a molecule 30 that has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art 49 including: the production of hybrid hybridomas; disulfide exchange; chemical cross-linking; addition of peptide linkers between two monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line; and so forth.

The antibodies of this invention may also be human monoclonal antibodies produced by any of the several methods known in the art. For example, human monoclonal antibodies may be produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human" antibodies, by the expression of cloned human immunoglobulin genes, by phage-display, or by any other method known in the art.

In addition, it may be advantageous to couple the antibodies of this invention to toxins such as diphtheria, pseudomonas exotoxin, ricin A chain, gelonin, etc., or antibiotics such as penicillins, tetracyclines and chloramphenicol.

In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, 25 toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular application.

One of skill in the art will understand that antibodies directed against an aoHGE polypeptide may 30 have utility in therapeutic and prophylactic compositions and methods directed against human granulocytic ehrlichiosis and aoHGE infection. For 50 example, the level of aoHGE in infected ticks may be decreased by allowing them to feed on the blood of animals immunized with the aoHGE polypeptides of this invention.

The antibodies of this invention also have a variety of other uses. For example, they are useful as reagents to screen for expression of the aoHGE polypeptides, either in libraries constructed from aoHGE DNA or from other samples in which the proteins may be present. Moreover, by virtue of their specific binding affinities, the antibodies of this invention are also useful to purify or remove polypeptides from a given sample, to block or bind to specific epitopes on the polypeptides and to direct various molecules, such as toxins, to the surface of aoHGE.

To screen the aoHGE polypeptides and antibodies of this invention for their ability to confer protection against human granulocytic ehrlichiosis or their ability to lessen the severity of 20 aoHGE infection, laboratory mice are preferred as an animal model. Of course, while any animal that is susceptible to infection with aoHGE may be useful, mice are not only susceptible to aoHGE infection but are also afflicted with clinical symptoms of a disease 25 that is remarkably similar to human granulocytic ehrlichiosis in humans. Further, the humoral response of mice infected with aoHGE by tick transmission has been shown to be strongly similar to the human humoral response. Thus, by administering a particular aoHGE 30 polypeptide or anti-aoHGE polypeptide antibody to mice, one of skill in the art may determine without undue experimentation whether that polypeptide or antibody 51 would be useful in the methods and compositions claimed herein.

The administration of the aoHGE polypeptide or antibody of this invention to the animal may be accomplished by any of the methods disclosed herein or by a variety of other standard procedures. For a detailed discussion of such techniques, see Antibodies, A Laboratory Manual, supra. Preferably, if a polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).

Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more 20 administrations of the polypeptide, spread out over several weeks.

Once the aoHGE polypeptides or antibodies of this invention have been determined to be effective in the screening process, they may then be used in a therapeutically effective amount in pharmaceutical .00. compositions and methods to treat or prevent human granulocytic ehrlichiosis.

The pharmaceutical compositions of this invention may be in a variety of conventional depot 30 forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, 52 capsules, suppositories, injectable and infusible solutions. The preferred form depends upon the intended mode of administration and prophylactic application.

Such dosage forms may include pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. These carriers and adjuvants include, for example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol. Adjuvants for topical or gel base 'forms may be selected from the group consisting of 20 sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, S. polyethylene glycol, and wood wax alcohols.

The vaccines and compositions of this invention may also include other components or be 25 subject to other treatments during preparation to enhance their immunogenic character or to improve their tolerance in patients.

Compositions comprising an antibody of this invention may be administered by a variety of dosage 30 forms and regimens similar to those used for other passive immunotherapies and well known to those of skill in the art. Generally, the aoHGE polypeptides 53 may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides the vaccine against hepatitis).

Any pharmaceutically acceptable dosage route, including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the polypeptide or antibody composition For example, the composition may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly including paravertebrally and periarticularly subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes. Preferably, the compositions of the invention o.are in the form of a unit dose and will usually be 20 administered to the patient intramuscularly.

The aoHGE polypeptides or antibodies of this invention may be administered to the patient at one time or over a series of treatments. The most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the exoected infection, previous therapy, the patient's health status and response to immunization, and the judgment 30 of the treating physician.

For example, in an immunocompetern-: patient, the more highly irmunogenic the pclyzep::de, the lower 54 the dosage and necessary number of immunizations.

Similarly, the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant. Generally, the dosage will consist of 10 ug to 100 mg of the purified polypeptide, and preferably, the dosage will consist of 10-1000 ug. Generally, the dosage for an antibody will be 0.5 mg-3.0 g.

In a preferred embodiment of this invention, the aoHGE polypeptide is administered with an adjuvant, in order to increase its immunogenicity. Useful adjuvants include RIBI, and ISCOM, simple metal salts such. as aluminum hydroxide, and oil based adjuvants such as complete and incomplete Freund's adjuvant.

When an oil based adjuvant is used, the polypeptide is usually is administered in an emulsion with the adjuvant.

In yet another preferred embodiment, E.coli expressing proteins comprising an aoHGE polypeptide are administered orally to non-human animals according to methods known in the art, to decrease or lessen the severity of aoHGE infection. For example, a palatable regimen of bacteria expressing an aoHGE polypeptide, alone or in the form of a fusion protein or multimeric protein, may be administered with animal food to be 25 consumed by wild mice or other animals that harbor aoHGE.

Ingestion of such bacteria may induce an immune response comprising both humoral and cellmediated components. See J.C. Sadoff et al., "Oral 30 Salmonella Typhimurium Vaccine Expressing S"Circumsporozoite Protein Protects Against Malaria", ,Science 240, pp. 336-38 (1988) and K.S. Kim et al., 55 "Immunization Of Chickens With Live Escherichia coli Expressing Eimeria acervulina Merozoite Recombinant Antigen Induces Partial Protection Against Coccidiosis", Inf. Immun., 57, pp. 2434-40 (1989); M.

Dunne et al., "Oral Vaccination Against Human granulocytic ehrlichiosis Using Salmonella Expressing OspA," Inf. and Immun., 63:1611 (1995); E. Fikrig et al., "Protection of Mice From Lyme Borreliosis By Oral Vaccination With Escherichia coli Expressing OspA," J.

Infec. Dis., 164:1224 (1991).

Moreover, the level of aoHGE infection in ticks feeding on such animals may be lessened or eliminated, thus inhibiting transmission to the next animal.

According to yet anocher embodiment, the antibodies of this invention as well as the aoHGE polypeptides of this invention, and the DNA sequences encoding them are useful as diagnostic agents for detecting infection with aoHGE. The polypeptides are 20 capable of binding to antibody molecules produced in animals, including humans, that are infected with aoHGE, and the antibodies are capable of binding to aoHGE or an-igeLns thereof.

Such diagnostic agents may be included in a 25 kit which may also comprise instructions for use and o other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound.

For example, the pol-peptide or antibody may be labeled with a detection means that allows for the detection of 30 the polypeptide when it is bound to an antibody, or for the detection of the antibody when it is bound to aoHGE or an antigen thereof.

56 The detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as 1251 or 5Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as 11C, 150, or 1N. Binding may also be detected by other methods, for example via avidin-biotin complexes.

The linking of the detection means is well known in the art. For instance, monoclonal antibody molecules produced by a hybridoma can be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.

The diagnostic kits of the present invention 20 may be used to detect the presence of a quantity of aoHGE or anti-aoHGE antibodies in a body fluid sample o *oo such as serum, plasma or urine. Thus, in preferred embodiments, an aoHGE polypeptide or an antibody of the present invention is bound to a solid support typically 25 by adsorption from an aqueous medium. Useful solid matrices are well known in the art, and include crosslinked dextran; agarose; polystyrene; polyvinylchloride; cross-linked polyacrylamide; nitrocellulose or nylon-based materials; tubes, plates 30 or the wells of microtiter plates. The polypeptides or antibodies of the present invention may be used as 57 diagnostic agents in solution form or as a substantially dry powder, in lyophilized form.

aoHGE polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole aoHGE and thus may alleviate such pitfalls as false positive and false negative results.

In particular, one of skill in the art would understand that aoHGE polypeptides of this invention that are selectively expressed in the infected host and not in cultured aoHGE, and antibodies directed against such polypeptides, allow detection of antigens and antibodies in samples that are undetectable by diagnostic methods using lysates of cultured spirochetes as the antigen.

One skilled in the art will realize that it may also be advantageous in the preparation of detection reagents to utilize epitopes from more than one aoHGE protein and antibodies directed against such 20 epitopes. It may be particularly advantageous to use epitopes of aoHGE polypeptides that elicit antibodies early in aoHGE infection in combination with epitopes from other aoHGE polypeptides that elicit antibodies that occur in the later stages of human granulocytic 25 ehrichiosis. Diagnostic reagents containing multiple epitopes which are reactive with an:ibodies appearing at different tir-s are useful to detect the presence of anti-aoHGE antibodies throughout the course of infection and to diagnose human granulocytic 3 ehrlichiosis at all stages.

The skilled artisan also will realize that it may be advar.aaecs preare a =a-nosti kit 58 comprising diagnostic reagents to detect aoHGE as well as other pathogens found in the same tick vector, for example, Borrelia burgdorferi and Babesia microti, and instructions for their use.

The polypeptides and antibodies of the present invention, and compositions and methods comprising them, may also be useful for detection, prevention, and treatment of other infections caused by rickettsia which may contain surface proteins sharing amino acid sequence or conformational similarities with the aoHGE polypeptides of the present invention, for example, Ehrlichia equi and Ehrlichia phagocytophila.

In order that this invention may be better understood, the following examples are set forth.

These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

Example I Development of an Animal Model for HGE- 20 We established the laboratory mouse as an animal model in which to investigate the pathogenesis of HGE and to screen the aoHGE polypeptides and •antibodies of the present invention for their ability Sto elicit an immune response effective to treat or protect against aoHGE infection and/or human granulocytic ehrlichiosis. We chose to use mice because of the extensive immunologic, biologic and genetic parameters available for manipulation.

We examined the susceptibility of various Se 30 s:rains cf mice :o infec:ion with the NCR- isolate c-f 59 the HGE agent. We inoculated the mice via tick-borne infection or syringe inoculatin by several different routes. Barthold et al., J. Inf.Dis., (in press).] We chose mice having maximum genetic disparity and representing different H-2 haplotypes. The.mice used for these studies included C3H/HeJ, C3H/HeN and C3H/Smn.CIcrHsd/scid mice, purchased from the Jackson Laboratory (Bar Harbor, NCI Animal Production Program, Frederick Cancer Research Center (Federick, MD), and Harlan Sprague Dawley, Inc. (Indianapolis, IN), respectively. Pregnant outbred Crl:CD-1(ICR) mice were purchased from Charles River Breeding Laboratories (Wilmington, MA).

To examine the course of tick-borne aoHGE infection, we placed 5 aoHGE-infected nymphal ticks on naive C3H mice and allowed them to feed to repletion.

All of the mice became infected, having visible morulae in peripheral blood smear:- at 5-10 days after tick feeding. We necropsied 4 mice with verified infection 20 and 4 age-matched control mice at days 5, 10, 17 and 24 after tick feeding.

All infected mice exhibited transient splenomegaly and we were able to culture aoHGE from peripheral blood and spleen from all mice on days 17 25 and 24. All infected mice also developed detectable antibodies to aoHGE by day Infected mice developed transient hematologic aberrations similar to those described in human HGE including leukopenia, with a reduction in total 30 eukocytes, granulocytes and lymphocytes, and anemia.

Morulae were found only in granulocytes.

60 At all time points, there was marked hematopoiesis in spleens and bone marrow of all infected mice and the lungs of most infected mice showed perivascular lymphoid nodules indicative of antigenic stimulation.

To assess the susceptibility of mice to syringe inoculation, we inoculated 3-5 week old mice both intraperitoneally and subcutaneously with 0.1 ml of serial dilutions (undiluted, 1:10, 1:100 and 1:000) of blood from aoHGE-infected SCID mice granulocytes with morulae). We collected blood from the mice on days 7, 14, 17 and 21 after inoculation and examined peripheral blood smears for morulae to establish aoHGE infection. Mice inoculated by both routes became infected, although they appeared to be more susceptible to infection by i.p. inoculation.

Xenodiagnosis (occurrence of aoHGE infection in uninfected ticks which feed on infected mice) demonstrated that mice remained persistently infected S. 20 for up to 55 days.

In a separate experiment, we necropsied mice infected with aoHGE by syringe inoculation at days 10, 30 and 60 after inoculation. We assessed infection by visualization of morulae, culture, PCR and mouse 25 infectivity at each time point. All of the mice remained infected at 30 days. At EC days, no infection was detectable by the morulae, PCR or culture assays.

However, one mouse remained infected at 60 days, based on a mouse infectivity assay.

The results of these studies showed that mice, upon tick or syringe inoculation, become infected and develop clinical symptoms of a disease that is 61 remarkably similar to HGE in humans. Mice remained persistently infected for at least 55 days after inoculation, and had a 100% correlation between infection, seroconversion, and disease.

We therefore chose the laboratory mouse as our animal model for HGE in humans because it is a fully immunocompetent adult host that 1) is susceptible to aoHGE infection with small numbers of organisms, 2) develops persistent infection and 3) develops a 100% incidence of hematological symptoms of HGE.

Example II Infant Mouse Infectivity Assay Based on our observation that the mouse infectivity assay appeared to be the most sensitive assay for detecting the presence of infectious aoHGE, we compared the sensitivity of the assay in mice of various ages. We inoculated groups of 4-5 mice at 1 day, 3 days 5 days, 1 week and 3 weeks of age by i.p.

injection with 0.1 ml of infected SCID mouse blood and assessed infection by hematocrit, spleen weight, 20 morulae and PCR at 10 days after inoculation.

W2 c.-covered that mice inoculated at 1 day and 3 days had a significantly higher percent of granulocytes with morulae than older mice. All infected mice had increased spleen weights.

We next assessed the sensitivity of our infant mouse infectivity assay. We inoculated groups of 4 one day old mice i.p. with serial dilutions (undiluted, 1:10 and 1:100) of infected SCID mouse blood. Mice became infected at all inoculum doses as 30 determined by increased spleen weight and visualization zf n or'lae.

P 1 62 Based on our results, our infant mouse infectivity assay is significantly more sensitive than the currently used assays for diagnosing aoHGE infection.

Example III Isolation and Sequencing of Immunoaenic aoHGE PolvypeDtide To identify aoHGE polypeptides that elicit a humoral response in an infected animal, including humans, we probed lysates of aoHGE-infected HL-60 cells with sera from patients infected with aoHGE and from mice experimentally infected with aoHGE.

A. Preparation of aoHGE-Infected HL-60 Cells We grew aoHGE isolate NCH-1 in HL-60 cells according to the previously published methods of Goodman et al., with some modifications Goodman et al., "Direct Cultivation of the Causative Agent of Human Granulocytic Ehrlichiosis," N. Engl. J. Med., 334, pp. 209-215 (1996)].

Briefly, we cultured HL-60 cells (American 20 Type Culture Collection 240-CCL) in Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum, with no added antibiotic, maintained at 37°C with 5% carbon dioxide.

We inoculated 10 ml HL-60 cells grown to a density of 1 x 10' cells per ml in 70 ml culture flasks with 50,l blood or splenic tissue from mice infected with aoHGE isolate NCH-1. We maintained cell density between 5 x 106 and 1.5 x 10' cells per ml by feeding the cells twice a week. Usually we removed and discarded 5 ml of the culture and added an equal amount o o: ~resh medium. If te cell ccun- was below 5 x 4* 1> 63 per ml, we added fresh HL-60 cells to a final concentration of 1 x 106. We confirmed infection of the cells by light. microscopy of cultured cells stained with DiffQuick (Baxter, Miami, FL).

B. Patient Sera We obtained sera from aoHGE patients from L.

Magnarelli at the Connecticut Agricultural Experimental Station and S. Telford at Harvard. We obtained 26 convalescent and/or acute serum samples from 18 patients with confirmed aoHGE infection based on the identification of granulocytic morulae in the peripheral blood smear. All patients had fever, headache, malaise and leukopenia and/or thrombocytopenia with or without anemia. In most cases, the human aoHGE sera were obtained 2-8 weeks after the diagnosis of infection.

C. Mousp _$rf We prepared anti-aoHGE antiserum as follows.

We inoculated naive mice intraperitoneally with 20 peripheral blood from an aoHGE patient. We allowed uninfected I. scapularis ticks Telford, Harvard School of Tropical Public Health, Cambridge, MA) to feed to rep.et.n on infected mice. We placed engorged ticks in mesh-covered vials containing moist plaster of 25 Paris and held them at room temperature until molting nto nymphs as described Telford et al., "Perpetuation Of The Agent Of Human Granulocytic Ehrlichiosis In A Deer Tick-Rodent Cycle," Proc. Natl.

Acad. Sci. USA, 93, pp.

6 209-6214 (1996)].

We infected three to four week old random sex C3H specific pathogen free mice (Jackson Laboratories, Bar Harbor, ME) by placing five hardened nymphs on each F p 64 mouse and allowing the ticks to feed to repletion. We made daily blood smears for 1 week starting on day five after the ticks had engorged. All mice had verified granulocytic morulae in one or more of the daily blood smears at 5-10 days after tick feeding.

We were able to culture aoHGE in HL-60 cells inoculated as described supra with peripheral blood and pooled splenic homogenates from infected mice at 17 and 24 days after infection. We obtained serum 10, 17 and 24 days after tick feeding.

Those of skill in the art will understand that anti-serum to other isolates of aoHGE, such as the Yale isolate, R01, the Minnesota isolate, or other isolates, may be prepared in the same way.

D. Immunoblot Analysis We prepared lysates for immunoblotting as follows. We pelleted HL-60 cells infected with aoHGEisolate NCH-1 at 2000 rpm and washed twice in PBS. We resuspended the washed cells at one tenth of the 20 original volume, heated to 100"C for 5 min., mixed with loading buffer and placed the cells on a 10% SDSpolyacrylamide minigel (Hoeffer). We loaded 25 ug of total protein in each lane.

We separated the proteins by electrophoresis 25 and transferred to nitrocellulose. We blocked the strips with 5% dry milk for 1 hour and incubated with a 1 1 00 dilution of the patient sera (1:500 for mouse sera) for 1 hr. We detected bound antibody using S• alkaline phosphatase-conjugated F!ab')2 anti-human or 30 anti-mouse immunoglobulin M or G (Sigma) as secondary S* antibodies and NBT-BCIP.

*o t r 65 We used lysates of uninfected HL-60 cells, prepared in an identical fashion as a control. As an additional control, we probed lysates of uninfected and aoHGE NCH-l-infected HL-60 cells with sera from volunteers who did not have aoHGE infection.

As shown in Figure 9, antibodies in the human anti-aoHGE antisera reacted with aoHGE proteins having molecular weights of 40, 44, 65, 80, 94, 105, 110, 115 and 125 kDa.

The murine sera additionally reacted with aoHGE proteins with molecular weights of 25, 34 and kDa and proteins with molecular weights between 40 and 44 kDa. Sera from mice infected by tick bite, but not sera from mice infected by syringe, reacted with an kDa aoHGE protein.

Example IV Characterization of aoHGE Proteins To characterize the immunogenic aoHGE proteins that are recognized by antibodies in sera of aoHGE infected individuals, we sequenced proteins 20 identified in immunoblots conducted as described in Example III.

To perform the protein sequencing, first we purified aoHGE from infected HL-6C cells prepared as described in Example III, as fcllows.

25 We purified the aoHGE by renografin density gradient centrifugation as described by Hanson et a., with some modifications Hanson et al., "Some Characteristics of Heavy and Light Bands of Rickettsia Sprowazekii on Renografin Gradients," Infect. Immun., 3 pp. 596-6C4 1981); S.M. Chen e- al., p 66 "Identification of the Antigenic Constituents of Ehrlichia chaffeensis," Am.J.Trop.Med.Hyg., 50, pp. 52- 58 (1994)].

Briefly, we cultured aoHGE (NCH-1 isolate) in HL-60 cells, centrifuged 1000 ml of aoHGE-infected

HL-

cells (at least 70% infected cells) at 1500 rpm for min. and resuspended in PBS-glucose and centrifuged again using the same conditions.

We lysed the HL-60 cells by shearing with a 2 1-gauge needle, pelleted the cellular debris at 2000 rpm for 10 min., collected the supernatant and incubated it with RNase and DNase (final concentration ug/ml) to remove any human DNA. Using 42% and discontinuous renografin gradient (Hypaque 76, Nycomed Inc., we ultracentrifuged at 22,000 rpm for min. at 4 0 C in a swing bucket rotor (Beckman, Fullerton, CA). We collected aoHGE in a band at he and 42% renografin interface. We collected the interface band in a sterile pipette and dissolved in SPGN sucrose, 3.7 nmM KHPO, and 5 mM 1-glutamine), pelleted at 13,000 rpm and resuspended in SPGN at a concentration of 2 mg/ul. We stored the suspension at 0

C.

The critical steps were adequate lysis of the 25 HL-60 cells while leaving the aoHGE cells intact so that subsequent incubation with excess RNase and DNase (to eliminate HL-60 RNA and DNA) does not affect the S: aoHGE.

We isolated aoHGE proteins as a single band 3 0 on SDS-polyacrylamide gels as previously described [j.

SanmbrooK et al., Molecular Cloninq: A Laborat-ory go99 67 Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), p. 18.60 ff.].

Briefly, we dissolved the purified aoHGE in sample buffer 2-mercaptoethanol, 10% glycerol, 2% SDS and 0.8% bromophenol blue in 6,25 mM Tris buffer, pH 6.8) and heated for 10 mins. at 100°C. We electrophoresed 20 pg per lane of pure aoHGE with molecular mass standards (Bio-Rad Laboratories, Hercules, CA) and stained with Comassie Blue.

We isolated by excision the 80-kDa band for automated amino-terminal peptide sequencing by high performance liquid chromatography (HPLC) at the Yale Protein Purification and Analysis Facility. The Nterminal amino acid sequence of the 80-kDa protein, which we designated the 80-1 polypeptide, is set forth in SEQ ID NO: 7. We conducted a search of GenBank with the Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, WI). Our search revealed that the 80-kDa aoHGE polypeptide is 20 most likely a member of the HSP-70 heat shock protein S. family. The N-terminal amino acid sequence is homologous with the N-terminal sequences of B.

*burgdorferi, E. coli (ECDNAK), and human (HSHSP70) heat shock protein 70. The most closely homologous protein 25 identified in the database is the E. burgdorferi HSP- We also isolated by excision the 44-kDa band.

Our attempts to sequence the amino-terminal peptide were unsuccessful, suggesting that the amino-terminal may be blocked. To circumvent this problem, we generated internal fragments of the 44-kDa aoHGE protein by trypsir digestion. The amino acid sequence P p 68 of one fragment, designated the 44-1 polypeptide, is set forth in SEQ ID NO: 5. We designated a second fragment of the 44 kDa aoHGE NCH-1 protein as the 44-2 polypeptide. The amino acid sequence of the 44-2 polypeptide is set forth in SEQ ID NO: 6. Figure 6 illustrates the A search of the Genbank Database revealed that the 44-1 polypeptide is approximately homologous with a region from amino acid 130-138 of the major surface protein 2 (MSP-2) of Anaplasma marginale.

The 44-2 polypeptide is homologous with a region of MSP-2 from amino acid 362-372. The MSP-2 protein is encoded by a gene which is one member of a large family of genes with a high degree of homology in A. marginale genome. A. marginale is an important veterinary erythroparasitic pathogen and MSP-2 may confer protection against A. marginale infection Palmer et al., "The Immunoprotective Anaplasma marginale Major Surface Protein 2 Is Encoded by A Polymorphic Multigene 20 Family," Infec. Immun., 62, pp. 3808-3816 (1994); G. H.

Palmer et al., "Molecular Basis For Vaccine Development oo Against Anaplasmosis and Babesiosis," Vet. Parasitol., S" 57, pp. 233-253 (1995)]. Based on its homology with MSP-2, we believe that the 44-kDa aoHGE protein contains protective epitopes.

Those of skill in the art would uncerstand br that other aoHGE polypeptides, from The NCH-1 isolate and from other strains of aoHGE, which are useful for the detection, treatment or prevention of human 30 granulocytic ehrlichiosis or for the study of the pathenogenesis of the disease may be isolated and 0.

i. 69 sequenced without undue experimentation according to the methods described herein.

Example V Isolation of the DNA Encoding the 44 kDa Protein To obtain the complete sequence of the 44 kDa aoHGE protein obtained as described in Example IV, and the DNA encoding that protein, we use the amino acid sequences of the 44-1 and 44-2 aoHGE NCH-1 polypeptide to design synthetic degenerate oligonucleotide primers for PCR amplification of larger regions of the DNA encoding the 44 kDa aoHGE NCH-1 protein from genomic aoHGE DNA.

Those of skill in the art will appreciate that genomic aoHGE DNA may be isolated according to any of a variety of methods known in the art. See, for example, J. Sambrook et al., supra.

The skilled artisan will further appreciate that PCR amplification of the regions of the DNA encoding the 44 kDa aoHGE polypeptide may be performed 20 by any of a variety of methods known in the art.

S* "The °CR product may be isolated and purified according to any methods known in the art, for example, isolating the product by agaraose gel electrophoresis and purifying using gene clean (BIO 101) according to the manufacturer's instructions.

Using these amplified products, we screen a genomic or cDNA library of aoHGE DNA to obtain the gene encoding the 44-kDa polypeptide. We then sequence the Isolated DNA by any method known in the art, for 30 example, the dideoxy chain termination method.

oe* 0 70 Alternatively, synthetic degenerate oligonucleotide primers may be used directly to screen genomic or cDNA libraries of aoHGE.

Those of skill in the art would be able to isolate the DNA encoding other aoHGE polypeptides without undue experimentation using the methods described herein.

Example VI Identification of Immunodominant aoHGE PolvpeDtides To identify immunodominant aoHGE polypeptides, we performed an immunoblot of lysates of aoHGE NCH-1 infected HL-60 cells proteins, prepared as described in Example III, using twenty sera from 13 patients with documented E. chaffeensis infection. Dr.

J.G. Olson (CDC, Atlanta, GA) kindly provided the patient sera. None of the E. chaffeensis sera reacted with the 40, 44, 65 and 80 kDa aoHGE proteins. One sera out of the twenty reacted weakly with the 110 kDa aoHGE protein and another was reactive with a 120-kDa 20 aoHGE protein. In contrast, sera from eighteen patients infected with aoHGE reacted with all the aoHGE proteins. These results suggest that the 40, 44 and kDa aoHGE polypeptides are immunodominant aoHGE Spolypeptides.

We selected E. chaffeenss because it is the causative agent of the other major human ehrlichiosis, human monocytic ehrlichiosis. However, one of skill in the art will understand that antisera from other bacterial infections may also be used. For example, 3c scme cross-reactivty between aoHGE antigens and sera ro.m patien:s with a history of Lyme disease have been V 71 reported Wormser et al., "False-positive Lyme Disease Serology in Human Granulocytic Ehrlichiosis," Lancet, 347, pp. 981-982 (1996).

Those of skill in the art will further understand that other methods well known in the art, such as IFA or ELISA may also be used to identify immunodominant aoHGE proteins and immunodominant regions of aoHGE proteins without undue experimentation using the methods described herein.

Example VII Construction of An aoHGE Genomic Expression Library We constructed an aoHGE genomic DNA expression library in Lambda ZAP II (Stratagene) according to previously published methods (La Jolla, CA) Lam et al., Inf. Immun., 62, pp. 290-298 (1994) Briefly, we extracted the genomic DNA from the purified aoHGE by phenol/chloroform extraction.

To construct the library, we randomly sheared ug of genomic aoHGE DNA, blunt-ended with S1 20 nuclease, and methylated the EcoRl sites with EcoRl methylase. W- then ligated EcoRl linkers to the ends of the DNA molecules, digested with EcoRl and purified the fragments over a sucrose gradient. We isolated fragments of 1 to 9 kb and ligated overnight at 5°C in a 1:1 molar ratio with EcoRl digested, phosphatase treated Lambda ZAP II arms. We plated the packaged DNA with BB4 cells, incubated overnight and isolated the plaques.

Those of skill in the art would understand 30 tat aoHGE genomic DNA expression libraries of other 72 isolates of aoHGE may be constructed without undue experimentation according the methods described herein.

A. Screening of the Exrression Library We screened the expression library with serum from mice and humans infected with aoHGE to identify genes encoding aoHGE antigens that elicit antibody responses in both humans and mice. Such antigens are potentially useful as diagnostic reagents and in vaccines.

To screen the library, we used the picoBlue Immunoscreening Kit (Stratagene). We induced protein production from the recombinant plaques with 10mM

IPTG

and transferred the proteins to duplicate plaque lifts on nitrocellulose filters according to methods well known in the art.

We incubated one set of plaque lifts with the human serum from a patient admitted to the Yale-New Haven Hospital and diagnosed as having aoHGE infection.

We probed the other set with mice antiserum prepared as 20 described in Example III. After washing, we incubated the filters with a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-human or anti-mouse IgG antibody (Organon Teknika Corp., West Chester,

PA),

and used nitro blue tetrazolium (NBT) (Stratagene) and 25 5-bromo-4-chloro-3-indolyl phosphate

(BCIP)

(Stratagene) for color development. We identified seven clones that reacted with both human and mouse aoHGE antisera for further study.

We also screened the aoHGE expression library with sera from mice hyperimmunized with purified heatkilled aoHGE, prepared as described in Example III. We

I'

73 identified two additional clones, which we designated clone eM3 and clone eM4.

Example VIII Cloning of Immunoaenic aoHGE Genes Screening of an aoHGE NCH-1 genomic expression library, prepared as described in Example VII, revealed seven clones that reacted with human and mouse antisera.

We excised the pBluescript plasmid from two of the clones, clones E6 and E7, by infection of XL1- Blue E. coli cells and rescued with R408 helper phage according to the manufacturer's instructions. Using the recovered plasmid, we used T3 and T7 universal primers to obtain initial sequences of the plasmids.

From that initial sequence of 100-300 bp, we made new primers which we used to extend the sequences 100-300 bp at a time until we obtained the entire sequence.

Alternatively, one of skill in the art could readily generate a nested set of deletions in the DNA insert with the Erase-A-Base System (Promega, Madison, 20 WI) using SmaI to generate the 5' blunt end and BstXI to generate a 3' overhang), and then sequence the subclones using, the Sequenase Kit (United States Biochemical Corp., Cleveland, OH) and reconstruct the entire sequence using MacVector (International Biotechnology, Inc., New Haven, CT).

The n"-leotide sequence of the plasmid inserts from clones E6 and E7 were determined by the Yale Protein Purification and Analysis Facility using the Circumvent Thermal Cycle Dideoxy DNA sequencing kit 30 (New England Biolabs). Conditions for denaturation, 74 annealing and extension were: 94" C for 30 sec., 550 C for 20 sec, and 72" C for 20 sec., respectively.

Analysis of the DNA sequence of the insert from clone E6 revealed that we had isolated a clone containing at least one complete open reading frame.

The DNA sequence of clone E6 is set forth in SEQ ID NO: 1. The deduced amino acid sequence is set forth in SEQ ID NO: 2. We designated the first complete open reading frame We designated the protein encoded by the gene "E6".

Similarly, analysis of the DNA sequence of the insert from clone E7 revealed a partial open reading frame having the DNA sequence set forth in SEQ ID NO: 3. The deduced amino acid sequence is set forth in SEQ ID NO: 4. We designated the partial open reading frame "e7" and the antigen encoded by the gene "E7".

We conducted a search of GenBank (date) with the Genetics Computer Group Program (University of 20 Wisconsin Biotechnology Center, Madison, WI) Our :search revealed that we had isolated two novel aoHGE antigens. The sequence of e6 showed some homology with A. marginale MSP-2.

Using the same techniques, we isolated and 25 sequenced the inserts from clones eM3 and eM4.

Analysis of the DNA sequence of the insert from clone eM3 revealed that we had isolated a partial reading frame. Using probes derived from the DNA sequence of clone eM3, we isolated a complete open reading frame 30 from the genomic aoHGE library. Analysis of the DNA sequence revealed that it encoded the 44-kDa protein isolated as described in Example I' Figure 6 75 illustrates the location of the 44-1 and 44-2 polypeptides. The DNA and amino acid sequences of the 44-kDa protein are set forth in SEQ ID NOS: 10 and 11, respectively. The DNA sequence of clone eM4 is set forth in SEQ ID NO: 12. The deduced amino acid sequence is set forth in Figure 12.

Using oligonucleotide primers based on the DNA sequence of clone eM3, we amplified 5 additional DNA sequences using aoHGE genomic DNA as the template.

Those sequences, which we designated E5-3A, E5-3B, E5-5B and ES-6, are set forth in Figures 13-17, respectively. We discovered that sequences E5-3B, and E5-6, have regions of substantial homology with regions of the 44-kDa protein. As seen in Figures 18- 20, nucleotides 400-600 and 900-1300, approximately, of the 44-kDa protein, define regions of homology among the sequences.

Based on -our discovery of these conserved sequences and of the homology between the 44-kDa 20 protein and the MSP-2 protein in A. marginale, which is encoded by one member of a large family of genes, we believe that we have discovered a novel aoHGE gene family which we have designated the "44-kDa" gene family. The skilled artisan will recognize that, using 25 techniques that are well known in the art, one can readily synthesize probes and primers derived from the sequences disclosed herein and obtain DNA sequences of other cross-hyb-idizing members of the novel 44-kDa gene family of this invention.

SExample X Exo f th Ede 5 30 Example IX ExoressiOn of the E PlvppeDide o S p 76 To express the aoHGE genes of this invention, we utilized the pGEX-2T vector, which is capable of directing expression of cloned inserts as glutathione S-transferase fusion proteins [see J. Sears et al., "Molecular Mapping of OspA-Mediated Immunity to Lyme Borreliosis", J. Immuna.,, 147, pp. 1995-2000 (1991)] The vector also contains a thrombin cleavage site immediately following the GT protein, thus, allowing the recovery of recombinant proteins without the GT fusion partner.

We first used PCR to amplify the e6 gene lacking the sequences encoding the hydrophobic leader peptides. We chose to delete that sequence to ensure that the polypeptide would be expressed as soluble fusion protein rather than as a lipoprotein, which would be anchored to the cell membrane or might aggregate elsewhere in the cell during or after biosynthesis.

To facilitate subcloning, we amplified the e6 20 gene using a 5' primer with an additional EcoRl site and a 3' primer with a XhoI site (SEQ ID NOS: 8 and 9).

We used 50 ng of plasmid DNA excised from initial phage colonies using the R408 helper phage as a template for the genes.

We performed the PCR for 30 cycles with Initial template denaturation at 94 0 C for 1 minute, annealing at 55 0 C for 1 minute and extension at 72C for 2 minutes.

We digested the amplified gene products with 30 EcoRI and XhoI and cloned into the corresponding sites n the PMX plasmid. We then used -he ligation mixture to transform Escherichia coli DH5E according to methods I s 77 well known to those of skill in the art. We isolated colonies containing the recombinant plasmid on Luria broth plates supplemented with ampicillin.

We induced expression of the e6 gene as a glutathione S-transferase fusion protein by growing the transformed bacteria to logarithmic phase and adding 1 mM isopropyl-l-thi-beta-galactoside (IPTG) for 3 hours.

Alternatively, we subclone the aoHGE gene into the pETl5b vector, which is capable of directing expression of cloned inserts as fusion proteins with a series of six amino terminal histidines.

Alternatively, the skilled artisan will appreciated that the aoHGE polypeptides of this invention may be recombinantly expressed without a fusion partner using techniques well known in the art.

One of skill in the art could readily express the other aoHGE polypeptides of this invention without undue experimentation following the above-described techniques.

Example X Purification of Recombinant Fusion Proteins After inducing protein expression as described in Example IX, we place the E. coli in 25 phosphate buffered saline (PBS) with 1% Triton and subject them to sonication. We purify the glutathione S-transferase-aoHGE polypeptide fusion protein (GT-E6) from cell lysates as follows.

We separate the cell supernatant and pellet 30 by centrifugation at 800 rpm for 8 mins. and pass the supernatant containing the recombinant fusion proteins •over a glutathione--Sepharose 4B column (Pharmacia) 78 according to the manufacturer's instructions. We elute the fusion protein from the column using a solution containing excess glutathione and quantify using the Bradford assay.

In addition, to purify the aoHGE proteins without the glutathione S-transferase, we load the glutathione S-transferase fusion proteins over the glutathione-Sepharose 4B column, add thrombin to cleave the recombinant aoHGE protein from the GT and incubate io overnight at room temperature. We then elute the proteins with 50 mM Tris-CaCl 2 -NaC1, treat the eluent with anti-thrombin beads and centrifuge at 13,000 rpm.

To purify recombinant fusion proteins expressed in the pET15b vector system, we pass the supernatant containing the recombinant fusion proteins over a nickel column and we elute the fusion protein from the column with EDTA.

To purify aoHGE antigens that are not soluble in E. coli, we add SDS up to a concentration of 0.1% to 20 the lysate to enhance solubility.

One of skill in the art would understand that S• other aoHGE polypeptides of this invention may be readily purified without undue experimentation using these procedures.

S. 25 Example XI Preparation Of Antibodies Directed Against The Recombinant aoHGE olvoDerides Of This Invention We generate antibodies directed against the aoHGE polypeptides of this invention as follows. We 30 immunize mice (Frederick Cancer Research Center, Frederick, MD) subcutaneously with the aoHGE fusion 79 protein expressed as described in Example IX in complete Freund's adjuvant (CFA) and boost with the same amount of antigen in incomplete Freund's adjuvant (IFA) at 14 and 28 days according to published protocols. We immunize control mice in the same manner with recombinant glutathione S-transferase.

Fourteen days after the last boost, we collect sera from the immunized animals and use it to hybridize to Western blots of SDS-PAGE gels of recombinant aoHGE polypeptides. We detect antibodies elicited by the recombinant aoHGE polypeptides by immunoblotting and by ELISA.

Alternatively, a skilled artisan will recognize that antibodies directed against aoHGE polypeptides of this invention can be obtained by immunizing mice with cells expressing a DNA sequence encoding an aoHGE polypeptide of this invention.

Example XII Detection of Anti-aoHGE Polypeptide Antibodies By TFA and ELISA We detected anti-aoHGE antibodies in the plasma of .ic- with tick-oorne aoHGE infection by indirect immunofluorescence assay (IFA) and ELISA as follows.

We collected plasma from nice infected as described in Example III, at days 5, 17 and 24 after tick-feeding. We then used :te plasma in ELISA and indirect immunofluorescence assay (IFA) with aoHGEinfected HL-60 cells prepared as described supra as antigen.

30 A. IA i.

II

80 We suspended infected and uninfected (control) HL-60 cells in culture medium, washed 2x in PBS, air-dried and fixed in cold acetone (-20"C for min.) on 12-well Teflon®-coated multiwell slides

(CEL-

LINE, Hewfield, NJ). We placed twenty ul volumes of serial 2-fold dilutions of plasma form individual infected and control mice (starting at 1:40 dilution) in the wells of antigen-coated slides, incubated in humidified chambers at 37C for 30 min., washed 3x in PBS, then flooded with fluorescein-conjugated goat anti-mouse polyvalent immunoglobulin (Sigma, St. Louis, MO) at 1:100 dilution. We then incubated, washed, airdried the slides, coverslipped over PBS-glycerol and examined under a fluorescence microscope. We included negative control antigen (uninfected HL-60 cells) and negative control plasma (uninfected mice) each time we performed the assay.

B. EISA To detect and quantify the specific IgM and 20 IgG response to aoHGE, we used a modification of a previously described indirect ELISA [Rikihisa, Y. et al., "Clinical Histopathological and Immunological Responses of Ponies to Ehrlichia sennetsu and Subsequent Ehrlichia risticii Challenge," Infect.

25 Immun., 56: (1988)].

Briefly, we coated duplicate sets of 96-well microtiter plates (corning Glass Works, Corning,

NY)

w ith 1 x 1 0 aoHGE-infected or uninfected (control)

HL-

60 cells. We fixed the plates in 10% neutral buffered formalin, washed with PBS-Tween 20, then blocked for 1 hnr. with 200 !l blocking buffer (3 gelatin in PBS, SC.51 Tw'een 20). We added 100 ul of serial 2-fcld w o.

81 dilutions of plasma samples in PBS-1% BSA, starting at 1:80, to each antigen-coated well and incubated for 1 hour at room temperature. We then washed the plates 3 times with PBST and incubated with 100 ml of horseradish peroxidase-conjugated goat anti-mouse polyvalent immunoglobulin (Sigma, St. Louis, MO) diluted 1:12,000 in PBS-1% BSA as above.

Finally, we added the substrate, 0.4% 3,3',5,5'-tetramethylbenzidine (TMB) in an organic base and 0.02% hydrogen peroxide in a citric acid buffer (Kirkegard and Perry Laboratories, Inc., Gaithersburg, mD) to each well, incubated 10 min. in darkness at then stopped the reaction by adding an equal volume of lN HCL. We measured the optical density (OD) of each well at 450 nm with a UV max ELISA reader (Dynatech Laboratories, Alexandria, VA).

We determined the signal to noise ratio and signal minus noise (S N) value for each dilution of every plasma sample by dividing and subtracting 2C respectively, the OD of the well with infected cells by the OD of the well with uninfected cells. We determined the cut-off point for each dilution by testing normal mouse sera and determining means and standard deviations (3 standard deviations above the means). Within-test variablility was approximately 15% when different batches of cul:ure were used.

o *As s'-^wn in Figure 8, we detected anti-aoHGE antibodies in the plasma of aoHGE infected mice by IFA and ELISA as early as 10 days after infection. The 30 response peaked at 17 days and diminished by 24 days.

Example Ac::ve irrzatiortn Of Mice (I (I 82 With E. coli Expressing Recombinant aoHGE PolpDeptides To determine if recombinant aoHGE NCH-1 polypeptides are able to elicit an immune response that is protective against human granulocytic ehrlichiosis, we induce expression of the protein in a culture of E.

coli as described sLra.

We then inject mice with live L, _L1 expressing an aoHGE NCH-1 protein and boost for a period of weeks. As a control, we inject mice with E.

call transformed with the vector, pDC197-12. We bleed the mice after the last boost and prepare an immunoblot, as described supra, to determine if the mice are synthesizing antibody against the recombinant aoHGE polypeptide.

We then challenge the mice with the various isolates of aoHGE to determine if active immunization elicits a protective immune response against a range of aoHGE isolates. We then sacrifice the mice and evaluate for infection and disease as described sur.

e identify protective aoHGE polypeptides by their ability to prevent aoHGE infection or disease.

Those of skill in the art would be able to identify protective aoHGE polypeptides from other isolates according to the methods described herein.

Example XIV Active Immunization Of Mice With Purified Recombinant aoHGE Pol yoep*ieds To demonstrate that the immune response generated by immunization with purified recombinant aoHGE polypeptides is sufficienr tc fully protect U I 83 against subsequent infection and the clinical manifestations of disease, we immunize mice with purified recombinant aoHGE polypeptides prepared as described in Example 10, and boost periodically. As a control, we inject mice with purified glutathione Stransferase. After the final boost, we bleed the mice and prepare an immunoblot as described in Example III, to determine if the mice are synthesizing antibody against the recombinant protein. We then challenge the mice with various isolates of aoHGE and evaluate them for infection and disease. We identify protective recombinant aoHGE polypeptides by their ability to prevent aoHGE infection and disease.

Example XV Identification of aoHGE Polypeptide Fragments That Elicit Protective Antibody Production B Cell EpitoDes One way to identify regions of aoHGE proteins that contain protective B-cell epitopes is to determine which regions of the protein are recognized by 20 monoclonal antibodies that confer protection against aoHGE infection.

We begin by producing fragments of the aoHGE S* protein. First, we PCR-amplify portions of the gene using oligonucleotide primers containing EcoRI and 25 BamHl sites. We then clone these fragments into DGEX- 2T in frame with the glutathione S-transferase protein.

oooeo SWe then transfc-" E.coli with the recombinant plasmids, and induce expression of the aoHGE polypeptide fragments as glutathione S-transferase fusion proteins.

Next, we prepare an immunoblot with whole cell extracts from E.coli expressing either the aoHGE fragment-glutathione S-transferase fusion protei s, or

Q

84 the full length aoHGE polypeptide-glutathione

S-

transferase fusion protein. We then incubate the immunoblot with a monoclonal antibody previously shown to confer protection against aoHGE infection (see Example

XVIII).

Binding of the protective monoclonal antibody to a fragment indicates that the fragment contains a protective (B cell) epitope. This example does not necessarily imply that the epitope recognized by the monoclonal antibody is the only protective epitope in the aoHGE protein. Nor does it imply that the region encoding the B-cell epitope recognized by the monoclonal antibody does not also contain a T-cell epitope. However, it does illustrate one method that may be used to identify protective epitopes of aoHGE proteins.

AAnother way to identify regions of aoHGE proteins that contain B cell epitopes is to use aoHGE polypeptide fusion proteins to absorb antibodies from 20 protective polyclonal serum. The various

T

7 -aoHGE or aoHGE-glutathione S-transferase fusion proteins are coupled to CnBr activated Sepharore in order to construct a column, using standard techniques.

We prepare polyclonal anti-aoHGE antiserum as in Example

III.

We then pass the serum over the aoHGE Spoypeptide-fusion protein column, to absorb antibodies which recognize the fusion protein. The residual serum is then used to passively immunize mice, as described 30 u ra.

We challenge the immunized mice with aoHGE, sacrifice and examine the tissues and blood for 85 infection and disease. We are able to determine which fusion proteins are able to elicit protective antibodies, because polyclonal rabbit serum containing antibodies which recognize such fusion proteins containing B cell epitopes will be depleted of the ability to confer protection to passively immunized mice.

Once we have localized various epitopes to regions of the fusion proteins, we conduct further analyses using short synthetic peptides of 5-35 amino acids. The use of synthetic peptides allows us to further define each epitope, while eliminating variables contributed by the non-aoHGE portion of the fusion protein.

Example XIV Active Immunization With Purified. Heat-killed aoHGE To determine whether the aoHGE polypeptides of this invention were able to elicit an immune response that is effective to protect against aoHGE 20 infection, we actively hyperimmunized five C3H mice subcutaneously in the back with 15 gg purified, -killed aoHGE, prepared as described in Example IV, in complete Freund's adjuvant (CFA) and boosted twice with an identical amount of antigen in incomplete Freund's 25 adjuvant (FA) at bi-monthly intervals. Prior to immunization, we dialyzed the purified aoHGE against ?BS, and treated at 56"C for 1 hour. We immunized five control mice with 10 gg BSA in an identical fashion.

Fourteen days after the final boost, we bled the mice and examined the sera from each animal for aoHGE antibodies by probing lysates of aoHGE-infected cells in im-unoblot as described in Example I.

II

86 aoHGE-immunized mice had high titers of aoHGE-specific antibodies, detectable by immunoblot at a serum dilution of at least 1:2,000.

We challenged the actively immunized and sham immunized mice by syringe and tick-borne inoculation.

For the syringe inoculation, we challenged mice actively immunized with purified, -killed aoHGE with 100 ul of blood from a mouse that had been infected with aoHGE for 2 weeks. We then sacrificed the mice days after challenge and evaluated for aoHGE infection by PCR using aoHGE specific 16S ribosomal DNA primers Pancholi et al., "Ixodes danmmini as a Potential Vector of Human Granulocytic Ehrlichiosis," J. Inf.

Dis., 172, pp. 1007-12 (1995) All 5 control mice developed aoHGE infection, based on aoHGE-specific DNA in the blood. In contrast, no aoHGE DNA could be detected in the blood of 4 of mice vaccinated with purified, -killed aoHGE.

For the tick inoculation, we placed 3-4 20 aoHGE-infected I. dammini nymphs which had fed to repletion on CD-1 mice that had been infected for 2 weeks with aoHGE. The aoHGE infection rate of the ticks was 85% as determined by visual inspection of the salivary glands using the Feulgen reaction

(S.R.

Telford et al., Proc. Natl. Acad. Sci. USA, 93, pp.

6209-6214, supra]. The ticks were allowed to engorge to repletion on immunized and control mice.

Fourteen days after the ticks had fallen off the immunized animals, we killed the mice with CO, and *30 obtained blood by cardiac exsanguination. We examined olood smears which were air dried and stained with -ffg.k p200 high power fields/smear) for the presence 87 of morulae and calculated the percentage of aoHGEinfected neutrophils. We considered the presence of 1 or more definitive morulae as a positive.

In addition, at necropsy, we inoculated 100ul of anticoagulated blood from each mouse into culture flasks containing 5 ml of 5 X 105 to 1 X 106 HL-60 cells per ml. We determined aoHGE infection of the cells at 2, 3, 4 and 6 weeks after inoculation.

Finally, mice were examined for aoHGE by PCR using aoHGE specific 16S rDNA, supra.

AS shown in Table 1, 5 of 9 control mice had morulae in peripheral neutrophils, 6 of 9 control mice were culture positive and 9 of 9 control mice were PCR positive for aoHGE DNA in the blood.

In contrast, no morulae were detected in blood smears of the immunized mice (Fisher Exact Test, P 0.015 compared with control mice)-and aoHGE could not be cultured from any of the immunized mice (Fisher Exact Test, P 0.005, compared with control mice).

20 Five of nine immunized mice were PCR positive (Fisher Exact Test, P 0.041 compared to control mice).

The p-sit',e PCR rcZults may, in part, be explained by the extreme sensitivity of the assay. In a quantitative PCR study, we detected product using 5 X 25 1 0 6 g of aoHGE DNA was used as the template. aoHGE has a chromosome that migrates at approximately 700 kb in pulse-field gel electrophoresis. By estimating that 1 mole of aoHGE has a molecular mass of approximately 4.6 X 10' g (7.0 X 105 bp X 660 g/bp), we calculate that 1 aoHGE has an approximate molecular mass of 7.6 X 10 16 g (4.6 X 10- g/mole 6.02 X 12C crganisms/mole), we -88 estimate that the POCR assay can detect a single aoHGE organism.

The data indicate that active immunization with purified, killed aoHGE in CFA elicits a protective immune response.

TABLE 1 Irmunogen Mouse Ctue Mor"I ,1lei

PCR

Faxper.imen t 1 CFA, control I+ F2 3 4 aoHGE-iysates

S

6 7 7 Experiment 2 CF-control I .4 aoHGE-ivsates6 T 7 a a a a a *9 1 10 D 1 L 89 Example XVII Identification of aoHGE Polypeptide Epitopes That Elicit Cross-Protective Antibodies Antibodies elicited in the actively immunized mice that are directed against epitopes that are shared among various isolates of aoHGE, will confer protection against infection with these various isolates of aoHGE.

To determine which epitopes of aoHGE polypeptides are able to elicit such antibodies, we immunize mice with the various aoHGE polypeptide fusion proteins, and challenge the mice with various isolates of aoHGE as described, ura. We inoculate C3H mice with various aoHGE isolates to determine which are infective and then inoculate the various infective isolates into mice which have been actively immunized with aoHGE polypeptide fusion proteins. We examine the mice for signs of infection and disease. We design a vaccine around the epitopes that are shown to confer protection S.*oo against infection with many different isolates of 20 aoHGE.

To determine the longevity of protection, we immunize mice with aoHGE polypeptide fusion protein and boost. We then infect the mice with aoHGE and evaluate for infection and disease at 6 months after challenge.

25 Example XVIII Decrease In Spirochete Load In i-cks £eedinc On Imm.unized Animals Previous studies with B. burgdorferi have shown that immunization of mice with recombinant OspA can eliminate the spirochetes from ticks feeding on the immunized animals Fikrig et al., "Elimination of 90 Borrelia burgdorferi from vector ticks feeding on OspAimmunized mice", Proc. Natl. Acad. Sci., 89, pp. 5418- 5421 (1992)]. Thus, to determine if aoHGE are killed when infected ticks feed on animals immunized with the aoHGE polypeptides of this invention, we conduct the following experiment.

We place ticks, infected with aoHGE as described in Example III, on mice immunized with GT (control), with aoHGE-GT fusion proteins. After feeding to repletion, the ticks are allowed to naturally detach over water. Approximately ten days post-repletion, we homogenize individual ticks in PBS and spot aliquots on slides. We allow the slides to air-dry, fix in cold acetone and assay by direct or indirect immunofluorescence.

For the direct immunofluorescence assay, we incubate the slides with FITC-conjugated anti-aoHGE antiserum, mount under a coverslip and examine on a Zeiss Axioscop@ Fluorescent Microscope. We quantify 20 the aoHGE by counting the number of fluorescing cells in approximately 20 fields per slide.

One of skill in the art would understand that the effect of immunization with other aoHGE polypeptides of this invention on aoHGE in ticks can be readily determined without undue experimentation using the methods taught herein.

Example XIX Passive Immunization of Mice To investigate whether passive immunization with anti-aoHGE antserum can confer protection against aoHGE infetic and HGE, we perfcrmed the following aoHGE immunizarion study.

I'

91 We produced polyclonal mouse anti-aoHGE antiserum by inoculating C3H mice with 15 ug of heatkilled aoHGE lysate prepared as described in Example IV in complete Freund's adjuvant (CFA) and boosted twice with the same preparation in IFA. We immunized control mice with normal serum. Two weeks after the last boost, we bled the animals for serum.

We then passively immunized 3-5 naive mice intradermally with 200 ul aoHGE antiserum diluted in PBS in three separate experiments. Control mice were passively immunized with normal mouse serum. One day following passive immunization, we challenged immunized and control mice with aoHGE by intraperitoneal inoculation with 50 pl of blood from mice that had been infected with NCH-1 isolate two weeks earlier, an by tick transmission using 3-4 aoHGE infected ticks. Mice were boosted with 200 ul of aoHGE antiserum diluted 1:5 with PBS on days 4, 8 and 12 after challenge.

20 On day 14 after challenge, we sacrificed the mice and analyzed their blood and tissues for signs of aoHGE infection and disease. Specifically, we evaluated 100 pu of anticoagulated blood in a coulter counter (Antech Diagnostics, Farmingdale, NY) for leukopenia. We also removed and immediately weighed the whole spleen from each animal at necropsy. We assessed infection by examination of peripheral blood smears for morulae, culture in HL-60 cells and by PCR using 165 rDNA aoHGE-specific primers.

30 As set forth in Table 2, morulae were detected in blood smears of 7 of 11 control mice compared to i of 12 immur.-zed mice (Fisher Exact Test, 92 P =0.008). Likewise, we recovered aoHGE by culture from 7 of 11 control mice compared to 1 of 12 immunized mice.

Using PCR, aoHGE was amplified from blood in 10 of 11 control mice compared to 4 of 12 immunized mice (Fisher Exact Test, P= 0.001). However, we determined that immunized mice that were PCR positive had fewer aoHGE in peripheral blood than the control mice. Using serial dilution PCR, amplified DNA was discernible in serum from control mice at a dilution of 10'-108 whereas product could only be obtained from serum of immunized mice up to a dilution of 10'. As noted, supra, we estimate that the PCR assay can detect a single aoHGE organism. Accordingly, passive immunization either conferred complete protection or lessened the severity of aoHGE infection.

In terms of clinical symptoms, the control mice but not the protected mice exhibited neutropenia (462 cell/m n 280 SD compared 3,240 cells/mm 1,340 20 SD) and splenomegaly (0.27 g 0.05 SD compared to O.12g 0.03 SD).

e e 0 93 TABLE 2 Iuuge(aH -calne) JMouse culture Morulae PC [ixper~iment I (tick-borne) NMS, control aoHGE-antisera .Exper.i.ment 2 (tick-borne) NMS, control aoHGE-antisera E~xperlment. 3 'syringe nocula NMS. control aoHGE-an:ilsera

N

pI j 94 Example XX Passive Immunization With Anti-aoHGE Monoclonal Antibodies To determine if immunity to aoHGE infection and disorders caused by such infection can be conferred by passive immunization with an anti-aoHGE monoclonal antibody, we prepare anti-aoHGE monoclonal antibodies by fusion of spleen cells from mice infected with aoHGE to mouse P3X63Ag8 myeloma cells, according to methods well known to those of skill in the art. We then determine the isotypes of the monoclonals, and select antibodies reactive with aoHGE for aoHGE immunization studies.

Those of skill in the art would understand that monoclonal antibodies directed against other isolates of aoHGE and against individual aoHGE proteins may be generated according to the methods described herein.

We then passively immunize mice with supernatant from monoclonal antibody producing cells, and challenge the animals with aoHGE. We then sacrifice the mice and examine the blood and tissues for signs of aoHGE infection and disease.

Example XXI Passive Immunization With Antibodies Directed Against S25 Recomb:nant aoHGE PolvpeDtides To determine if antiserum from animals immunized with recombinant aoHGE polypeptides confers protection, we passively immunize mice with serum from mice immunized with the recombinant aoHGE polypeptide 0 30 cr wi:h E. col- expressing a recombinant acHGE zclypeptide as describoedz n xample We then 95 challenge the passively immunized mice aoHGE shortly after the immunization as desribed, supra. We then sacrifice the mice and examine the blood and tissues for signs of aoHGE infection and disease.

One of skill in the art would understand that to detect a protective effect, one can vary the experimental conditions. For example, one could obtain antiserum by immunization with a recombinant polypeptide without GT, collect antiserum at a different time point when the titer is higher, passively immunize with more antiserum, decrease the aoHGE challenge dose, or other means known in the art.

Example XXII Determination of Protective Etoe We construct recombinant genes which will express fragments of the aoHGE polypeptides in order to determine which fragments contain protective epitopes.

First, we produce overlapping 200-300 bp fragments S. which encompass the entire nucleotide sequence of each of the genes, either by restriction enzyme digestion, 20 or by amplification of specific sequences of using PCR and oligonucleotide primers containing restriction endonuclease recognition sequences, as described supra.

We then clone these fragments into an appropriate expression vector, preferably a vector from which the fragments will be expressed as fusion proteins, in order to facilitate purification and increase stability. For example, the gene fragments could be cloned into pGEMEX (Promega, Madison, WS) and expressed as T7 gene 10 fusion proteins. Such proteins would be insoluble and thus easily purified by recovery of the insoluble pellet fraction followed by 96 solubilization in denaturants such as urea.

Alternatively, the fragments could be expressed as glutathione S-transferase fusion proteins as described above. We then transform appropriate host cells and induce expression of the fragments.

One way to identify fragments that contain protective B-cell epitopes is to use the individual purified fragments to actively immunize mice, as described above. After challenge of the mice with o0 aoHGE, we determine the presence of infection by blood and spleen cultures in HL-60 cells and by examination of peripheral blood smears for granulocytic morulae.

Another technique to identify protective epitopes is to use the various fragments to immunize mice, allow ticks infected with aoHGE to feed on the mice, and then determine whether the immune response elicited by the fragments is sufficient to cause a decrease in the level of aoHGE in the ticks. Any epitopes which elicit such a response, even if they are 20 not sufficient by themselves to confer protection against subsequent infection with aoHGE, may be useful In a multicomponent vaccine.

Once we have localized various epitopes to particular regions of the fusion proteins, we conduct further analyses using short synthetic peptides of 5-35 amino acids. The use of synthetic peptides allows us to further define each epitope, while eliminating any variables contributed by the non-aoHGE portion of the fusion protein.

f (I I 97 Example XXIII Preparation of a MulticomDonent Vaccine We determine which of the protective epitopes is able to elicit antibodies that will protect against subsequent infection with isolates of aoHGE other than the isolate from which the protective polypeptide was cloned. We then design a vaccine around those epitopes. If none of the protective epitopes is able to confer protection against infection with other isolates of aoHGE, it may be particularly advantageous to isolate the corresponding aoHGE polypeptides from those isolates. A multicomponent vaccine may then be constructed that comprises multiple epitopes from several different aoHGE isolates. Such a vaccine will, thus, elicit antibodies that will confer protection against a variety of different isolates.

A skilled artisan would appreciate that with .knowledge of the protective epitopes as determined by the methods described herein, one can readily design a mrulticomponent vaccine comprising those epitopes and 20 epitopes that have been shown to be protective against other diseases, Particularly other diseases known to be transmitted by the I. scapularis tick, such as Lyme disease and babesiosis.

Example XXIV Identification of T cell eitDes Stimulation in animals of a humoral immune response containing high titer neutralizing antibodies will be facilitated by antigens containing both T cell and B cell epitopes. To identify those polypeptides containing T cell epitopes, we infect mice with aoHGE in complete Freund's adjuvant, as described supra.

Shortly after priming, we harvest the lymph nodes and Lu 98 generate in vitro T cell lines. These T cell lines are then cloned using limiting dilution and soft agar techniques. We use these T cell clones to determine which polypeptides contain T cell epitopes. The T cell clones are stimulated with the various polypeptides and syngeneic antigen presenting cells. Exposure of the T cell clones to the polypeptides that contain T cell epitopes in the presence of antigen presenting cells causes the T cells to proliferate, which we measure by H-Thymidine incorporation. We also measure lymphokine production by the stimulated T cell clones by standard methods.

To determine T cell epitopes of the polypeptides recognized by human T cells, we isolate

T

cell clones from aoHGE-infected patients of multiple HLA types. T cell epitopes are identified by stimulating the clones with the various polypeptides S.*and measuring H-Thymidine incorporation. The various T cell epitopes are then correlated with Class II HLA 20 antigens such as DR, DP, and DQ. The correlation is :o performed by utilization of B lymphoblastoid cell lines expressing various HLA genes. When a given T cell clone is mixed with the appropriate B lymphoblastoid cell line and an aoHGE polypeptide, the B cell will be able to present the polypeptide to the cell.

Proliferation is then measured by 3-Thymidine ncorporation.

Alternatively, T cell epitopes may be identified by adoptive transfer of T cells from mice 30 immunized with various of the aoHGE polypeptides of this invention to naive mice, according to methods well known to those of skill in the art. [See, for example, 99 M.S. DeSouza et al., "Long-Term Study of Cell-Mediated Responses to Borrelia burgdorferi in the Laboratory Mouse", Infect. Immun., 61, pp. 1814-22 (1993)].

We then synthesize a multicomponent vaccine based on different T cell epitopes. Such a vaccine is useful to elicit T cell responses in a broad spectrum of patients with different HLA types.

We also identify stimulating T cell epitopes in other immunogenic aoHGE polypeptides or in non-aoHGE polypeptides and design multicomponent vaccines based on these epitopes in conjunction with B cell and T cell epitopes from the aoHGE polypeptides of this invention.

Example XXV Construction of fusion proteins ComDrising T and B cell epitoes After identifying T cell epitopes of the aoHGE polypeptides, we construct recombinant proteins comprising these epitopes as well as the B cell epitopes recognized by neutralizing antibodies. These fusion proteins, by virtue of containing both T cell 2 0 and B cell epitopes, permit antigen presentation to T cells by B cells expressing surface immunoglobulin.

These T cells in turn stimulate B cells that express surface immunoglobin, leading to the production of high titer neutralizing antibodies.

We also construct fusic. proteins from the aoHGE polypeptides by linking regions of the polypeptides determined to contain B cell epitopes to strong T cell epitopes of other antigens. We Ssynthesize an oligonucleotide homologous to amino acids 30 120 to 140 of the Hepatitis B virus core antigen. This region of the core an.tgen has been shown to contain a

I

100 strong T cell epitope Millich, et al., Ijra The oligonucleotide is then ligated to the 5' and 3' ends of segments of DNA encoding the B cell epitopes recognized by neutralizing antibodies. The recombinant DNA molecules are then used to express a fusion protein comprising a B cell epitope from the aoHGE polypeptide and a T cell epitope from the core antigen, thus enhancing the immunogenicity of the polypeptide.

We also construct fusion proteins comprising epitopes of the aoHGE polypeptides as well as epitopes of the tetanus toxoid protein.

We also construct a plasmid containing the B cell epitopes of various of the aoHGE polypeptides incorporated into the flagellin protein of Salmonella Bacterial flagellin are potent stimulators of cellular and humoral responses, and can be used as vectors for protective antigens Newton, C. Jacob,

B.

Stocker, "Immune Response To Cholera Toxin Epitope Inserted In Salmonella Flagellin", Sci5en, 244, 20 PP. 70-72 (1989)].

.i We cleave the cloned H 1-d flagellin gene of SSalmonella muenchens at a unique Eco RV site in the hypervariable region. We then insert blunt ended DNAs encoding protective B cell epitopes of the polypeptides 25 using T4 DNA ligase. The recombinant plasmids are then used to transform non-flagellate strains of Salmonella for use as a vaccine. We immunize mice with live and formalin killed bacteria and assayed for antibody production. In addition, we test spleen cells for 30 proliferative cellular responses to the peptide of nterest. Finally we challenge the mice immunized with this agent with aoHGE as described 101 We also construct fusion proteins comprising B cell epitopes from one of the aoHGE polypeptides and T cell epitopes from a different aoHGE polypeptide or other immunogenic aoHGE polypeptides. Additionally, we construct fusion proteins comprising T cell epitopes from aoHGE polypeptides and B cell epitopes from an aoHGE polypeptide and/or other immunogenic aoHGE polypeptides. Construction of these fusion proteins is accomplished by recombinant DNA techniques well known to those of skill in the art. Fusion proteins and antibodies directed against them, are used in methods and composition to detect, treat, and prevent human granulocytic ehrlichiosis as caused by infection with aoHGE.

Example XXVI Construction Of An aoHGE Fusion Protein From aoHGE Polypeptides Of Different Isolates :I We identify protective epitopes within aoHGE p: polypeptides from an aoHGE isolate other than NCH-1 by 20 producing overlapping fragments of the protein and testing each the for presence of T cell and B cell epitopes, and/or for the ability to confer protection against aoHGE infection and disease in our animal model system. We then select the fragments which encode both protective epitopes and amino acid sequences that differ from each other and use these fragments to construct aoHGE polypeptide fusion proteins comprising protective epitopes from both isolates. Such fusion proteins confer protection against a broad range of 30 aoHGE isolates.

102 Example XXVII Oral Immunization Of Mice With aoHGE PolvepDtide Fusion Proteins To determine whether oral vaccination with an aoHGE polypeptide is sufficient to protect mice from infection and disease as caused by aoHGE, we culture

E.

coli harboring the aoHGE polypeptide-containign plasmid at 30"C. We induce expression of the fusion protein by raising the temperature to 42*C for 2 hours, then harvest the bacteria by centrifugation and resuspend in PBS at a concentration of 1 x 10 9 bacteria/ml.

We use 0.1 ml of this suspension to orally inoculate mice. Inoculation may be performed by gavage using a ball tipped metal needle. We boost the mice with the same amount of bacteria on days 10, 20, 30 and 40. We inoculate control mice in a similar fashion with bacteria lacking the pl9 7 -aoHGE polypeptide plasmid. We bleed the mice 7 days after :th- second and fourth boosts and conduct immunoblots on extracts of aoHGE, as described in Example I, to detect and 20 quantify antibodies against the aoHGE polypeptide.

Fourteen days after the last boost, we challenge the mice by inoculation with aoHGE and evaluate for infection and disease.

While we have described a number of embodiments of this invention, it is apparent that our basic constructions may be altered to provide other embodiments which utilize the processes and products of S* this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the 30 appended claims, rather than by the specific embodiments which have been presented by way of example.

Page(s) (23are claims pages they appear after the sequence listing 103 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Yale University STREET: 451 College Street CITY: New Haven STATE: CT COUNTRY: USA ZIP: 06520 (ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR THE PREVENTION AND DIAGNOSIS OF HUMAN GRANULOCYTIC

EHRLICHIOSIS

(iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT Unassigned FILING DATE: Concurrently Herewith

CLASSIFICATION:

(vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/027,180 FILING DATE: 01-OCT-1996 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1669 base pairs eae*: TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 357..974 e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTCGGGTACA GGTTTTTATA TTTGGAGCTC TTGTACTGTG TTTACCCCGG GATTTATTAT I: TGGGTAGGCT TGATATTCAG GTTCTATCAT CGCAGCTATT CATGGCGCTA TTACAGATAA 120 ATTTGGAATT TTGTANATTG GTTATCTAGT ATTCTATTAT TGGATTCTA AGGTAAGACA .180 TAGTGCACA7 7GCT7TGAGC AC7AATCG-A TTCC7GTCG T3AATTATTC ATCTAAACCA 240 104 ATACTAATTA CAATGCTGCA ATAGTTTTTA GCTATTTAAC CCCGATATCT

ATTGCATTGC

GGCAGTATTA TTAGAAAAAG ATGGAGCAGA ATATCTACTA GCTAAGGAGT

TAGCTT

ATG ATG TTG TTA CTG GAC AGA CTG ATA AGC TTG CTG CTG CTC TTG CCA Met Met Leu Leu Leu Asp Arg Leu Ile Ser Leu Leu Leu Leu Leu Pro 1 510 AGA CCT CCG GGA MAG ATA TCG TTC AGT TTG CTA AGG CGG TGG AGA TTT Arg Pro Pro Gly Lys Ile Ser Phe Ser Leu Leu Arg Arg Trp Arg Phe 25 CTG CTC CTA AGA TCG ATG AGA AGG TTT GTG CGA CGA MAG ATC GCG MAG Leu Leu Leu Arg Ser Met Arg Arg Phe Val Arq Arg Lys Ile Ala Lys 40 GTG GGA GTG GTA ATA MAT ACG GTG TTT ACG GAG CGA CTA CCG ATG ATT Vai Gly Val Val Ile Asn Thr Val Phe Thr Gin Arg Leu Pro Met Ile 55 CAT CAA CAT ATC CAA GGG CAC GAT GTG GTG CTG CAG GGC ACA ATA GTC His Gin His Ile Gin Giy His Asp Val Val Leu Gin Gly Thr Ile Val 70 75 300 356 404 452 500 548 596 AGA GTG GMA GTC CMA Arg Val Giu Val Gin GCA CTC CAC AGG TTT TGC ATG ACT TTG CGG AGA Aia Leu His Arg P MAA CTC TGT GGA MAT GGT AGT MAG MAC Lys

GGA

Gi y

GAC

Asp TT A Leu 145

CTT

Leu Pro

TCC

Se r Le u

ACT

Thr

CTA

Leu 13 0

GC

Al a

CTA

Le u

GG'T

Gly AT G Me t Cys

CCA

Pro 115

ACA

Thr

A

Lys

CTT

Lej

GMA

GI u

CAA

Gin Gly 100

MAG

Lys

MAG

ACT

Th r

TGT

C ys

AC--

Thr 180

TGT

C ys

CCA

Pro

CTC

Leu

ATT

lie

GTT

Val1 165

CAT

His

CAT

GMA

Gin

MAC

As n

GMA

150

TTT

Phe

AAT

As n

CGT

ACT

T.h r

TCT

Ser 135

GGG

Gi y

GGA

Gly

ACT

Th r

ATT

Asn Gly Ser L ys

MAT

Asn 120

GAC

Asp

GGT

Gly

TTT

Ph e

ACT

Th r

GOA

Asn 105

GAC

Asp

GMA

Ci u

GAG

Gin

TTA

Len

AAT

Asn 185

T

T

A

A

L

V

4 1*

AC

~he Cys Met 90 GG CCC ACA rp Pro Thr AC GCC AMA sn Ala Lys ALA ACC ATA ys Thr Ile 140 TT GTG GMA al. Val Glu 155 :A ACG ATT r Thr Ile 70 G TAT CTC ~r Tyr Leu ;G AGO ATC :g Arg lie Thr Len TCA AGC Ser Ser 110 GCT GTC Ala Vai 125 GTA GCA Val Ala ATC AGG Ile Arg GCG MAG Aia Lys ATA CAC Ile His 190 AGG AGA Arg Arg 205 Arg

GGC

Gly

GCT

Ala

GGG

Gly

GCG

Ala

GCA

Al a 175

GAC

Asp Arg

ACT

Thr

GGA

Gly

TTA

Le u

TTT

Phe 160

GCA

Al a

ACA

Thr 644 692 740 788 836 884 932 His Arg Ile Ala Arg Ai 200 195 -AMTATC-ACA ATTTCCAAAT ATTOATCTTO

GGATAAAT:

CTAAZCC2SC CATAGAAG37 3AAGACAC7A CCCA~cG..;: TAGTAACGAC

CCCAACACT

=TAAGTTAA TAATTCT:GC 1034 1094

CTCTCTAATT

ATCGATTCAT

ATACCTTAAT

AA.AAACCCAC

AAAGTCCTAT

AGTGTTCATG

TATACAATGG

GTCAGATTCG

GGGCTATTGA

CTATGGCAGC

CACAACTCAT

TGCCGCTTTA

CAAGTCCCAA

GATAATAGTC

TCCCATTACT

AAGGTCCACG

TTCGCAACAT

CAACGTGTGT

TATGGCGATT

TGGAGCTCCC

TCGTGTTGCG

ATCACGACCT

CGCACTTATG

CGTTGAACCA

TAATACAACA

CAAAGATGGC-

ATAGTTTAGA

TTTGATTTCG

ATTTACG TAG

CCCGGGCATG

105

TTAGGTTAGG

AAACTCCACG

CAAATG CAMT

ACTATGGTCC

ACTCTTCAAT

GCGCACGAAC

TACATTATCG

GGCATTGTTC

TCTTAAGGCC

GGTTA

CAGAAAATAG GACTTATGGG ACAATGAGTC CTCAAAGTAC GTCGTAGATT

ATAGATTCCT

TATTCAATAG

ATCCTATAGT

CACACGCGAA

AGAAAACTGA

CAGTTGAAAA

CCACAATACG

GATAAGACGT

CTTATAGAAC

ATAAACGGGA

ACAAGACTGT

TTAAGCTATA GTTCGAATAG 1154 1214 1274 1334 1394 1454 1514 1574 1634 1669 INFORM.ATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 206 amino acids TYPE: amino aciai TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Met Leu Leu Leu Asp Arg Leu Ile Leu Leu Leu Leu 1.

Arg Pro Pro Gly 20 Ile Ser Phe Ser 25 Phe Leu Arg Arg Leu Pro Arg Phe Ala Lys Leu Leu Leu Val Gly Val 50 Arg Ser Met Arg Val Arg Arg Val Ile Asn Phe Thr Glu Pro Met lie Gin His 7le Gin Asp Val Val Leu Cys Gly Thr Ile Arg Val. Gilu Vai Leu His Arg Met -tr Leu Lys Leu Cys Gly Thr Pro 115s Asp Leu Thr Gly Ser Lys As n 105 Asp Pro Thr Ser Arg Arg Gly Thr Ala Gly Pro Glu Thr Asn Ala Lys Lys Leu Asn Giu Lys Thr 130 Leu Ala 145 Ala Giy Leu Lvs T~ :1e G' -,IVal 155 Ara Ala 106 Leu Leu Leu Cys Val Phe Gly Fne Leu Ser Thr Ile Ala Lys Ala Ala 165 170 175 Pro Gly Giu Thr His Asn Thr Thr Asn Thr Tyr Leu Ile His Asp Thr 180 185 190 Ser Met Gin Cys His Arg Ile Ala Arg Arg Arg Ile Arg Arg 195 200 205 INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1753 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (Ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1. .1407 (xi) SEQUENCE DESCRIPTION: SEQ :D NO:3: CCT GAG ATA CAT TTA GAT TTG TAT TGG OCA CTA AMA AAT CCA TTC AAA 48 Pro Glu Iie His Leu Asp Leu Tyr Trp Ala Leu Lys Asn Pro Phe Lys 1 5 10 AGT OGA TAC AAA GAC ATA TTT CAA MAT ACO CCT ATT ATG TTT TAT ATA 96 Ser Giy Tyr Lys Asp Ile Phe Gin Asn Thr Pro Ile Met Phe Tyr Ile 25 T AO IC O ATA GMA MG TTT AGA AAA. MG OTO ACT GAG CTT TAC AGA AAG 144 Tyr Asn Giu Lys Phe Arq Lys Lys Val Thr Glu Leu Tyr Arg Lys 0 35 40 Z7T MT7 T- MT TAT ACA GMA GGA ATA AGO CAG AGT AAA MT MAT AAA 192 o o 1eu Asn Ph e Asn Tyr Thr Giu Gly Ile Arg Gin Ser Lys Asn Asn Lys 50 55 o MAT TAT TTA ATT TTT TAT AAA MT MAC TGC CMA TAT TTA TAT GMA GTA 240 Asn Tyr Leu Ile Phe Tyr Lys Asn Asn Cys Gin Tyr Leu Tyr Giu Val o 65 70 75 oCAA AAA A-TA OAT TCT CCA AAACA T A CT77T ATT 288 Sl'rn Lvs Tle Asp Ser Pro Lys Ser Asn Val Giu Thr Leu Ile Tyr Phe *85 TAT GAG ATC AMA GMA ACT TAT OAT MAT CMA GMA CTA AAA MT TTT TTA 336 o. *o:.yr Glu le Lys Giu Thr Tyr Asp Asn Gin Giu Leu Lys Asn Phe Leu 100 105 110 OTT TAT 7TA MAG OCO TTG GMA MC AAC TTA CAC AGC ATT MAA ATA CMA 384 L.eu Tyr Leu Lys Al a Leu Giu Asn Asn Leu His Ser 'le Lys Ile Gin AAT CTA Asn Leu 130 TTT AAC Phe Asn 145 AGA CTC Arg Leu ATA AAT lie Asn

GAA

Glu

TCC

Ser

AAA

Lys

AAA

Lys

GGA

Gly

CTT

Leu

GAT

Asp

CAC

His

TCC

Ser

AAA

Lys

TAT

Tyr 165

CAA

AAA

Lys

GAG

Glu 150

CAA

Gin

GAT

CTT ACC Leu Thr 135 CAA GAG Gin Glu ATC AAC Ile Asn GAA ATC 107 ACC GAA CTA Thr Glu Leu CCA ATA ATA Pro Ile Ile 155 GAA AAA AGC Glu Lys Ser 170 ATT AAA AAT

CTA

Leu 140

AAT

Asn

TTA

Leu

TCA

Ser

GAG

Glu

TTT

Phe

AGA

Arg

GAA

Glu ATT CCA Ile Pro CAA AAC Gin Asn GAA TTT Glu Phe 175 TCA ATT Ser Ile

AAA

Lys

AAA

Lys 160

TTA

Leu

GTG

Val 432 480 180 Gin Asp Giu Ile Ile Lys Asn 185 190 CCT AAA MAT TTA GAA TAC AAT ATG GMA GGA AAT TTC ACG CTA TCT CAC Pro Lys Asn Leu GIu Tyr 195

GAT

Asp

TTT

Phe 225

AAA

Lys

TCA

Ser

GCT

a

ACT

Thr

ATA

lie 305 Leu

ACT

Thr

CA

Gin 210

AAA

Lys

GTT

Val

TTT

Phe Phe

ATC

Ile 290

GAC

Asp

GAA

G1u

A.AT

Asn

TAC

Tyr

GAT

Asp

TCA

Ser

TCA

Ser

AAC

Asn 275

TCT

Ser

OTA

Leu

TAT

Tyr

TTA

Leu

AAC

Asn

AAA

Lys

TCA

Ser

TTC

Phe 260

TCT

Ser

AGC

Ser

GAT

Asp

TCA

Ser

AM

Lys 340

ATC

Ile

AAA

Lys

A

Lys 245

TCA

Ser

GCC

Ala

TTC

Phe

AAA

Lys

GCT

Ala 325

ATA

Ile

AAA

Lys

GTT

Val 230

AAA

Lys

AAT

Asn

AAA

Lys

TTT

Phe

AAA

Lys 310

CTT

Leu

ACA

Thr Asn

TTC

Phe 215

GAA

Glu

GAA

Glu

GAA

Glu

AAA

Lys

ATA

Ile 295

ACA

Thr

GAA

GIL

GCA

Ala Met 200

GAA

Glu

TTT

Phe

CTA

Leu

AAA

Lys

TCA

Ser 280

TCG

Ser

TTA

Leu

ATA

lie

AAA

Lys Glu

AAT

Asn

TTA

Leu

ATA

Ile

ATT

ile 265

ACA

Thr

ATT

Ile

AAA

Lys

CCT

Pro

TAC

Tyr 345 Gly

GGA

Gly

AAC

Asn

AAA

Lys 250

TTA

Leu

ATT

Ile

AA

Lys

AAA

Lys

GTT

Va I 330

AGC

Ser Asn

AAA

Lys

ACA

Thr 235

GAA

Glu

GGA

Gly

GAT

Asp

ATA

Ile

TGOC

Cys 315

TTT

Phe

GAT

Asp Phe

TTA

Leu 220

TCT

Ser

GCA

Ala

ATA

Ile

TTT

Phe

AAA

Lyss 300

MAT

Asn GM A Glu

CTT

Leu

AGA

Arg

TCT

Ser

AAA

Lys

TTT

Phe 285

IGG

Trp Fro

GAT

Asp

ACC

Thr

ATT

Ile

CAA

Gin 270

ATA

Ile

CCT

Pro

GA-

AsD

GCA

Ala

ACT

Thr 350

TAT

Tyr

GAA

Glu 255

TAT

Tyr

GAT

Asp

TCT

Ser

TAT

AAA

Lys 335

TAT

Tyr

TTT

Phe 240

AGT

Ser

TTA

Leu

GAG

Glu

AAA

Lys

C-T

Leu 320

GGC

Giy

GAA

Glu Asn Lys Ile Lys 624 672 720 768 816 864 912 960 1008 1056 1104 AA ATA ATA ATA ACT Lys :le lie Ile Thr 355 AAA A-AC MTA; C:C AAA GGC TAC ATT MT GGC ACA Lys Asn Asn-. Pro Lys Gly Tyr :1e Asn Gly Thr 360 365 -108 GAA TTT TTG ATA TCT AMA GGA MAT GAT AAA AAC AGC M-C TTT TTT ATA Giu Phe Leu Ile Ser Lys Gly*Asn Asp Lys Asn Ser Asn Phe Phe Ile 370 375 380 AGC TTT TTA MAT GTT GMA AAM CAT ATC ATT CAT ACA ATT MAT TAT AAA Ser Phe Leu Asn Val Giu Lys His Ile Ilie His Thr Ile Asn Tyr Lys 385 390 395 400 ATT GMA AMA ATA MAT TCT MAG AMA TGG TTA ATT TTA MAT ATA GGG GGT Ile Glu Lys Ile Asn Ser Lys Lys Trp Leu Ile Leu Asn Ile Giy Gly 405 410 415 TCT TAT MAC ACA GTC MAG ATC CMA GAT GTA ATA MAT TAC TCT CMA ACA Ser Tyr Asn Thr Val Lys Ile Gin Asp Vai Ile Asn Tyr Ser Gin Thr 420 425 430 CTA MAT TTA ATG ATA CTA CCA TTA AAT MAT MT TTT GAT MAC AAA ATA Leu Asn Leu Met Ile Leu Pro Leu Asn Asn Asn Phe Asp Asn Lys Ile 435 440 445 AMA CTG MAT TCA AMA ATA AMA MT TTA ATT TTT TAT ACT MAT ATA AMA Lys Leu Asn Ser Lys Ile Lys Asn Leu Ile Phe Tyr Thr Asn Ile Lys 450 455 460 AA -,AT GMA MT AMA TAAATMATM GTAGTAAAAT ATTMATMCT

GGGTATAA

Lys Tyr Giu Asn Lys 1152 1200 1248 1296 1344 1392 1447 1507 1567 1627 1687 1747 1753 a baa.

a a a.

465~

TTATCCTMAG

ATATGMACGA

ATCCTGTATT

ACAGMATCAT

ATAACAAACT

A TAT G MAGMCATAA AAAGTATTTA ATC-TTTMATT

TAMACAA

CMACATMATA GACGTACATT CCGCATTGGA

AAMAGTCGGC

ATTGAA.AT TTMACATCAG MATTGGGMAT

GAMAGCATCT

TTTATACATA GCATCAAACC CAAAAGMATA

CTTTACAGCA

TATMMAGAA ATTCOAMGCC TATCAAA.AGC AAC3GTATAT

AGGTATMATC

ATTACAMACG

CATTCGAGMA

AM-GMAGTTT

AACACATTMA

INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 469 amino acids TYPE: amino acid TOPOLOGY: iinear fii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ :D NO:4: Pro Giu Ile His Leu Asp Leu Tyr Trp Ala Leu Lys Asn Pro Phe Lys 1 510 Ser Gly Tyr Lys Asp Ile Phe Gin Asr. Thr Pro ile met Phe Tyr Ile 25 :yr Asn 1le Giu Lys Phe Arq L7yS Lys Val Thr c-lu Leu Tyr Arq Lys 40 C. 11 J 109 Len Asn Phe Asn Tyr Thr Giu Gly Ile Arg Gin Ser Lys Asn Asn Lys 55 Asn Gin Tyr Leu Asn Phe 145 Arg Ile Pro Asp Phe 225 1,ys Ser Ala Thr ile 305 Leu Thr Lys Tyr Lys G1u Tyr eu 130 As r.

Leu Asn Lvs Gin 210 Lys Va I Pe Phe Ile 290 Aso Glu Asn Leu Ile Ile Leu 115 Glu Ser Lvs L s Asn 195 Tyr Asp Ser Ser As n 275 Ser Leu Tyr Zeu :le 255 Ile Asp Lys 100 Lys Gly Leu Asp h is 180 Leu Asn Lys Ser P-e 260 Ser Ser Asp Ser Lys 340 Phe Ser Glu Ala Ser Lys Tyr 165 Gln Glu Ile Lys Lys 245 Ser Ala Phe Lys Ala 325 Ile Thr Tyr 70 Pro Thr Leu Lys Glu 150 Gin Asp Tyr Lys Val 230 Lys Asn -ys Phe Lys 310 Len Thr Lys Lys Lys Tyr Glu Len 135 Gin Ile Glu Asn Phe 215 Glu Glu Glu Ile 295 Thr Glu Ala Asn Asn Ser Asp Asn 120 Thr Glu Asn Ile Met 200 Glu Phe Len Lys Ser 280 Ser Leu Ile Lys Asn 360 Asn Asn Asn 105 Asn Thr Pro Glu Ile 185 Glu Asn Leu Ile Ile 265 Thr Ile Lys Pro Tyr 345 Pro Cys VaI 90 Gin Leu Glu Ile Lys 170 Lys Gly Giy Asn Lys 250 Len Ile Lys Lys Val 330 Ser Lys Gin 75 Glu Glu His Leu Ile 155 Ser Asn Asn Lys Thr 235 Glu Gly Asp Ile Cys 315 Phe Asp Gly Tyr Thr Len Ser Len 140 Asn Leu Ser Phe Leu 220 Ser Ala Ile Phe Lys 300 Asr.

Glu Len Tyr Len Len Lys Ile 125 Glu Phe Arg Glu Thr 205 Asn Arg Ser Lys Pne 285 Trp Pro I le Asp le 36S Tyr Ile Asn 110 Lys Ile Gln Glu Ser 190 Leu Lys rhr Ile Gln 270 Ile Pro Asp Ala Thr 350 Asn Glu Tyr Phe Ile Pro Asn Phe 175 Ile Ser Ile Tyr Glu 255 Tyr Asp Ser Tyr Lys 335 Tyr Gly Val Phe Len Gin Lys Lys 160 Len Va1 His Lys Phe 240 Ser Len Glu Lys Len 320 Gly Glu Thr 0 i.

110 Glu Phe Leu Ile Ser Lys Gly Asn Asp Lys Asn Ser Asn Phe Phe Ile 370 375 380 Ser Phe Leu Asn Val Glu Lys His Ile Ile His Thr Ile Asn Tyr Lys 385 390 395 400 Ile Glu Lys Ile Asn Ser Lys Lys Trp Leu Ile Leu Asn Ile Gly Gly 405 410 415 Ser Tyr Asn Thr Val Lys Ile Gln Asp Val Ile Asn Tyr Ser Gin Thr 420 425 430 Leu Asn Leu Met Ile Leu Pro Leu Asn Asn Asn Phe Asp Asn Lys Ile 435 440 445 Lys Leu Asn Ser Lys Ile Lys Asn Leu Ile Phe Tyr Thr Asn Ile Lys 450 455 460 Lys Tyr Glu Asn Lys 465 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID 0* Xaa Val Gly Asp Gly Val Tyr Asp Asp Leu Pro Ala Gin Arg 1 5 2) INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear ;11) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val Glu Leu Glu Ile Gly Tyr Glu Arg i 2) INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids 111 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Xaa Xaa Glu Ile Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Xaa Xaa 1 5 10 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CACCTCGAGG GAATTCTGAT GCATTTA 27 INFORMATION FOR SEQ ID NO:9: i(i) SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "p_'mer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 0; CACGAATTCC CTCCGGGAAA GATATCG 27 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1334 base pairs TYPE: nucleic acid STRANDEDNESS: sinale TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic! 112 (ix) FEATURE: NAME/KEY:

COS

LOCATION: 361. .420 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAATTCCTGA AAAGTATGAG AAAAGGAAAG ATAATCTTAG GAAGCGTAAT

GATGTCTATG

GCTATAGTCA TGGCTGGGAA TGATGTTAGG GCTCATGATG ACGTTAGCGC

TTTGGAAACT

GGTGGTGCGG GATATTTCTA TGTTGGTTTG GATTACAGTC CAGCGTTTAG

CAAGATAAGA

GATTTTAGTA TAAGGGAGAG TAACGGAGAG ACTAAGGCAG TATATCCATA

CTTAAAGGAT

GGAAAGAGTG TAAAGCTAGA GTCACACAAG TTTGACTGGA ACACACCTGA

TCCTCGGATT

GGGTTTAAGG ACAACATGCT TGTAGCTATG GAAGGCAGTG TTGGTTATGG

TATTGGTGGT

GCC AGG GTT GAG CTT GAG ATT GGT TAC GAG CGC TTC MAG ACC MAG GGT Ala Arg Val Glu Leu Giu Ile Gly Tyr Glu Arg Phe Lys Thr Lys Gly 1 5 10 ATT AGA GAT AGT GGTAGTAAGG AAGATGAAGC TGATACAGTA

TATCTACTAG

Ile Arg Asp Ser CTAAGGAGTT AGCTTATGAT GTTGTTACTG GACAGACTGA TAAGCTTACC

GCTGCTCTTG

o o.

0 0 000.

a 0.

a

CCAAGACCTC

ATATCGAAAA

CT-TCGGT-AAC

CCTCCTACGC

TCAGTGCMAC

GCNATGCAGG

TAGTCAAGGA

TTGAAGGGGG

C.TTGTTATGA

GTGG7AACTT

AGGCTGGGTT

ATCGTGTTG-T

CTAGTCCGC

GTGGGGAATT

CGGTAAAGAT

GAAGGTTTGC

TGCTMATAGC

CAGCAATAGT

GCTAAGTGGT

CACTCCGGAA

AT TAACC CCC

CGAGGTTGTT

TCTTCTTAGT

CGTGGGGGTT

GAGTTATCAG

GGGAGATGGT

GGGTCGTACT

TGGTGTTAGG

ATCGTTCAGT

AGGACGAAGA

TCGGATGCAG

TCTCATGGGG

GATGGCAGTG

CCCGTTCAAA

GAAGAAAAAA

GAGATCAGGG

GAAGGTTTAG

GTTGATGGCC

CTC'TCTCCTG

GTTTATGATrG

AAGGATACTG

TTTGCTTTTT

TTGCTAAGGC

AGAATGGGGG

CGAAAGCGGA

GCACTGGTGA

TGAACTGGCC

ACGATAACGC

CCATAGTGGC

CGGTTTCTTC

GCGTTGTT-C

ATATCACTCC

PLAATATCTGZ-

A'TCTITCCGGC

CTATTGCTAA

A.AGC

CGTGGAGATT

TTCTCGTTAT

TGTAGCTGTG

GGAGACTTTA

NGCGTCGAAA

GGCAGCTGTA

AGGGTTACTA

TACTTC'TGTG

7TAGC7GC-

TA.AGCTTC

TTTTGCAGGT

TCAACGTCTT

CTTCTCCATG

TCCTCCCCTA

AGTAAGTATG

TGTAGTGCAG

AAGAACTTTG

AAGGCGGAAA

GCGAAGGACC

GCTAAAACTA

ATGGTCAATG

GTTGG7CTCG TATAGA77AA

GGTTTCTACC

GTAGATGATA

GCTTATGTCG

120 180 240 300 360 408 460 520 580 640 700 760 820 880 940 1000 1060 1120 1180 1240 1300 1334 2) INFORMATION FOR SEQ ID NO:11: 113- SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Ala Arg Val Glu Leu Glu Ile Gly Tyr Glu Arg Phe Lys Thr Lys Gly 1 5 10 Ile Arq Asp Ser INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 2957 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATGCAGAAGA GCAAAGTTCT ATGCTTAGCG GCGGATTGCC AGGGATAGTG CTGATGAAGA 0 0 0 0*00*0 6 0 0 00 0 0

ACACGAGGAG

CCTACCGGTG

GAAGAGGAGC

GCTGAGGATC

TGTCCTACTG

GTGAAAGAGG

ATTGCTGAGG

CCTACTGGCG

AAAGAGGAGC

GCTGAGGATC

AGTGATGAGT

GCTTCAAGTA

GATAGAGATG

GTAGAAGGG7 C=7=

TTTGAAGTAA

CTCACAAATC

AAAGTCCTGC

AAGAGGGGAT

GCGCTCACAA

AGCAAAGACC

ATCAAGAGGG

CTCACAAATC

AAAGACC-TGC

AAGAGGGGAT

TGAATCTAGA

AAGATGAGGA

ATGTAGCTGA

T-GGGGCGTT

77GGAGAAGA

TATTGTATTG

TCATGGGGGA

GCTTGATGTG

AGGGGTAAAA

AT CTCAT GGG

TGCGCTTGAT

GATAAA.AGTA

TCATGGGGGA

GOTTGATGTG

AGGGGTAAAA

GOATGGAAAT

'AATGTTGCA

AGGTAATGAT

TGATGACAGC

A GAG CGTGCC

ATTGTGACTG

CACAAGAAGT

ATAAGTAGTG

GGTGGCTGCA

GGACACAAGA

GTGATAAGTA

GATTGCGACT

CACAAGAAGT

ATAAGTAGTG

GGTGGCTGCA

AAGGAGGTTG

GAGTATGGTT

ATTTACCCGT

TCCGATGGTG

GAAGAAGAT 7

CGGGCGACGA

NGTCTCATCC

AATTGCCTAA

ATGCTGGAGA

AGTTGTCTCA

GTGAATTGCC

GCGGAGATGA

TGTCTCATCC

AA-TGCCTAA

ATGC-TGGAGA

CGGTTTACGG

CTCT TGGTGA

GGTGGGAAGA

AGGGTTACTA

-7GATGTGTA

TAGCGATTGT

TGTAGAGGTG

GGATGACATT

TGATAGCGAT

TCCTGTAGAG

TAAGGATGAC

TTGCGATTGT

TGTAGAGC'rG

GGATGACAT

TGATTGCCCT

AGAGGAAGGT

TGTTGGCGCC

ATATGATGTC

CTCCGAAAGT

TCAAGATCCA

120 180 240 300 36.0 420 480 540 600 660 720 78 0 840 900 960 00 00 0 0

S

00 0 0 0@ 114 GTAGAAGTGG

ATGATGAGGG

GCGGACTCTC TGACAGATTG ACTACTTCTT CTAGTGCTGG

AGTTGCTGAT

GGAAGAAGAA

AAAACAGGAT

GACAAAAGAT

AAACGGAAGT

GACGCTCTCT

CCCGAGTTGA

GAAAAGATAA

GATGTGACGG

AGCTATCGGA

AGACAGCGAA

TAACCAATAA

CCTGTTCCGA TGGTTATGCA ATCCAATGTG .9 '9 9 C 9*99 *9 9 a 9

I:

94 @9 9 .a 9094e* 9 996 9 c*.

ACTTTGTAT;

AAGAAGCGT'

AAGTCTCA-Aj

TGGAGTGCTC

GGTGAAGGAC

AAGCTGTGT.A

GATGTGGAAA

TATGAGGTAG

ATAGCAAAGG

AATGGTGGCG

ATGAATCTGC-

A.ACAAGCATT

CGTAGTACAA~

GGGTGTTGGC

GAAAACGGTG

AAGGTCACAA

CTTGCTAGTT

AAAGATCAAG

GAAGGATTTA

CAGAA'-TATA

TAGGTGTGTA

CSTTGTAGCA

CC-TATGC-C

kAAGTTGAGA', r' CTGGACGTC( k. CTAGAACGAC

;CAGTGGAATC

GGCAGCCCAG

AGGATAATAG

AGACAATAAC

CGAGAGTTGT

ATTTAATTGT

ATAGTTTTCT

CTACACAAAG

ACGTGGAAGG

GTACATCTCA

AAAGCGATAA

CAGTAATGAT

ATAAAGCTC'C

TGCAATATAA

TGTTGACTGT

GAAAACGTGC

CAACG'rCCTC AA-TT TGTAT

CACCCCGAC

r' GTGGGCAGGT

AGGTCAGTAT

;TAACAGAAAAT

CATAAACAAG

CGATCATAGC

CCGCTCTAGC

TGCGCAAAGT

TGGGGGAGGC

TTATTACAGG

GGCGAACAAT

CGTCACTAGA

GCGTAGTCTA

GGAAAGAGAT

AAACGGACAA

AACGTGCGAG(

ATCTCA6AGCA(

AAGCTTGTGT

ATTGAAAGGC

CAGGCTTATGC

AAAAGCAGCC C TCTTGCTATA

C

ATCAATT-CT I1 ACCGCCACAT C

TCTTCTGAC

GGTGACTAT

A TTGAGATT

AAAGTGTTG

AGTAGCAAT

TATGAGCAAi

AATGATGGC;

GGTGAAGCTC

GCAATGCTGC

TCACAATCCC

GCAGAGCATC

GCAGTTTCTA

A.CGGGGCAAT

rCTTCTCAGG

%GCAGTGTTA

kCAATCACAG kACGCTATGT

ATGATGAGT

CGAAGCGAC

LAGAACACCG

CGATCTTTT

GTTGGGGTA

GTGGACCAC

AAGACACCC

TTGACAACA

AATTTATTG

TGGCTACCC

GAGCAGAAA

-GTCATGCT

CTT CGCCG

ATTTAGAGGC

TGCCAAATGA

AAGCTTTAGT

CTGAGTTGAA

GTTGCATTTG

TGAGGTCAAC

AAAACCGAGA

CATCTGAGAC

TGTTGGATTT

AGGGAAGCAA

A.CTCGTTAAT(

kAGGGGGTGAC CTGGTACTGA TGCGTGGCCA

GCTCAAATTC

TTGGTGAGCC

GAGATGTATT

TTACGAAATT

TTCTCAAGGG

TTTGTACGTG

CAT TTATTCC

CAGAGATACC

CACCTAGTAC

GATAAAGAAA

AGT TCAAGCC tCATTGTTGA

CCCGATGTCT

T TACGSTAGCT

TACAATTGC

TC CT CTGCT C

CAT'GGCGCC

TCACTCTTGC

GCAGTATGAG

GGCTAATGAC

CTCATTCGAG

ACTACTAGAG

GAAAGAAACG

TGGTATGGGT

TGGAGTAGGA

TAGTGGATCA

AATATGAACT

CGTGGA'I'GCT

GTAGATCTCT

GGCATACCTT

CTCGCTGGCA

AATATTTAAA

GTCCCTGTGT

ACTGGCGTTA

TGATTCTGGT

TGGCGATGTT

GGGGGAACAA

AAGCATTCAG

GCAGGCGAGG

ATATAGTTAT

GAATAAGTGT

TGCCTTAGGT

TGAGAAGTTG

CAAACCATGG

:CTGGAAATA

ACTGTACTG

1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 S. 94, 9 4 9 eq 4, 9*

S.

115- TCCGTCTCTT CAGCATACCT CTGCCTTAGC AAAGTGTTGG CCTACAGCAT CATGTATATA 2820 TTTTTTATAT TGAGTGCAAA GAAGGCATGA AAGAGGACAT ACAGAAAGTA AGCGGTTTCG 2880 TGTTTTTCAA GGACGGTCAT ACGCTAGGGT ATAATGCAAA GAAGTATTTG ATACTCTATT 2940 CACTGTCGCA CAGGTGT 2957

Claims (35)

1. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a polypeptide of the agent of human granulocytic ehrlichiosis (aoHGE), wherein said aoHGE polypeptide is selected from the group consisting of: (a) (b) (c) (d) (e) (f) (g) shown in (h) the E6 polypeptide of SEQ ID NO: 2; the E7 polypeptide of SEQ ID NO: 4; the 44-kDa polypeptide of SEQ ID NO 11; the 44-1 polypeptide of SEQ ID NO: the 44-2 polypeptide of SEQ ID NO: 6; the 80-1 polypeptide of SEQ ID NO: 7; the eM4 polypeptide having the amino acid sequence Figure 12; an 80 kD polypeptide comprising the polypeptide serotypic variants of any one of the polypeptides of fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of a derivative of any one of the polypeptides of said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of a polypeptide that is immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with any one of the polypeptides of a polypeptide that is capable of eliciting antibodies that are immunologically reactive with aoHGE and any one of the polypeptides of and -117- a polypeptide that is immunologically reactive with antibodies elicited by immunization with any one of the polypeptides of

2. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes an aoHGE polypeptide, wherein said polypeptide is selected from the group consisting of: a 40 kDa, 44 kDa, 65kDa, 80 kDa, 94 kDa, 105 kDa, 110 kDa, 115 kDa, and 125 kDa polypeptide which appears as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, serotypic variants, fragments and derivatives thereof.

3. The DNA molecule according to claim 1 or claim 2, wherein said polypeptide comprises a protective epitope.

4. A DNA molecule comprising a DNA sequence encoding an fusion protein, wherein the fusion protein comprises an aoHGE polypeptide encoded by a DNA molecule according to any one of claims 1, 2 or 5 A DNA molecule comprising a DNA sequence encoding a multimeric protein, which multimeric protein comprises an aoHGE polypeptide encoded by a DNA molecule according to any one of claims 1, 2 or

6. An expression vector comprising a DNA molecule according to any one of claims

7. A host cell transformed with a DNA molecule according to any one of claims -118-

8. The host cell according to claim 7, wherein said DNA molecule is integrated into the genome of said host cell.

9. The host cell according to claim 7 or 8, wherein said host cell is selected from the group consisting of: strains of E. coli; Pseudomonas, Bacillus; Streptomyces; yeast, fungi; animal cells, including human cells in tissue culture; plant cells; and insect cells. A polypeptide encoded by a DNA molecule according to any one of claims

11. A method for producing a polypeptide according to claim 10, comprising the step of culturing a host cell according to any one of claims 7-9.

12. An aoHGE polypeptide selected from the group consisting of: 1 the E6 polypeptide of SEQ ID NO: 2; the E7 polypeptide of SEQ ID NO: 4; the 44-kDa polypeptide of SEQ ID NO 11; the 44-1 polypeptide of SEQ ID NO: the 44-2 polypeptide of SEQ ID NO: 6; the 80-1 polypeptide of SEQ ID NO: 7; the eM4 polypeptide having the amino acid sequence shown in Figure 12; an 80 kD polypeptide comprising the polypeptide serotypic variants of any one of the polypeptides of -119- fragments comprising at least 8 amino acids taken as a block from any one of the polypeptides of a derivative of any one of the polypeptides of said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of a polypeptide that is immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with any one of the polypeptides of a polypeptide that is capable of eliciting antibodies that are immunologically reactive with aoHGE and any one of the polypeptides of and a polypeptide that is immunologically reactive with antibodies elicited by immunization with any one of the polypeptides of

13. An aoHGE polypeptide selected from the group consisting of: a 40 kDa, 44 kDa, 65kDa, 80 kDa, 94 kDa, 105 kDa, 110 kDa, 115 kDa, and 125 kDa polypeptide which appears as a single band on a Western blot after reacting with sera from an animal infected with aoHGE, serotypic variants, fragments and derivatives thereof.

14. The aoHGE polypeptide according to any one of claims 12, 13 or 41, wherein said polypeptide comprises a protective epitope.

15. A fusion protein comprising an aoHGE polypeptide according to any one of claims 12, 13 or 41.

16. The fusion protein according to claim 15, wherein said fusion protein comprises two or more aoHGE 0I (I -120- polypeptides, each derived from a different isolate of aoHGE.

17. A multimeric protein comprising an aoHGE polypeptide according to any one of claims 12, 13 or 41.

18. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a component selected from the group consisting of: a polypeptide according to any one of claims 12-14; a fusion protein according to claim or 16 and a multimeric protein according to claim 17.

19. The pharmaceutical composition according to claim 18, wherein the component is crosslinked to an immunogenic carrier. The pharmaceutical composition according to claim 18 or 19, further comprising at least one additional immunogenic aoHGE polypeptide.

21. The pharmaceutical composition according to claim 18 or 19, further comprising at least one additional non-aoHGE polypeptide.

22. A method for treating or preventing aoHGE infection or HGE comprising the step of administering to a I subject a pharmaceutical composition according to any one of claims 18-21.

23. A diagnostic kit comprising a component selected from the group consisting of: a polypeptide according to any one of claims 12, 13 or 41; a fusion protein according to any one of claims 15 or 16; and a multimeric protein -121- according to claim 17, and also comprising a means for detecting binding of said component to an antibody.

24. An antibody that binds to a polypeptide according to any one of claims 12, 13 or 41. The antibody according to claim 24 which is polyclonal.

26. The antibody according to claim 24 which is monoclonal.

27. A diagnostic kit comprising an antibody according to any one of claims 24-26.

28. A method for detecting aoHGE infection comprising the step of contacting a body fluid of a suspected infected mammalian host with a polypeptide according to any one of claims 12, 13 or 41,; a fusion protein according to claim or 16; and a multimeric protein according to claim 17.

29. A pharmaceutical composition comprising an antibody according to any one of claims 24-26. A vaccine comprising a polyclonal anti-aoHGE antibody.

31. A vaccine comprising a monoclonal anti-aoHGE antibody.

32. A method for detecting aoHGE infection comprising the step of contacting a body fluid of a mammalian host with an antibody according to any one of claims 24-26. -122-

33. A method for treating or preventing aoHGE infection, comprising administering to a subject an antibody according to any one of claims 24-26, a pharmaceutical composition according to claim 29, or a vaccine according to claim 30 or 31.

34. A method for detecting the presence of aoHGE in a biological sample, comprising the step of subjecting the sample to PCR DNA analysis using a primer derived from a DNA molecule according to any one of claims 1, 2 or A diagnostic kit for detecting the presence of aoHGE in a sample, wherein the kit comprises a primer derived from a DNA molecule according to claim 1 or claim 2.

36. A method for detecting the presence of aoHGE in a sample, comprising the step of inoculating an infant laboratory mouse with said sample and detecting aoHGE infection in the mouse.

37. The method according to claim 36, wherein said infant mouse is no more than 5 days old. ooo

38. The method according to claim 36, wherein said infant mouse is 1 day old.

39. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence selected from the group consisting of: the E5-3A sequence set forth in Figure 13, the E5-3B sequence set forth in Figure 14, the E5-5A sequence set forth in Figure 15, the E5-5B sequence set forth in Figure 16, and the E5-6 sequence set forth in Figure 17. -123- An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which hybridizes under stringent conditions to a DNA sequence selected from the group consisting of: the DNA sequence of SEQ ID NO: 10 and a DNA sequence according to claim 39.

41. A polypeptide encoded by a DNA molecule according to claim

42. The use of a component selected from the group consisting of: a polypeptide according to any one of claims 12-14; a fusion protein according to claim 15 or 16; a multimeric protein according to claim 17; and an antibody according to any one of claims 24-26; in the manufacture of a medicament. DATED this 2 3 rd Day of October 2001 Yale University By their Patent Attorneys CULLEN CO 0: *o g