WO1998014584A2 - Compositions et procedes destines a la prevention et au diagnostic de l'ehrlichiose granulocytaire humaine - Google Patents

Compositions et procedes destines a la prevention et au diagnostic de l'ehrlichiose granulocytaire humaine Download PDF

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Publication number
WO1998014584A2
WO1998014584A2 PCT/US1997/017675 US9717675W WO9814584A2 WO 1998014584 A2 WO1998014584 A2 WO 1998014584A2 US 9717675 W US9717675 W US 9717675W WO 9814584 A2 WO9814584 A2 WO 9814584A2
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Prior art keywords
aohge
polypeptide
polypeptides
kda
antibodies
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PCT/US1997/017675
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English (en)
Inventor
Erol Fikrig
Stephen W. Barthold
Jacob Ijdo
Wei Sun
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Yale University
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Application filed by Yale University filed Critical Yale University
Priority to AU47416/97A priority Critical patent/AU4741697A/en
Priority to EP97909914A priority patent/EP0932680A2/fr
Priority to JP10516827A priority patent/JP2001502528A/ja
Priority to CA002268013A priority patent/CA2268013A1/fr
Publication of WO1998014584A2 publication Critical patent/WO1998014584A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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) .
  • HGE human granulocytic ehrlichiosis
  • this invention relates to polypeptides and DNA sequences which encode them, from the agent of HGE, referred to herein as "aoHGE".
  • aoHGE agent of HGE
  • 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 which is effective to prevent or lessen the severity, for some period of time, of aoHGE infection.
  • 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.
  • 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" [S.R. Telford et al., "Perpetuation of the Agent of Human Granulocytic Ehrlichiosis In a Deer Tick- Rodent Cycle," Proc . Na tl . Acad. Sci . USA, 93, pp. 6209-6214 (1996)].
  • the Ehrlichia which causes human granulocytic ehrlichiosis will be referred to herein as "aoHGE.”
  • the tick vector has been shown to be Ixodes scapulari s (also referred to as Ixodes dammini ) in the Ixodes ri cinus complex [S. et al . , Proc . Na tl . 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.
  • 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 mi croti , the agent of babesiosis, and Borrelia burgdorferi , the agent of Lyme disease.
  • a greater understanding of human granulocytic ehrlichiosis may provide insight into clinical symptoms that result in misdiagnosis of other tick-borne infections, most notably Lyme disease.
  • 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 those DNA sequences, unicellular hosts transformed with those DNA seq ent s 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 advantageously used in methods and means for the diagnosis of aoHGE infection and human granulocytic ehrlichiosis .
  • diagnostic means and methods characterized by aoHGE polypeptides, DNA sequences encoding them or antibodies directed against these polypeptides. These means and 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.
  • this invention also provides methods for the identification and isolation of additional aoHGE polypeptides, as well as compositions and methods comprising such polypeptides.
  • 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) .
  • 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: 5) .
  • 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: 10) .
  • 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: 7) .
  • 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 aoHGE-HL-60 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.
  • 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 e ⁇ gene (SEQ ID NOS: 8 and 9) .
  • the underlined portion of the 3' primer indicates the inserted Xhol site.
  • the underlined portion of the 5' 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) .
  • Figure 12A-B depicts the amino acid sequence of the eM4 polypeptide of aoHGE isolate NCH-1 (SEQ ID NO: 12).
  • 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: 10) .
  • 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: 10) .
  • 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: 10) .
  • Figure 16 depicts the DNA sequence designated 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: 10) .
  • 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: 10) .
  • Figure 18 is a matrix plot depicting a region of homology between approximately nucleotides 200-400 and 600-1000 the E5-3B DNA sequence and approximately 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.
  • 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 related disorders ⁇ aused by aoHGE infection.
  • this invention provides a 40-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.
  • this invention provides a 44-kDa aoHGE polypeptide and fragments 44-1 and 44-2 thereof, ind compositions and methods comprising the polypeptide and fragments.
  • this invention provides a 65-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide. 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.
  • this invention provides a 105-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.
  • this invention provides a 110-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.
  • this invention provides a 115-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide.
  • this invention provides a 125-kDa aoHGE polypeptide and compositions and methods comprising the polypeptide. In another embodiment, this invention provides an E6 polypeptide and compositions and methods comprising the polypeptide.
  • this invention provides an E7 polypeptide encoded and compositions and methods comprising the polypeptide.
  • this invention provides an eM4 polypeptide encoded and compositions and methods comprising the polypeptide.
  • this invention provides an E5-3A, E5-3B, E5-5A, E5-5B and E5-6 DNA ssequences, and compositions and methods comprising them.
  • the preferred compositions and methods of each of the aforementioned embodiments are characterized by immunogenic aoHGE polypeptides.
  • an "immunogenic aoHGE polypeptide" is any aoHGE polypeptide that, when administered to an animal, is capable of eliciting a corresponding antibody.
  • immunogenic aoHGE polypeptides are intended to include additional aoHGE polypeptides which may be identified according to the methods disclosed herein.
  • 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.
  • 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 of this invention.
  • aoHGE polypeptides provided by this invention are substantially free of an Ehrli chia 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 prevention of aoHGE infection.
  • a polypeptide that is "substantially free of an Ehrli chia bacterium or 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 m si tu hybridization with aoHGE specific probes or by any other method for detecting aoHGE infection.
  • it is a polypeptide that is detectable as a single band on an immunoblot probed with polyclonal anti-aoHGE anti-serum.
  • this invention provides immunodominant aoHGE polypeptides.
  • 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.
  • an "immunodominant region" of an aoHGE polypeptide denotes a region of an aoHGE 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.
  • 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 than the level of reactivity with serum from patients infected with aoHGE .
  • the immunodominant polypeptides would be bound at a level 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.
  • 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.
  • this invention relates to diagnostic means and methods characterized by the aoHGE polypeptides, DNA sequences or antibodies of the invention.
  • This invention further provides an immunocompetent nonhuman, mammalian model for human HGE.
  • the laborato__, mouse model, described herein, is characterized by clinical features that closely mimic HGE in humans.
  • the mouse model is useful for selecting the preferred aoHGE polypeptides and antibodies of this invention that are effective to 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.
  • the infant mice are 5 days old or less.
  • the mice are 3 days old or less.
  • the mice are 1 day of age.
  • an "aoHGE polypeptide” is a polypeptide encoded by a DNA sequence of aoHGE.
  • aoHGE polypeptides include the 40, 44, 65, 80, 94, 110, 115, or 125-kDa polypeptide expressed by aoHGE, as described in Example I, infra, an E6, E7 or eM4 polypeptide or fragments or derivatives thereof.
  • an "aoHGE polypeptide” includes polypeptides encoded by a DNA sequence of any organism that causes HGE.
  • 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:
  • 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 (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and the 40-kDa aoHGE polypeptide of (a) or
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 40-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • a "44-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting 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 (a) -(f);
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a polypeptide of (a) -(f);
  • a "65-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting of:
  • a derivative of a 65-kDa aoHGE polypeptide of (a) or (b) said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b) ;
  • 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 (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and the 65-kDa aoHGE polypeptide of (a) or
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 65-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • an "80-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting of:
  • a "94-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chi a bacterium or fragments thereof and which is selected from the group consisting 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 94-kDa aoHGE polypeptide of (a) or (b) or (c) ;
  • aoHGE polypeptides than are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 94-kDa aoHGE polypeptide of (a) or (b) or (c) ; and (f) aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 94-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • a "105-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting of:
  • 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 (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 105-kDa aoHGE polypeptide of (a) or
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 105-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • a "110-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting 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 110-kDa aoHGE polypeptide of (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 110-kDa aoHGE polypeptide of (a) or
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 110-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • a "115-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of
  • Ehrli chia bacterium or fragments thereof which is selected from the group consisting of: (a) a 115-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;
  • a derivative of a 115-kDa aoHGE polypeptide of (a) or (b) said derivative being at least 80% identical in ammo acid sequence to the corresponding polypeptide of (a) or (b) ;
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 115-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • a "125-kDa aoHGE polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting 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 125-kDa aoHGE polypeptide of (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a 125-kDa aoHGE polypeptide of (a) or
  • aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a 125-kDa aoHGE polypeptide of (a) or (b) or (c) .
  • an "E6 polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting of:
  • a derivative of an E6 polypeptide of (a) or (b) said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a) or (b);
  • aoHGE polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with an E6 polypeptide of (a) or (b) or (c) ;
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and an E6 polypeptide of (a) or (b) or (c) ;
  • an "E7 polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting of:
  • aoHGE polypeptide ⁇ that are immunologically reactive with antibodies generated by infection of a mammalian host with aoHGE, which antibodies are immunologically reactive with an E7 polypeptide of (a) - (c);
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and an E7 polypeptide of (a) -(c); and (g) aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with an E7 polypeptide of (a) -(c).
  • an "eM4 polypeptide” denotes a polypeptide which is substantially free of Ehrli chia bacterium or fragments thereof and which is selected from the group consisting 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 (a) or (b) or (c);
  • aoHGE polypeptides that are capable of eliciting antibodies that are immunologically reactive with aoHGE and a polypeptide of (a) or (b) or (c) ; and (f) aoHGE polypeptides that are immunologically reactive with antibodies elicited by immunization with a polypeptide of (a) or (b) or (c) .
  • a "serotypic variant" of an aoHGE polypeptide of this invention also referred to 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 Tm, to any portion of the DNA sequence encoding the aoHGE polypeptide disclosed herein.
  • 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.
  • 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 hepatic enzyme levels, gastro-intestinal or pulmonary hemorrhaging and other disorders caused by aoHGE infection.
  • aoHGE infection including fever, myalgia, arthralgia, anemia, leukocytopenia, thrombocytopenia, neutropenia, elevated hepatic enzyme levels, gastro-intestinal or pulmonary hemorrhaging and other disorders caused by aoHGE infection.
  • it may be evidenced by a decrease in the level of aoHGE in infected ticks which have fed on treated animals.
  • 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 aoHGE isolates and perhaps from other rickettsia as well, which are useful in the methods and compositions of this invention.
  • 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.
  • a "protective epitope” is (1) an epitope which is recognized by a protective antibody, and/or (2) 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.
  • 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 hepatic enzyme levels, gastro-intestinal or pulmonary hemorrhaging, and other related disorders.
  • a protective epitope may comprise a T cell epitope, a B cell epitope, or combinations thereof.
  • a "protective antibody” is an antibody that confers protection, for some period of time, against aoHGE infection or any one of the physiological disorders associated with aoHGE infection or HGE.
  • 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 irrvrtiunostimulatory molecules.
  • a T cell epitope may also be an epitope recognized by cytotoxic T cells that may affect intracellular aoHGE infection.
  • a strong T cell epitope is a T cell epitope which elicits a strong T cell response.
  • B cell epitope is the simplest spatial conformation of an antigen which reacts with a specific antibody.
  • 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 the severity, for some period of time, of aoHGE infection.
  • an “an anti-aoHGE polypeptide antibody, " also referred to as “an antibody of this invention, " is an antibody directed against an aoHGE polypeptide of this invention.
  • an antibody of this invention may be directed against a 40-kDa, 44-kDa, 65-kDa, 80-kDa, 94-KDa, 110-kDa, 115- kDa, 125-kDa polypeptide expressed by aoHGE, as described in Example I, infra, an E6, E7 or eM4 polypeptide, or a fragment, derivative or serotypic variant of the aforementioned polypeptides.
  • An anti- aoHGE polypeptide antibody of this invention includes antibodies directed against polypeptides expressed by aoHGE, or fragments or derivatives thereof, that are immunologically cross-reactive with any one of the aforementioned polypeptides.
  • an anti-aoHGE polypeptide antibody of this invention includes antibodies directed against other aoHGE polypeptides identified according to methods taught 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 F(v), 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 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. In addition, because at least some, if not all of the aoHGE polypeptides disclosed herein are protective surface proteins of aoHGE, they are particularly useful in single and multicomponent vaccines against human granulocytic ehrlichiosis.
  • multicomponent vaccines are preferred because such vaccines may be formulated to more closely resemble the immunogens presented by replication- competent 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.
  • compositions and methods of this invention comprise aoHGE polypeptides having enhanced immunogenicity.
  • Such polypeptides may result 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.
  • aoHGE polypeptides of this invention may be modified by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS.
  • the polypeptides are small, chemically synthesized polypeptides, it may be desirable to couple them to an immunogenic carrier.
  • the coupling must not interfere with the ability of either the polypeptide or the carrier to function appropriately.
  • Useful immunogenic carriers are well known in the art.
  • examples of such carriers are keyhole limpet hemocyanm (KLH) ; albumins such as bovme serum albumin (BSA) and ovalbumm, PPD (purified protein derivative of tuberculin) ; red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
  • KLH keyhole limpet hemocyanm
  • BSA bovme serum albumin
  • PPD purified protein derivative of tuberculin
  • red blood cells tetanus toxoid
  • cholera toxoid agarose beads
  • activated carbon or bentonite.
  • Modification of the ammo 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 lmmunogenicity or alter their biochemical properties.
  • the polypeptides may also be prepared with the objective of increasing stability or rendering the molecules more amenable to purification and preparation.
  • One such technique is to express the polypeptides as fusion proteins comprising other aoHGE or non-aoHGE sequences.
  • derivatives of the aoHGE polypeptides may be prepared by a variety of methods, including by m vi tro 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.
  • 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.
  • conservative substitution is preferred, e.g., 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, e.g., by causing changes in charge, conformation and other biological properties.
  • substitutions would include for example, substitution 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 for a residue having a net negative charge.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
  • the immunogenicity, immunodominance and/or protectiveness of a derivative of this invention can be readily determined using methods disclosed in the Examples.
  • the aoHGE polypeptides disclosed herein are prepared as part of a larger fusion protein.
  • an aoHGE polypeptide of this invention may be fused at its N- terminus or C-terminus to a different immunogenic aoHGE polypeptide, to a non-aoHGE polypeptide or to combinations thereof, to produce fusion proteins comprising the aoHGE polypeptide.
  • 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.
  • fusion proteins are constructed which comprise one or more of the aoHGE polypeptides fused to other aoHGE polypeptides. Such fusion proteins are particularly effective in the prevention, treatment and diagnosis of human granulocytic ehrlichiosis as caused by a wide spectrum of aoHGE isolates.
  • 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.
  • moieties such as immunoglobulin domains
  • Such fusions may be prepared without undue experimentation according to 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 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.
  • the fusion proteins comprising the aoHGE polypeptides be produced at the DNA level, e.g., 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.
  • 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 lipids and carbohydrates.
  • the multimeric proteins will consist of multipxe T or B cell epitopes or combinations thereof repeated within the same molecule, either randomly, or with spacers (amino acid or otherwise) between them.
  • aoHGE is incorporated into a vaccine. As disclosed in
  • Examples and , animals immunized with such a vaccine produce antibodies that confer protection against aoHGE infection.
  • an aoHGE polypeptide of this invention which is also a protective aoHGE polypeptide is incorporated into a single component vaccine.
  • aoHGE polypeptides of this invention which are also protective aoHGE polypeptides are incorporated into a multicomponent vaccine comprising other protective aoHGE polypeptides.
  • 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 m which the various components are covalently associated. Alternatively, it may contain multiple individual components.
  • a multicomponent vaccine may be prepared comprising two or more of the aoHGE polypeptides, wherein each polypeptide is expressed and purified from independent cell cultures and the polypeptides are combined prior to or during formulation.
  • a multicomponent vaccine may be prepared from heterodimers or tetramers wherein the polypeptides have been fused to immunoglobulin chains or portions thereof.
  • a vaccine could comprise, for example, a 44-kDa aoHGE polypeptide fused to an 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.
  • the host cell selected should be capable of assembling the two chains appropriately.
  • the heavy and light chain fusions could be produced from separate cell lines and allowed to associate after purification.
  • the multicomponent vaccine will comprise numerous T cell and B cell epitopes of protective aoHGE polypeptides.
  • aoHGE polypeptides of this invention may be administered to an animal via a 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, upon ingesting the liposome, would digest the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response.
  • the liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those 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.
  • 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 .
  • 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 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.
  • a method which comprises the steps of treating an animal with a multicomponent vaccine comprising a therapeutically effective amount of an aoHGE polypeptide, or a fusion protein or multimeric protein comprising such polypeptide in a manner sufficient to prevent or lessen the severity, for some period of time, of aoHGE infection.
  • 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.
  • 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 differentiate into antibody producing plasma cells.
  • a closed autocatalytic circuit exists which will result in the amplification of both B and T cell responses, leading ultimately to production of a strong immune response which includes high titer antibodies against the aoHGE polypeptide.
  • T H 1 T-helper cells type 1
  • T H 2 T-helper cells type 2
  • T H 1 or T H 2 cells may also be favored by the mode of administration of the polypeptide.
  • aoHGE polypeptides may be administered in certain doses or with particular adjuvants and immunomodulators, for example with interferon-gamma or interleuken-12 (T H 1 response) or interleukin-4 or interleuken-10 (T H 2 response) .
  • 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 (1) remove protective antibodies from polyclonal antiserum directed against the polypeptide or (2) elicit an immune response which is effective to prevent or lessen the severity of aoHGE infection.
  • 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.
  • the polypeptides that contain T cell epitopes may be identified in vi tro by testing them for their ability to stimulate proliferation and/or 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 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 .
  • 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) .
  • 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.
  • aoHGE polypeptides of this invention may be prepared by recombinant means, chemical means, or combinations thereof.
  • 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, e.g., using PCR and oligonucleotide primers derived from the sequences herein disclosed.
  • 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 invention.
  • the DNA sequences of this 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 m 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. An ammo terminal methionine may or may not be present on the expressed polypeptides of this invention. f Lue terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques. 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 SV40, bovme papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retroviruses including lentiviruses .
  • Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E.
  • coli including pBluesc ⁇ pt, pGEX-2T, pUC vectors, col El, pCRl, pBR322, pMB9 and their derivatives, pET- 15, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g. ⁇ GTIO and ⁇ GTll, and other phages .
  • Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof.
  • Useful vectors for insect cells include pVL 941.
  • 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.
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC 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 k ase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ - matmg system and other constitutive and mducible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the early and late promoters of SV40 or adenovirus the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promote
  • DNA sequences encoding the aoHGE polypeptides of this invention are cloned in the expression vector lambda ZAP II (Stratagene, La Jolla, CA) , in which expression from the lac promoter may be induced by IPTG.
  • 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.
  • 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.
  • the host in selecting a vector, the host must be considered because 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 expression of any other proteins encoded by the vector, such as antibiotic or other selection markers, should also be considered.
  • 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 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.
  • 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 inorganic ligands or monoclonal antibodies) ; size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis ; and the like.
  • liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like
  • affinity chromatography such as with inorganic ligands or monoclonal antibodies
  • size exclusion chromatography such as with inorganic ligands or monoclonal antibodies
  • immobilized metal chelate chromatography immobilized metal chelate chromatography
  • gel electrophoresis and the like.
  • 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. Mer ⁇ field, "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. Memhofer, 4 The Peptides : Analysis, Synthesis, Biol ogy; Modern Techniques Of Peptide And Ammo Acid Analysi s, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Sp ⁇ nger-Verlag (1984).
  • these synthetic methods comprise the sequential addition of one or more ammo acid residues to a growing peptide chain.
  • peptide coupling agents are used to facilitate this reaction.
  • peptide coupling agents suitable for the uses described herein see M. Bodansky, supra .
  • a suitable, selectively removable protecting group is utilized for ammo acids containing a reactive side g ⁇ oup, e.g., lysine.
  • 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.
  • compositions and re granulocytic e r..: 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 th.._ invention.
  • the antibodies of this invention also hav- variety of other uses. For example, they are use- reagents to screen for expression of the aoHGE polypeptides, either in libraries constructed from aoHGE DNA or from other samples in which the protei- may be present.
  • the antibodies of this mventio- are also useful to purify or remove polypeptides frc- given sample, to block or bind to specific epitopes z - the polypeptides and to direct various molecules, sue as toxins, to the surface of aoHGE .
  • mice are preferred as ar animal model.
  • any animal that is susceptible to infection with aoHGE nay be useful mice are not only susceptible to aoHGE infection but are also afflicted with clinical symptoms of a d ⁇ sea_ that is remarkably similar to human granulocytic ehrlichiosis in humans.
  • the humoral r esDO" of mice infected with aoHGE by tick transmission has been shown to be strongly similar to the human hjrc response.
  • a polypeptide is use:, . _::ere with a pharmaceutically c z z z _, 3 z.c as complete or incomplete Fre building::' I muramyl dipeptides) or I ⁇ 3I ⁇ .g complexes ) .
  • Such adjuvants ma ;p: ⁇ e from rapid dispersal by sea::a-.
  • tr.a the immunization sche:_- ⁇ .. :r more administrations cf ⁇ " :cread out over several weeks.
  • _ e - - ⁇ D or antibodies of this invention have : - to e effective in the screening prc: ⁇ . : ⁇ e used in a therapeutically erf-: pharmaceutical compositions and ⁇ - _ prevent human granulocytic ehrl_ ⁇ invention may be _ "er.tional depot forms.
  • Such dosage forms may include ,:e_ ⁇ t ⁇ cally acceptable carriers and adjuvants : . . are known to those of skill in the art.
  • eietable fatty acids water, salts or e_ trcyt s such as protamine sulfate, disodium hyiroge ⁇ phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyr roiidone, cellulose-based substances, and polyethylene glycol.
  • Adjuvants for topical or gel base fcrr.s may be selected from the group consisting of so ⁇ -:n ⁇ carboxymethylcellulose, polyacrylates, pc-_. oxyethylene-polyoxypropylene-block polymers, pc_.ethyiene glycol, and wood wax alcohols.
  • the vaccines and compositions of this invention may also include other components or be s c:e:t to other treatments during preparation to er.r.ar.oe their immunogenic character or to improve their tc_ .ra ee in patients.
  • compositions comprising an antibody of this in .::::: may be administered by a. variety of dosage ::. - and regimens similar to those used for other p . . :z irrmunotherapies and well known to those of
  • the aoHGE polypeptides may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g., the vaccine against hepatitis) .
  • Any pharmaceutically acceptable dosage route including parenteral, intravenous, intramuscular, mtralesional or subcutaneous injection, may be used to administer the polypeptide or antibody composition.
  • 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, mtra-articularly, trasynovially, mtrathecally, mtralesionally, periostally or by oral or topical routes.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient intramuscularly.
  • 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 lmmunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician.
  • the more highly immunogenic the polypeptide, the lower ⁇ or of immunizations, ecessary treatment time will ⁇ e -5 administered with an osa ⁇ 'lli consist of 10 ⁇ g - . -y eptide, and preferably, -1000 ⁇ g.
  • an osa ⁇ 'lli consist of 10 ⁇ g - . -y eptide, and preferably, -1000 ⁇ g.
  • Useful I5C0M, simple metal salts ⁇ oil based adjuvants - " round's adjuvant.
  • -s ea the polypeptide -Ision with the
  • E. coli excressmg proteins corner- :_ng an aoriGE polypeptide are administered orally to nc-"-man -nimals according to "etno ⁇ s -nnown _n tne art, decrease or lessen the :e'er-ty of ao- ' C ⁇ infect- Fir example, a palatable re:. er.
  • oa::er ⁇ a expre _ng an aoHGE polypeptide, a-ine or -n ::e form of a --5 ion protein or multimeric protein, may oe aomm_ste: - .Mtn animal food to be oon:-me ⁇ cy ⁇ __ ⁇ mice or "er animals that harbor
  • .ot e -mbodiment
  • the antibodies of :;.:.- on as well as the aoHGE polypeptides of :n. .tio.n, and the DNA sequences encoding them are . .5 diagnostic agents for detecting infect::: oHG ⁇ .
  • the polypeptides are capable of bindm: co ⁇ y molecules produced in animals, including tnat are infected with aoHGE, and the an:: are oapabie of binding to aoHGE or an_.ige..s :
  • agents may be included in a kit which may also e instructions for use and other appropriate ; , preferably a means for detecting when tne tioe or antibody is bound.
  • the • :e :r antibody may be labeled with a detection m- .: allows for the detection of the polypeptide :•.
  • the detection means may be a fluorescent labeling agent such as fluorescem 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 125I or Cr 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 C, 0, or N. Binding may also be detected by other methods, for example via avid -biotm complexes.
  • FIC fluorescem isocyanate
  • FITC fluorescein isothiocyanate
  • an enzyme such as horseradish peroxidase (HRP) , glucose oxidase or the like, a radioactive element such as 125I or Cr that produces gamma ray emissions, or a radioactive element that emits posi
  • monoclonal antibody molecules produced by a hybridoma can be metabolically labeled by incorporation of radioisotope-conta mg ammo acids m 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 may be used to detect the presence of a quantity of aoHGE or anti-aoHGE antibodies a oody fluid sample such as serum, plasma or urine.
  • a oody fluid sample such as serum, plasma or urine.
  • an aoHGE polypeptide or an antibody of the present invention is bound to a solid support typically by adsorption from an aqueous medium.
  • Useful solid matrices are well known in the art, and include crosslmked dextran; agarose; polystyrene; polyvmylchlo ⁇ de; cross-linked polyacrylamide; nitrocellulose or nylon-based materials; tubes, plates or the wells of microtiter plates.
  • polypeptides or antibodies of the present invention may be used as diagnostic agents in solution form or as a substantially dry powder, e.g., 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.
  • 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 m samples that are undetectable by diagnostic methods using lysates of cultured spirochetes as the antigen.
  • One skilled m 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 epitopes. It may be particularly advantageous to use epitopes of aoHGE polypeptides that elicit antibodies early in aoHGE infection m combination with epitopes from other aoHGE polypeptides that elicit antibodies that occur m the later stages of human granulocytic ehrlichiosis. Diagnostic reagents containing multiple epitopes which are reactive with antibodies appearing at different are useful to detect the presence of anti-aoHGE antibodies throughout the course of infection and to diagnose human granulocytic ehrlichiosis at all stages.
  • a diagnostic kit comprising diagnostic reagents to detect aoHGE as well as other pathogens found in the same tick vector, for example, Borrelia burgdorferi and Babesia mi croti , 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 ⁇ ckettsia which may contain surface proteins sharing ammo acid sequence or conformational similarities with the aoHGE polypeptides of the present invention, for example, Ehrlichia equi and Ehrli chia phagocytophila .
  • 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.
  • mice 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 to 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 lmmunologic, biologic and genetic parameters available for manipulation.
  • mice We examined the susceptibility of various strains of mice to infection with the NCH-1 isolate of the HGE agent. We inoculated the mice via tick-borne infection or syringe oculatin by several different routes. [S.W. Barthold et al . , J. Inf .Pis. , (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/sc ⁇ d mice, purchased from the Jackson Laboratory (Bar Harbor, ME.), NCI Animal Production Program, Frederick Cancer Research Center (Fede ⁇ ck, MD) , and Harlan Sprague Dawley, Inc. (Indianapolis,
  • CD-I mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) .
  • mice To examine the course of tick-borne aoHGE infection, we placed 5 aoHGE-mfected 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 smears at 5-10 days after tick feeding. We necropsied 4 mice with verified infection and 4 age-matched control mice at days 5, 10, 17 and 24 after tick feeding.
  • mice exhibited transient splenomegaly and we were able to culture aoHGE from peripheral blood and spleen from all mice on days 17 and 24. All infected mice also developed detectable antibodies to aoHGE by day 10.
  • Infected mice developed transient hematologic aberrations similar to those described in human HGE including leukopenia, with a reduction in total leukocytes, granulocytes and lymphocytes, and anemia. Morulae were found only granulocytes. 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.
  • mice 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-mfected SCID mice (10% granulocytes with morulae) .
  • aoHGE-mfected SCID mice 10% granulocytes with morulae
  • mice 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 l.p. inoculation.
  • Xenodiagnosis occurrence of aoHGE infection in unmfected ticks which feed on infected mice
  • mice remained persistently infected for up to 55 days.
  • mice infected with aoHGE by syringe inoculation at days 5, 10, 30 and 60 after inoculation.
  • mice remained persistently infected for at least 55 days after inoculation, and had a 100% correlation between infection, seroconversion, and disease.
  • 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 l.p. injection with 0.1 ml of infected SCID mouse blood and assessed infection by hematocrit, spleen weight, morulae and PCR at 10 days after inoculation. e ⁇ .:covered that mice moculated 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.
  • 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 .
  • HL-60 cells American Type Culture Collection 240-CCL
  • Iscove's modified Dulbecco's medium supplemented with 20% fetal bovme serum, with no added antibiotic, maintained at 37°C with 5% carbon dioxide.
  • C3H specific pathogen free mice Jackson Laboratories, Bar Harbor, ME
  • mice by placing five hardened nymphs on each mouse and allowing the ticks to feed to repletion.
  • antibodies m 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 35 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 80 kDa aoHGE protein.
  • aoHGE from infected HL-60 cells prepared as described in Example III, as follows.
  • aoHGE NCH-1 isolate
  • the critical steps were adequate lysis of the 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 aoHGE.
  • the N- terminal amino acid sequence of the 80-kDa protein which we designated the 80-1 polypeptide, is set forth in SEQ ID NO: 7.
  • GenBank Genetics Computer Group Program (University of Wisconsin Biotechnology Center, Madison, WI).
  • ECPNAK E. coli
  • HSP70 human heat shock protein 70.
  • the most closely homologous protein identified in the database is the B . burgdorferi HSP- 70.
  • a search of the Genbank Database revealed that the 44-1 polypeptide is approximately 70% homologous with a region from ammo acid 130-138 of the major surface protein 2 (MSP-2) of Anaplasma margmal e .
  • the 44-2 polypeptide is homologous with a region of MSP-2 from ammo 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 m A. margmal e genome.
  • A. margmale is an important veterinary erythroparasitic pathogen and MSP-2 may confer protection against A. margmale infection [G.H.
  • aoHGE polypeptides from the NCH-1 isolate and from other strains of aoHGE, which are useful for the detection, treatment or prevention of human granulocytic ehrlichiosis or for the study of the pathenogenesis of the disease may be isolated and sequenced without undue experimentation according to the methods described herein.
  • 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 .
  • PCR amplification of the regions of the DNA encoding the 44 kDa aoHGE polypeptide may be performed by any of a variety of methods known m the art.
  • ⁇ he D fR 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.
  • aoHGE polypeptides 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. chaff eensis infection. Dr. J.G. Olson (CDC, Atlanta, GA) kindly provided the patient sera. None of the E. chaff eensi s 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 aoHGE protein.
  • Immunoscreenmg Kit (Stratagene) . We induced protein production from the recombinant plaques with lOmM IPTG and transferred the proteins to duplicate plaque lifts on nitrocellulose filters according to methods well known in the art.
  • Example VIII Cloning of Immunogenic 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.
  • 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, WI) (e.g., using Smal to generate the 5' blunt end and BstXI to generate a 3' overhang), and then sequence the subclones using, e.g., the Sequenase Kit (United States Biochemical Corp., Cleveland, OH) and reconstruct the entire sequence using MacVector (International Biotechnology, Inc., New Haven, CT) .
  • Erase-A-Base System Promega, Madison, WI
  • Smal to generate the 5' blunt end and BstXI to generate a 3' overhang
  • sequence the subclones using, e.g., the Sequenase Kit (United States Biochemical Corp., Cleveland, OH) and reconstruct the entire sequence using MacVector (International Biotechnology, Inc., New Haven, CT) .
  • the 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 (New England Biolabs) . Conditions for denaturation, annealing and extension were: 94° C for 30 sec, 55° C for 20 sec, and 72° C for 20 sec, respectively.
  • the DNA sequence of clone E6 is set forth in SEQ ID NO: 1.
  • the deduced ammo acid sequence is set forth in SEQ ID NO: 2.
  • sequences E5-3A, E5-3B, E5- 5A, E5-5B and E5-6 are set forth in Figures 13-17, respectively.
  • sequences E5-3B, E5- 5B and E5-6 have regions of substantial homology with regions of the 44-kDa protein.
  • nucleotides 400-600 and 900-1300, approximately, of the 44-kDa protein define regions of homology among the sequences.
  • Example IX - Expression of the E6 Polypeptide 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. Immunol. , 147, pp. 1995-2000 (1991)].
  • the vector also contains a thrombm cleavage site immediately following the GT protein, thus, allowing the recovery of recombinant proteins without the GT fusion partner.
  • aoHGE polypeptides of this invention may be recombinantly expressed without a fusion partner using techniques well known m the art.
  • Example IX After inducing protein expression as described m Example IX, we place the E. coli in 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.
  • PBS phosphate buffered saline
  • GT-E6 glutathione S-transferase-aoHGE polypeptide fusion protein
  • mice We generate antibodies directed against the aoHGE polypeptides of this invention as follows.
  • mice Frrederick Cancer Research Center
  • 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.
  • I_FA indirect immunofluorescence assay
  • mice 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 supra . We identify protective aoHGE polypeptides by their ability to prevent aoHGE infection or disease.
  • mice with purified recombinant aoHGE polypeptides prepared as described in Example 10 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 S- transferase. 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.
  • protective recombinant aoHGE polypeptides by their ability to prevent aoHGE infection and disease.
  • 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 monoclonal antibodies that confer protection against aoHGE infection.
  • 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 .
  • Another way to identify regions of aoHGE proteins that contain B cell epitopes is to use aoHGE polypeptide fusion proteins to absorb antibodies from protective polyclonal serum.
  • the various T7-aoHGE or aoHGE-glutathione S-transferase fusion proteins are coupled to CnBr activated Sepharore in order to construct a column, using standard techniques.
  • aoHGE polypeptides of this invention were able to elicit an immune response that is effective to protect against aoHGE infection.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice 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 HL-60 cells in immunoblot as descrioed Example I.
  • aoHGE-immunized mice had high titers of aoHGE-specifIC antibodies, detectable by immunoblot at a serum dilution of at least 1:2,000.
  • mice actively immunized and sham immunized mice by syringe and tick-borne inoculation.
  • mice actively immunized with purified, -killed aoHGE with 100 ⁇ l of blood from a mouse that had been infected with aoHGE for 2 weeks.
  • mice 10 days after challenge and evaluated for aoHGE infection by PCR using aoHGE specific 16S ribosomal DNA primers [P. Pancholi et al . , "Ixodes dammmi as a Potential Vector of Human Granulocytic Ehrlichiosis, " J. Inf. Dis . , 172, pp.
  • mice developed aoHGE infection, based on aoHGE-specific DNA the blood. In contrast, no aoHGE DNA could be detected in the blood of 4 of 5 mice vaccinated with purified, -killed aoHGE .
  • tick inoculation we placed 3-4 aoHGE-mfected I. dammmi nymphs which had fed to repletion on CD-I 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 . Na tl . Acad. Sci . USA, 93, pp. 6209-6214, supra ] .
  • the ticks were allowed to engorge to repletion on immunized and control mice.
  • mice were examined for aoHGE by PCR using aoHGE specific 16S rDNA, supra .
  • aoHGE has a chromosome that migrates at approximately 700 kb in pulse-field gel electrophoresis .
  • 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, supra .
  • mice with aoHGE polypeptide fusion protein were immunized with aoHGE and boost.
  • ticks infected with aoHGE as described m 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 .
  • mice were passively immunized with normal mouse serum.
  • aoHGE antiserum diluted 1:5 PBS was administered to mice.
  • 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 ⁇ l 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 ⁇ l of aoHGE antiserum diluted 1:5 with PBS on days 4, 8 and 12 after challenge.
  • serial dilution PCR amplified DNA was discernible serum from control mice at a dilution of 10 3 -10 8 whereas product could only be obtained from serum of immunized mice up to a dilution of 10 3 .
  • the PCR assay can detect a single aoHGE organism. Accordingly, passive immunization either conferred complete protection or lessened the severity of aoHGE infection.
  • mice In terms of clinical symptoms, the control mice but not the protected mice exhibited neutropenia (462 cell/mm 3 ⁇ 280 SD compared 3,240 cells/mm 3 ⁇ 1,340 SD) and splenomegaly (0.27 g ⁇ 0.05 SD compared to 0.12g ⁇ 0.03 SD) .
  • 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 .
  • mice 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.
  • mice immunized with recombinant aoHGE polypeptides confers protection.
  • One of skill in the art would understand that to detect a protective effect, one can vary the experimental conditions.
  • 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.
  • 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 solubilization m denaturants such as urea.
  • 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 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 not sufficient by themselves to confer protection against subsequent infection with aoHGE, may be useful in a multicomponent vaccine.
  • the protective epitopes are 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.
  • Example XXIV Identification of T cell epitopes 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.
  • T cell lines Shortly after priming, we harvest the lymph nodes and generate m vi tro 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 3 H-Thymidme incorporation. We also measure lymphokine production by the stimulated T cell clones by standard methods .
  • T cell epitopes of the polypeptides recognized by human T cells we isolate T cell clones from aoHGE-infected patients of multiple
  • T cell epitopes are identified by stimulating the clones with the various polypeptides
  • T cell epitopes are then correlated with Class II HLA antigens such as DR, DP, and DQ.
  • Class II HLA antigens such as DR, DP, and DQ.
  • the correlation is performed by utilization of B lymphoblastoid cell lines expressing various HLA genes.
  • 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 T cell.
  • T cell epitopes may be identified by adoptive transfer of T cells from mice 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, 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)].
  • T and B cell epitopes 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 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 lmmunoglobin, leading to the production of high titer neutralizing antibodies. We also construct fusion 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.
  • oligonucleotide homologous to am o acids 120 to 140 of the Hepatitis B virus core antigen. This region of the core antigen has been shown to contain a strong T cell epitope [D.R. Millich, et al., supra 1.
  • 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 lmmunogenicity of the polypeptide.
  • fusion proteins comprising epitopes of the aoHGE polypeptides as well as epitopes of the tetanus toxoid protein.
  • Bacterial flagellm are potent stimulators of cellular and humoral responses, and can be used as vectors for protective antigens [S.M.C. Newton, C. Jacob, B. Stocker, "Immune Response To Cholera Toxin Epitope Inserted In Salmonella Flagellm", Science, 244, pp. 70-72 (1989) ] .
  • 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.
  • 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 .
  • mice 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 pl97-aoHGE polypeptide plasmid. We bleed the mice 7 days after the second and fourth boosts and conduct immunoblots on extracts of aoHGE, as described in Example I, to detect and 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.
  • AAT TAT TTA ATT TTT TAT AAA AAT AAC TGC CAA TAT TTA TAT GAA GTA 240 Asn Tyr Leu He Phe Tyr Lys Asn Asn Cys Gin Tyr Leu Tyr Glu Val 65 70 75 80
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE DNA (genomic) (ix) FEATURE:
  • GAAGAGGAGC AAAGTCCTGC GCTTGATGTG ATAAGTAGTG AATTGCCTAA GGATGACATT 240
  • TCTCCTGTGT TTGGAGAAGA AGAGCGTGCC GAAGAAGATT TTGATGTGTA TCAAGATCCA 960 GTAGAAGTGG ATGATGAGGG AGTTGCTGAT TCTTCTGAGG ATTTAGAGGC TGATTCTGGT 1020

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Abstract

L'invention concerne des procédés et compositions destinés à la prévention, au traitement et au diagnostic de l'ehrlichiose granulocytaire humaine, de même que des polypeptides de l'agent de cette ehrlichiose (aoHGE), des variants à type sérologique, fragments et dérivés des polypeptides, ainsi que des protéines de fusion et des protéines multimères comprenant ces polypeptides. L'invention concerne encore des vaccins comprenant les polypeptides de aoHGE seuls ou ajoutés à d'autres polypeptides de protection contre aoHGE, et elle concerne également des séquences d'ADN, des molécules d'ADN recombiné et des cellules hôtes transformées, utiles dans les compositions et procédés de l'invention, ainsi que des anticorps dirigés contre les polypeptides de aoHGE, et des trousses de diagnostic comprenant ces polypeptides ou anticorps.
PCT/US1997/017675 1996-10-01 1997-09-30 Compositions et procedes destines a la prevention et au diagnostic de l'ehrlichiose granulocytaire humaine WO1998014584A2 (fr)

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JP10516827A JP2001502528A (ja) 1996-10-01 1997-09-30 ヒト顆粒球エールリヒア症の予防および診断のための組成物ならびに方法
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US7807810B2 (en) 1999-04-08 2010-10-05 The Ohio State University Research Foundation Nucleic acids encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby
US9359407B2 (en) 2007-05-04 2016-06-07 The Ohio State University Research Foundation Ehrlichia ewingii proteins, nucleic acids, and methods of their use
US9861690B2 (en) 2007-05-04 2018-01-09 Ohio State Innovation Foundation Ehrlichia ewingii proteins, nucleic acids, and methods of their use
US10393741B2 (en) 2011-03-31 2019-08-27 Ohio State Innovation Foundation Compositions and methods for the detection of Anaplasma platys
US10227665B2 (en) 2012-01-26 2019-03-12 Luc Montagnier Detection of DNA sequences as risk factors for HIV infection

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AU4741697A (en) 1998-04-24
EP0932680A2 (fr) 1999-08-04
JP2001502528A (ja) 2001-02-27
CA2268013A1 (fr) 1998-04-09

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