Diagnosis of Bartonella using recombinant proteins

Abstract

Bartonellosis is an emerging group of infectious diseases caused by bacteria belonging to the genus Bartonella, which includes at least 22 named species of bacteria transmitted primarily by carriers (i.e., vectors) including fleas, pubic lice, nits, sand flies, ticks, and potentially mites and spiders. In some aspects, the present disclosure provides antigen-specific amino acid sequences of Bartonella.

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 386,990, filed December 12, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Aspects of the present disclosure provide diagnostic panels and methods for diagnosing infection with Bartonella.

[0003] Bartonellosis is an emerging group of infectious diseases caused by bacteria belonging to the genus Bartonella. The genus Bartonella includes at least 22 named species of bacteria that are primarily transmitted by reservoirs (i.e., vectors), including fleas, pubic lice, nits, sand flies, ticks, and potentially mites and spiders. Both domestic and wild animals can become infected with Bartonella (i.e., Bartonella spp.) by these vectors. At least 14 Bartonella spp. are involved in diseases that can be transmitted from animals to humans (i.e., zoonotic diseases). Of these zoonotic diseases, several can be transmitted to humans by companion animals (e.g., dogs and cats), typically via bites or scratches.

[0004] Human illnesses that have been identified as being caused by any of the Bartonella spp. include cat-scratch disease caused by Bartonella henselae, carrion disease caused by Bartonella bacilliformis, and trench fever caused by Bartonella quintana. Bartonella spp. have also been associated with diseases of the skin (i.e., rod-shaped angiomatosis), liver (i.e., peliosis hepatica), heart (i.e., endocarditis), eye (i.e., neuroretinopathy), blood (i.e., bacteremia), and brain (i.e., encephalitis).

[0005] Bartonella infections do not always cause overt disease. Many studies have detected clinically healthy individuals who test positive for Bartonella (i.e., seropositive) but have no history of typical Bartonella symptoms. Those who do become ill usually have mild illness that tends to resolve without treatment (i.e., self-limiting).

[0006] Bartonella was previously thought to cause serious infections in some individuals. Immunocompromised individuals, such as those receiving immunosuppressive treatment for cancer, organ transplant recipients, and those with human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS), are at increased risk of developing severe, life-threatening disease. Recent advances in diagnostic technology have facilitated the documentation of chronic bloodstream and skin infections caused by Bartonella in human blood donors and immunocompetent and immunocompromised individuals.

[0007] Prior to 1990, there was only one named species of Bartonella (i.e., B. bacilliformis), but there are now over 36 species, 17 of which are associated with an expanding spectrum of animal and human diseases.

[0008] The field of Bartonella research is still in its infancy, with many questions needing appropriate answers for patients. Advances in Bartonella research could significantly reduce the spectrum of chronic and debilitating animal and human diseases, thereby alleviating suffering worldwide. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Short Protocols in Molecular Biology, Second Edition, FM Ausubel, Ed., all John Wiley & Sons, NY, edition as of 2008 [Non-patent document 2] Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. [Non-patent document 3] Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease,” Healthcare (Basel) 2018 Aug 14;6(3):99. doi: 10.3390 / healthcare6030099 [Non-patent document 4] Liu S. et al, “Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease,” Healthcare (Basel) 2018 Aug 14;6(3):99. doi: 10.3390 / healthcare6030099 Summary of the Invention [Problem to be solved by the invention]

[0010] The present disclosure provides diagnostic panels and methods for diagnosing and treating bartonellosis resulting from infection with various Bartonella species.

[0011] A first aspect of the present disclosure relates to a diagnostic panel comprising individually identifiable labeled, tagged, or conjugated amino acid sequences comprising SEQ ID NOs: 1-41, and variants thereof that retain the immunological binding profile of the corresponding non-variant. In some embodiments of the first aspect, the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the first aspect, the amino acid sequences comprise one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes. In some embodiments of the first aspect, the amino acid sequences comprise one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

[0012] A second aspect of the present disclosure relates to a method for detecting infection with one or more Bartonella species, the method comprising contacting a biological sample obtained from a subject suspected of having a Bartonella infection with a diagnostic panel comprising an amino acid sequence comprising SEQ ID NOs: 1-41, or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant; and determining that the biological sample contains at least one of an IgM class antibody, an IgG class antibody, and an IgA class antibody, and that the biological sample is positive for Bartonella infection if a positive immune binding reaction with the IgM class antibody, IgG class antibody, or IgA class antibody is detected for at least one of SEQ ID NOs: 1-12 and at least one of SEQ ID NOs: 13-41, or if a positive immune binding reaction with the IgM class antibody, IgG class antibody, or IgA class antibody is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9-12. In some embodiments of the second aspect, the amino acid sequence is unlabeled, untagged, and unlinked. In some embodiments of the second aspect, the amino acid sequence comprises one or more labels selected from the group consisting of a stable isotope, a mass tag, and a fluorescent dye. In some embodiments of the second aspect, the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of an affinity tag, a chromatography tag, an epitope tag, an enzyme-conjugated tag, a fluorescent tag, and combinations thereof. In some embodiments of the second aspect, the amino acid sequence is bound to one or more substrates. In some embodiments of the second aspect, the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the second aspect, immunobinding of IgM class antibodies is detected using an anti-human IgM antibody linked to a detectable moiety. In some embodiments of the second aspect, immunobinding of IgG class antibodies is detected using an anti-human IgG antibody linked to a detectable moiety.In some embodiments of the second aspect, immune binding of IgA class antibodies is detected by use of an anti-human IgA antibody linked to a detectable moiety. In some embodiments of the second aspect, the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme. In some embodiments of the second aspect, the detectable moiety comprises alkaline phosphatase. In some embodiments of the second aspect, the detectable moiety comprises biotin. In some embodiments of the second aspect, the method comprises treating the subject for Bartonella infection in response to determining that the biological sample is positive for Bartonella infection. In some embodiments of the second aspect, the method includes determining that the biological sample is positive for Bartonella henselae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 13, 17, and 27; and determining that the biological sample is positive for Bartonella quintana infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 14, 18, and 28. and determining the biological sample to be positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 15, 19, and 29, and determining the biological sample to be positive for Bartonella elizabesae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 16, 20, and 30. In some embodiments of the second aspect, the method further comprises treating the subject for Bartonella henselae infection in response to determining the biological sample to be positive for Bartonella henselae infection. In some embodiments of the second aspect, the method further comprises treating the subject for Bartonella quintana infection in response to determining the biological sample to be positive for Bartonella quintana infection.In some embodiments of the second aspect, the method includes treating the subject for a Bartonella vinsonii infection in response to determining that the biological sample is positive for a Bartonella vinsonii infection. In some embodiments of the second aspect, the method includes treating the subject for a Bartonella elizabethoeae infection in response to determining that the biological sample is positive for a Bartonella elizabethoeae infection.

[0013] A third aspect of the present disclosure relates to a diagnostic panel comprising individually identifiable labeled, tagged, or conjugated amino acid sequences comprising one, two, three, or four of SEQ ID NOS: 1-4, one, two, three, or four of SEQ ID NOS: 5-8, and one, two, three, or four of SEQ ID NOS: 9-12, and variants thereof that retain the immunological binding profile of the corresponding non-variant. In some embodiments of the third aspect, the diagnostic panel comprises individually identifiable labeled, tagged, or conjugated amino acid sequences comprising one, two, three, or four of SEQ ID NOS: 1-12, and variants thereof that retain the immunological binding profile of the corresponding non-variant. In some embodiments of the third aspect, the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the third aspect, the amino acid sequences comprise one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes. In some embodiments of the third aspect, the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of an affinity tag, a chromatography tag, an epitope tag, an enzyme conjugate tag, a fluorescent tag, and combinations thereof.

[0014] A fourth aspect of the present disclosure relates to a method for detecting infection with one or more Bartonella bacteria, the method comprising the step of contacting a biological sample obtained from a subject suspected of having a Bartonella infection with a diagnostic panel comprising amino acid sequences including one, two, three, or four of SEQ ID NOs: 1-4; one, two, three, or four of SEQ ID NOs: 5-8, and one, two, three, or four of SEQ ID NOs: 9-12; or one, two, three, or four of SEQ ID NOs: 9-12, and variants thereof that retain the immunological binding profile of the corresponding non-variants; and if a positive immune binding reaction with IgM class antibodies, IgG class antibodies, or IgA class antibodies is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9-12, the biological sample is determined to be positive for Bartonella infection. In some embodiments of the fourth aspect, the diagnostic panel comprises SEQ ID NOS: 1-12, or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant, and the biological sample comprises at least one of an IgM class antibody, an IgG class antibody, and an IgA class antibody. If a positive immunological binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOS: 1-4, at least one of SEQ ID NOS: 5-8, and at least one of SEQ ID NOS: 9-12, the biological sample is determined to be positive for Bartonella infection. In some embodiments of the fourth aspect, the amino acid sequence is unlabeled, untagged, and unbinding. In some embodiments of the fourth aspect, the amino acid sequence comprises one or more labels selected from the group consisting of a stable isotope, a mass tag, and a fluorescent dye. In some embodiments of the fourth aspect, the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of an affinity tag, a chromatography tag, an epitope tag, an enzyme conjugate tag, a fluorescent tag, and combinations thereof. In some embodiments of the fourth aspect, the amino acid sequence is attached to one or more substrates.In some embodiments of the fourth aspect, the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the fourth aspect, immunobinding of IgM class antibodies is detected by using an anti-human IgM antibody linked to a detectable moiety. In some embodiments of the fourth aspect, immunobinding of IgG class antibodies is detected by using an anti-human IgG antibody linked to a detectable moiety. In some embodiments of the fourth aspect, immunobinding of IgA class antibodies is detected by using an anti-human IgA antibody linked to a detectable moiety. In some embodiments of the fourth aspect, the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme. In some embodiments of the fourth aspect, the detectable moiety comprises alkaline phosphatase. In some embodiments of the fourth aspect, the detectable moiety comprises biotin. In some embodiments of the fourth aspect, the method includes treating the subject for a Bartonella infection in response to determining that the biological sample is positive for a Bartonella infection.

[0015] A fifth aspect of the present disclosure relates to a diagnostic panel comprising individually identifiable labeled, tagged, or conjugated amino acid sequences, including SEQ ID NOs: 13-20 and 27-30, and variants thereof that retain the immunological binding profile of the corresponding non-variant. In some embodiments of the fifth aspect, the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the fifth aspect, the amino acid sequences comprise one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes. In some embodiments of the fifth aspect, the amino acid sequences comprise one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

[0016] A sixth aspect of the present disclosure relates to a method for detecting species-specific infection with Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and / or Bartonella elizabesae, the method comprising the steps of contacting a biological sample obtained from a subject determined to have a Bartonella infection with a diagnostic panel comprising amino acid sequences comprising SEQ ID NOs: 13-20 and 27-30, or one or more variants that retain the immunological binding profile of the corresponding non-variants, wherein the biological sample comprises at least one of an IgM class antibody, an IgG class antibody, and an IgA class antibody; and determining that the biological sample is infected with Bartonella henselae if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 13, 17, and 27. determining that the biological sample is positive for Bartonella quintana infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 14, 18, and 28; determining that the biological sample is positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 15, 19, and 29; and determining that the biological sample is positive for Bartonella elizabesae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 16, 20, and 30. In some embodiments of the sixth aspect, the amino acid sequence is an unlabeled, untagged, and non-binding sequence. In some embodiments of the sixth aspect, the amino acid sequence comprises one or more labels selected from the group consisting of a stable isotope, a mass tag, and a fluorescent dye. In some embodiments of the sixth aspect, the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of an affinity tag, a chromatography tag, an epitope tag, an enzyme conjugate tag, a fluorescent tag, and combinations thereof.In some embodiments of the sixth aspect, the amino acid sequence is bound to one or more substrates. In some embodiments of the sixth aspect, the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal. In some embodiments of the sixth aspect, immunobinding of IgM class antibodies is detected by using an anti-human IgM antibody linked to a detectable moiety. In some embodiments of the sixth aspect, immunobinding of IgG class antibodies is detected by using an anti-human IgG antibody linked to a detectable moiety. In some embodiments of the sixth aspect, immunobinding of IgA class antibodies is detected by using an anti-human IgA antibody linked to a detectable moiety. In some embodiments of the sixth aspect, the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme. In some embodiments of the sixth aspect, the detectable moiety comprises alkaline phosphatase. In some embodiments of the sixth aspect, the detectable moiety comprises biotin. In some embodiments of the sixth aspect, the method includes treating the subject for a Bartonella henselae infection in response to determining the biological sample as positive for a Bartonella henselae infection. In some embodiments of the sixth aspect, the method includes treating the subject for a Bartonella quintana infection in response to determining the biological sample as positive for a Bartonella quintana infection. In some embodiments of the sixth aspect, the method includes treating the subject for a Bartonella vinsonii infection in response to determining the biological sample as positive for a Bartonella vinsonii infection. In some embodiments of the sixth aspect, the method includes treating the subject for a Bartonella elisabethoe infection in response to determining the biological sample as positive for a Bartonella elisabethoe infection. [Brief explanation of the drawings]

[0017] [Figure 1]1 is an image showing the results of Bartonella immunoblots of IgM and IgG classes used to test nine human samples suspected of Bartonella infection. [Figure 2A-B] A comparative image of a Bartonella Western blot (FIG. 2A) and a Bartonella western blot of the present disclosure (FIG. 2B) is shown.

[0018] (Array description) SEQ ID NO: 1 - Amino acid sequence of Bartonella henselae htRA recombinant protein MVKKTFFTTLAAVSFSAALETALFFSGCGSSLWTTKAHANSVFSSLMQQQGFADIVSQVKPAVVSVQVKSNKKKKEWFFSDFFSTPGFDQLPDQHPLKKFFQDFYNRDKPSNKSLQRSHRLRPIA FGSGFFISSDGYIVTNNHVISDGTSYAVVLDDGTELNAKLIGTDPRTDLAVLKVNEKRKFSYVDFGDDSKLRVGDWVVAIGNPFGLGGTVTAGIVSARGRDIGTGVYDDFIQIDAAVNRGNSGGPT FDLNGKVVGVNTAIFSPSGGNVGIAFAIPAATAKQVVQQLIEKGLVQRGWLGVQIQPVTKEISDSIGLKEAKGALITDPLKGPAAKAGIKAGDVIISVNGEKINDVRDLAKRIANMSPGETVTLGV WKSGKEENIKVKLDSMPEDENMKDGSKYSNEHGNSDETLEDYGLIVAPSDDGVGLVVTDVDPDSDAADKGIRPGDVIVTVNNKSVKKVSDITDTIKNAQKLGRKAILLQVRTNDQNRFVALPIFKK

[0019] SEQ ID NO:2 - Amino acid sequence of Bartonella quintana htRA recombinant protein MVKKTFFPTLAAVGFSAALETALFFSGCGSSLWMTEARANSVFASLVQQQGFANIVSQVKPAVVSVQVKSNKKKKEWFFSDFFSSPGLDQLPDQHPLKKFFQEFYDRGKPSNKSLKRPHRLRPIA FGSGFFISSDGYLVTNNHVISDGTSYTVVLDDGTELNAKLIGTDARTDLAVLKVDEKRTFSYVDFGDDSKLRIGDWVVAIGNPFGLGGTVTAGIVSARGRDIGTGVYDDFIQIDAAVNRGNSGGPT FDLNGKVIGVNTAIFSLSGGNVGIAFAIPAAIAKQVVQQLIEKGSVQRGWLGVQIQPVTKEISDSIGLKEAKGALITDPLKGPAAKAGIKAGDVIISVNGEKINDVRDLAKRIANIKPGETVTLGV WKSGKEENIKVKLDTMPDDKVMKEDSKYSNGRNDSNETLEDYGLIVAPSDDGIGLVVTDVDSDSDAADKGIRPGDVIVTVNNKSVKKVSDITDTIKNAQKLGRKAILLQVRTNDQNRFVALPIFKK

[0020] SEQ ID NO: 3 - Amino acid sequence of Bartonella vinsonii htRA recombinant protein MVKKTFFKTLIAVSFSAALESALFFSGCGSSLWTTAAHANSVFTSFMQQPGFADIVSQVKPAVVSVQVKSNKKKDEWSFSNFFSGPGFDQLPDQHPLKRFFKEFYDLDKPKNKFPSHSHRLRPIA FGSGFFISSDGYIVTNKHVISDGTSYSVVLDDGTELNAKLIGSDQRTDLAVLKVNDKRKFSYVDFGDDSKLRVGDWVVAIGNPFGLGGTVTAGIVSARGRDIGTGVYDDFIQIDAAVNRGNSGGPT FDLNGKVVGVNTAIFSPSGGNVGIAFAIPAATVKQVVQQLIEKGSVQRGWLGVQIQPVTKEISDSIGLKEDKGALVTDPLKGPAEKAGIKAGDVIISVNGEKINDARDLAKRIANIRPGETVTLGV WRSGKEENIKVKLASMPEDEGKKEGSKYSNKRNDADETLEDYGLIVAPSDDVGLVVTDVDSDSDAADKGIRPGDVIITVNNKSVKKVSDITDTIKNAQKLGRKAILLQVRTNGQNRFVALPIFKK

[0021] SEQ ID NO: 4 - Amino acid sequence of Barsonella elizabethoe htRA recombinant protein MLKKTFFKTFVAVSFSAVLESALFFSGCTASLWTTKAHASSVFTSLVQQQGFADIVAQVKPAVVAVQVKSNKKKEDWFFSNFFSGPGIDQLPDQHPLKRLFKEFYDFDKPKNKFPHHSQRLRPIA FGSGFFISSDGYIVTNDHVISEGTSYSVVLDDGTELNAKLIGKDPKTDLAVLKVNDKRKFSYVDFGDDSKLRVGDWVVAIGNPFGLGGTVTAGIVSARGRDIGTSSYDDFIQIDAAVNRGNSGGPT FDLNGKVVGVNTAIFSPSGGNVGIAFAIPAGTAKQVVQQLIEKGSVQRGWLGVMIQPVTKEISDSIGLQEAKGALVTDPLKGPAAKAGIKAGDVIISVNDEKVNDSRDLAKRIANMSPQETVTLGI FRSGKEEKIKVKLAAMPEDEGKKESSKYLNERGNSDETLEDYGLIVAPSEDGLGLVVTDVDSDSDAADKGIRPGDVIVTVNNKSVKKTSDITDAIKNAQKLGRKAILLQVRTNDQNRFVALPIFKK

[0022] SEQ ID NO: 5 - Amino acid sequence of Bartonella henselae SucB recombinant protein MTTEIRVPTLGESVTEATVGKWFKKLGEAVAVDEPLIELETDKVTVEVPSPVAGKLSEIIAKEGDTVEVKALLGLVEAGAAGISQSFSPSATPIPEVPSELKQSSSSGAMQKDTMPPSPSAAKLMAENNIAKSNISGSGKRGQILKEDVLGVLEQEVKAPSVSAASSSASLVQEKHEERVRMTKLRQTIARRLKDAQNTAAML TTFNEVDMSAVMDLRKRYKDLFEKKHGVKLGFMGFFTKAVCHALKELPAVNAEIDGTDIVYKNYVNVGIAVGTDKGLVVPVVRHADQMSLAEIEKEIGRLGRLARDGKLAVSDMQGGTFTITNGGVYGSLMSTPILNAPQSGILGMHAIKERAMVVGGQIVIRPMMYLALSYDHRIVDGQEAVTFLVRVKESLEDPERLVLDL

[0023] SEQ ID NO: 6 - Amino acid sequence of Bartonella quintana SucB recombinant protein MTTGIRVPTLGESVTEATIGKWFKKLGEAVAVDEPLVELETDKVTVEVPSPVMGKLTEIIAKEGDIVEVNAVLGFVESGAAGISQSFSPSATSIPEAPSELEQSPSSSATPSGTMPPAPSAAKLMAENNIAKSDISGSGKRGQILKEDVLGALAQGTKASTSVATLTASSSSAAPIQEMREERVRMTKLRQTIARRLKDAQNTAA MLTTFNEVDMSAVMDLRKRYKDLFEKKHGVKLGFMGFFTKAVCHALKEFPTVNAEIDGTDIVYKNYVNAGIAVGTDKGLVVPVVRDADQMSLAEIEKEISRLGRLARDGKLAVSDMQGGTFTITNGGVYGSLMSTPILNAPQSGILGMHAIKERAMVVGGQIIICPMMYLALSYDHRIVDGQEAVTFLVRVKESLEDPERLVLDL

[0024] SEQ ID NO: 7 - Amino acid sequence of Bartonella vinsonii SucB recombinant protein MTTEIRVPTLGESVTEATVGKWFKKLGEAVAIDEPLVELETDKVTVEVPSPVAGKLFEIIAKEGDTVEVNALLGAVEAGAASVAKSPSSSETSVSAAPSELEQSSSSNTMPPAPSAAKLMAENNIAKSDILGSGKRGQILKEDVLNVLAQGVKTSPPAVSASSSTPVSVSSSAVAPVQEMREERVRMTKLRQTIARRLKDAQNTA AMLTTFNEVDMSAVMGLRKRYKDLFEKKHGVKLGFMGFFTKAVCHALKELPAVNAEIDGTDIIYKNYVNAGIAVGTDKGLVVPVVRDADQMSLAEIEKEIGRLGRLARDGKLAVSDMQGGTFTITNGGVYGSLMSTPILNAPQSGILGMHAIKERAMVVDGQIAIRPMMYLALSYDHRIVDGQEAVTFLVRVKESLEDPERLVLDL

[0025] SEQ ID NO:8 - Amino acid sequence of Bartonella elizabesae SucB recombinant protein MTTEIRVPTLGESVTEATVGKWFKKLGEAVAMDEPLVELETDKVTVEVPSPVAGKLSEIIAKEGDTVEVNALLGTVEAGAAGVTQSFSPSATSVPVASSESEKLASSNTMPPSPSAAKLMAENNIAKSDIAGSGKRGQILKEDVLGGLEQKTKTPTPSSSATGSSVVSVPETREERVRMTKLRQTIARRLKDAQNVAAMLT TFNEVDMSAVMDLRKRYKDLFEKKHGVKLGFMGFFTKAVCHALKELPAVNAEIDGTDILYKNYVNVGIAVGTDKGLVVPVVRDADQMSLAEIEKEIGRLGRLARDGKLAVSDMQGGTFTITNGGVYGSLMSTPILNAPQSGILGMHAIKERAMVVEGQVVIRPMMYLALSYDHRIVDGQEAVTFLVRVKESLEDPERLVLDL

[0026] SEQ ID NO: 9 - Amino acid sequence of Bartonella henselae N1pD / LppB recombinant protein MCLKVLGNVVRCNQGIALLAMIMVLVGCSSGMQRFANPFSYPTISNQPNISTTISTYTDSPMLQYGAGMIQSTELPPVEPVNDLSTYDNTAYNSPPQDGTSSPNSRIMGTPPRNLGTLSR SQMRNDPLFRQNSYIVQTGDTLLSIARQRGVSVEALKLVNGIRSNSIYIGQVLMIPSGRTAETSNVRNNDWGASKQSLSQSQSASSIRHKKYSSTEKAPITPKPSAQINRSNGEQNSSAQM SSLNYEKGVLDTVMNKDNGTTPQATGISKMRWPVRGRLLSQFGQKKGTTSRGIDIAVPEGSSVKAAENGIVIYASDGLKELGNVVMIRHENNIITIYGCNSKLVVTRGQRIRRGDEIAKSGVSGDVKTPRVYFEVRENSLPVDPIKYLEN

[0027] SEQ ID NO: 10 - Amino acid sequence of Bartonella quintana N1pD / LppB recombinant protein MYLKVLGKIFRCNQGITLLAMITILAGCSSGTQRFANIFSHTVSNRSNMSTTIPTDPGMLSYGGGVIQSTELPPVEPSNDSWADDSAPYNSPRQDGTLSPDGRIIGTPPRNLGTISRSQMNNTPLFRRNSYIVQSGDTLLSIARQIGISVEALKLANGISSNNSIYIGQVLMIPGRRTVATSNAQNDDRVTSTE PSSQSQVRSSIRYQKAPSTDKAPITAMPSSKINRSNAEQDSSTQMPSSNYKTDVLDTVMNTDDGVTPQATGISKMRWPVRGRLLSQFGQKKGTATSRGIDIAVPEGSSVKAAENGIVIYASDGLKELGNVVMIRHENDIITIYGYNSKLVVNRGQRIRRGDEIAKSGVSGDVKTPRVYFEVRKNSLPVDPIEYLEN

[0028] SEQ ID NO: 11 - Amino acid sequence of Bartonella vinsonii N1pD / LppB recombinant protein MCLKVLSNIFWRNFQGIFFLAMVTIFAGCSSGTQRFANIFPHRTESTHSNISTTMSADPQMLPYGGMIQSTELPPVESGSDSWINDNTAYNSTQPRGETSSDSRIMGTPPRDLGTLSRSQMGNSPIFRRNSYIVQSGDTLLSIARQIGINVEALKSANGIRNNSIYIGQVLMIPNSRTLATSNTTNDKRVVSTTE TSVQSQNTSAIRYKKASPINKTSATISSSAKINRSNIEKNSSKQMMQLNHEASLPDTITNTDSIVTPQATGISKMRWPVRGRLLSQFGQKKGTATNRGIDIAVPEGSSVKAAENGVVIYASDGLKELGNVVMIRHEDNIITIYGCNSRLVVNKGQRIRRGDEIAKSGISGNVKTPRVYFEVRKNAVPVDPTEYLEN

[0029] SEQ ID NO: 12 - Amino acid sequence of S. erythropoiesis N1pD / LppB recombinant protein MCLKALDNIFRHNFQRITFLAVVTIVAGCSSGSQRFSNIVSHHRVSSQSNIANSMSSDPGMLSYGGEMIQSTELPPVEPSDDSWIDDNSPQQDRGAPSNGRVMGAPPRNLGTLSRSQMDSSPIFRQNSYIVQSGDTLLSIARQVGVSVE ALKSANGINSNSIYIGQVLVLPSRRALATSKVPSYNDLAVSKAESSFQSQMTSSTKHKKASPIYKAPVTTPPSTQVNRSNSEKNSSKQMMQLNHESDLSSTVTRTDNIKTPQATGISKMRWPVRGRLLSQFGQKKGTIMNRGIDIAVPEG SSVKAAENGVVIYAGDGLKELGNVVMIRHENNIITIYGCNSQLVVNKGQRIRRGDEIAKSGVSGNVKTPRVYFEMRKNSLPVDPLKYLEN

[0030] SEQ ID NO: 13 - Amino acid sequence of Bartonella henselae 35 kDa recombinant protein MSNIYDWSLKADENAHSDNIINWAEGQPPSSVNDSARAMMQRVREYLADNGGSLDSTFIVNAEDKTTSIIVTTASPIEEYKNDIIIRFKAGGVNVGTTTVIVNNIEEKLLYKATNAGVTPLEGGELQRDAIYEMVYNKDVSMEDHGGWYLLNPTPLPPPKIET FPSGFIATFAMQEVPSGWLLCDGAVYERKDYPQLFKAIGDKWGKDSDTTFKVPDFRGMFLRGFDDGRGLDAGRQFADQQHDSIRSHTHIGTIEEAGEHTHKFQYYGVGWNSGDIGRRNPFYYRQSSIGVTQSAGAHTHNISLSSTGEAETRPVNATVVYAIKS

[0031] SEQ ID NO: 14 - Amino acid sequence of Bartonella quintana 35 kDa recombinant protein MSSIYDWSLEAAENAYADVNINWAEGQPPSSVNNSARVMMQRIKEYLLDNGGVMETQFTINEDKTSIRLITNSPIEAYMDGIVVRFKAQKQNRGITNIALKQLPMQPVYKITKSGIAPLKGQEIQIGGLYELVYICNIDGKNTDGWFLTNPVDKDNFPSGF IGTFAMEKLPDGWLVCDGKEYSRKEYADLFEALGETWGKGDGQTTFNVPDLRGMFLRGLDSGKGIDKKRSLGTKQEESFKAHTHEGTTERAGEHTHKYEVYTKSIDIIASTNGWEAMYNHPNKNKVWETRSAGAHEHKISLKKTGGDETRPVNVAVVYAVKA

[0032] SEQ ID NO: 15 - Amino acid sequence of Bartonella vinsonii 35 kDa recombinant protein MSNIYDWSLKADENANSDSIINWAEGQPPSSVNDSARAMMQRVREYLADSGGSIDSSFMVNVEDKTTFITLKTASLIEKYKNDIIIRFKSRGVNIGRTTITVNSIGEKPIYKATNAGIIPLEGGELQTDGIYEIVYNSNVSIEDYDGWFLLNPTPLPPPKVEP FPCGFIATFAMQEMPNGWLLCDGAVYKRKDYPQLFKAIGDKWGKDSNTTFKVPDFRGMFLRGFDNGRGLDSNRQFAKEQQDCIKSHEHVCTIERAGEHTHNFQYDGVGWSANDIGRRNPSYHYQTITGTTQSAGAHTHKVTISPTGERETRPVNTTVVYAIKS

[0033] SEQ ID NO: 16 - Amino acid sequence of Bartonella elizabesae 35 kDa recombinant protein MSNIYDWSLKADENAHSDNIINWSEGQPPSSVNDSARAMMQRVREYLADNGGSIDAGFMINAEDKTTLITLKTVSPIKKYNNDIIIRFKAHGVNVGKTTIKVNSMGEKPIYKATHTGVISLEGGELQTDGIYEMVYNGNISTEGRDGWYLLNPTSPKVESFPTGFIATFAMQEIPTGWL LCDGKAYKRKDYPQLFKAIGDKWGKDSDTTFKVPDFRGMFLRGFDDGRGLDDNRKFADEQQDSIKSHTHIGNIEESGEHTHNFQYKGVGWPTGNIGRLPNYYTYNATLQGTTDSAGAHTHKVTLSHTGETETRPVNTTVVYAIKS

[0034] SEQ ID NO: 17 - Amino acid sequence of Bartonella henselii 31 kDa recombinant protein MNIKSLMTTSVIALISASAAQAADVIVPHEVAPTVISAPAFSWTGFYIGGQVGNFSSKVEITDPNKKDKLFSKDDTPKPSGFMGGIYAGSNMDLGNNMILGVETDAVWADREDAKTSSAEAIGQDELETFRDSLKKANA AFAQGKTSDNVAAVDKHTDSLALKEKWSGATRVRIGFTAADRIMPYVAGGVSYAQVQAVSSTKVTQAADDAEIATAQLFDKTKTLVGFTLGGGVDFAMTDNVLLRAEYRYSDFGKKKFEKEGSEFSYKTNDFRVGVAYKF

[0035] SEQ ID NO: 18 - Amino acid sequence of Bartonella quintana 31 kDa recombinant protein MNIKSLTSVIASAQAADVIAAPVITPTFSWTGFYGGQIGNFTSDNKIKGLGKETKETTPQLSGIVGYAGNIDLGSGLILGVETDAIWADCEATKTSVPQANSLNDFKAAGITLKDKF SMSDHYTYKAKWSGATRARIGFSAFDRVMPYFAGVAYARMQGMKSVSGMNAAKNKLKGGLYDETKMVGFTVSGVDVAMTGNVLRGEYRISDFGKKFLNNTQEFNYKTNDFRVGVAYKF

[0036] SEQ ID NO: 19 - Amino acid sequence of Bartonella vinsonii 31 kDa recombinant protein MNIKSLITTSVIALASASAVQAADVIIPHEAAPVISAPAFSWTGFYIGGQVGSFSSKVEITEPSKNKLNKDLTPKPSGFMGGVYAGSNIELGNNLILGVETDAVWADREETKERFSHVLEADELDAFKGDLTQLKASP ATGKQIAAIKTGDKRTDSTLKEKWSGATRVRIGFAAADHIMPYVAGGVAYAQMQGTYTIKATEAKGNAEIATAKLFDATKTFVGYTLGGGVDIAMTDNVLLRAEYRYSDYGKKQFDRDKDEFSYKTNDFRVGVAYKF

[0037] SEQ ID NO: 20 - Amino acid sequence of Bartonella elizabethoe 31 kDa recombinant protein MNIKFLMATSVVTLVSVSAANAADVIVPHEVAPAVVTAPSFSWTGFYLGGQIGNFSSKTKVTIPGEDKELFKKDNTPSGFMGGIYAGSNVDLGSGLILGVETDAVWADREESKTARESTLTKKDATFFNDALKKAKVELTGQQKFI EDDQVTETHSYKEKWSGATRVRIGFAAVDRIMPYVAGGIAYAQVQGIQSVAGKKGERAKVEQKADGGEELPEGNKQTIDLTGGTWADDTKTMVGFTIGGGVDFAMTDNVLLRAEYRYSDFGKKFANDTREFNYKTNDFRVGVAYKF

[0038] SEQ ID NO: 21 - Amino acid sequence of Bartonella baciliformis 31 kDa recombinant protein MNIKCLVTASVCALISASAAQAADVMIPQEISPIISAPTFSWTGLYLGGQIGGLSGKHDFKAIGKDSEWPFANKDLKVSGFVGGLYAGSNIDLGSGLVLGVDTDIVWVDKEGKLSSNHKAETQDDADAFKQIFDENKIEVAKGQ IKELTQNFSLKEKWAGATRVRIGFGADRIMPYVSGGVAYTQVQAIGSAILKGTKDTGTEGGGGSASKAVRSEALDVLASGTITDEKKTLLGYTLGAGVDFAMTDNVILRTEYRYSDFGKKKFVKDAIETNYKTNDFRVGVAYKF

[0039] SEQ ID NO: 22 - Amino acid sequence of Bartonella claridiae 31 kDa recombinant protein MNIKCLVTASVCALISASAAQAADVMIPQEISPIISAPTFSWTGLYLGGQIGGLSGKHDFKAIGKDSEWPFANKDLKVSGFVGGLYAGSNIDLGSGLVLGVDTDIVWVDKEGKLSSNHKAETQDDADAFKQIFDENKIEVAKGQ IKELTQNFSLKEKWAGATRVRIGFGADRIMPYVSGGVAYTQVQAIGSAILKGTKDTGTEGGGGSASKAVRSEALDVLASGTITDEKKTLLGYTLGAGVDFAMTDNVILRTEYRYSDFGKKKFVKDAIETNYKTNDFRVGVAYKF

[0040] SEQ ID NO: 23 - Amino acid sequence of Bartonella grahami 31 kDa recombinant protein MNIKSLVTTSVIAMAAAASAAQAADVIIPRETAPAVISAAPSFSWTGFYIGGQVGNFSGKVDEFDSETKKKITDKDWTPKPSGFMGGIYVGSNVDLGNGLILGVETDAVWADREDSKTHSAVIGETGLAAFKNKLTAAEA TFASGKNIDNVTKDGKRVEGTSIKEKWSGATRVRIGFAAVDRILPYFAGGIAYTQLQVVNSLKAEGAADGAEIASAKMFDQSKTMVGFTVGGGVDFAMTDNVLLRAEYRYSDFGKKAFVKDEDKIAYKTNDFRVGVAYKF

[0041] SEQ ID NO: 24 - Amino acid sequence of Bartonella choerrae 31 kDa recombinant protein MNIKSLMTTSVIALLSASAVQAADVIVPHEIAPAVITAPTFSWTGFYIGGQVGNFSSKVEVTDPNTKNKLFSKDDAPKPSGFMGGVYAGSNIDLGNNVILGVETDAVWADREDSKTLSSKAIEETKLGEFKASLTKANA VVATGKKIDDIKKEDKHTDSLVLKEKWSGATRVRIGFAAADRIMPYVSGGVSYVQVQAVSSTKVTQAADGAEIVSAKFFDKTKTLVGFTLGGGVDFAMTDNVLLRAEYRYSDFGKKKFEKDGSELSYKTNDFRVGVAYKF

[0042] SEQ ID NO: 25 - Amino acid sequence of Bartonella localimae 31 kDa recombinant protein MNKHLVACSWTFIPVSMVQAADIMKPHQSQSYQPVPVISNNNFSWTGFYVGAQGFSGATLSPIQDSVEDRGQGFSIKSKDLIGIDMVWSGKTKVVFDVQDMSIKWAGMRVGFAMDRVMPPYVSGVSYVVQLQNVFTKRAKVRRRRERNTLPSDSPK FLDETKIRVGGGVGVDVAMTNVIMREYRSFGKFAKKFAKDLKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTK TKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTKTFRVGVAYKF

[0043] SEQ ID NO: 26 - Amino acid sequence of Bartonella washoeensis 31 kDa recombinant protein TKDETPQPSGFMGIYAGSNVDLNGLILGVETDAVWADRGETCTISVFSGNSSETSNAGASQTPNAGAEEKXISLKTTALVDAKIKNNFTFKWSGATRVVGFAAADRILPYVGIAYAQMQGIATVSGDASGKTVSTKESDTMTMVGFTIGGVAMTDNVLRAYRISDFGKFEKDEFHYKTNDFRVGVAYKF

[0044] SEQ ID NO: 27 - Amino acid sequence of Bartonella henselii 26 kDa recombinant protein MKKVIFKPLKsypvkmamialtllaaspithaeeskmKnatitvtatgenqatpdmaiinlavvtqdktaqkaladnnksmndiinafknngiqandlqtsglsiyqsnpnkdhekknngiv yhvsnsltvrirdlsnagkifdqamalgvnsvhgitftnantkpfyqearkkaiaeaaiekaktiaeaadvklgkiikinenddnyysrphlmskavnanytdttfssgelysvsvtivfaid

[0045] SEQ ID NO: 28 - Amino acid sequence of Bartonella quintana 26 kDa recombinant protein MTKIISQPLKSYSIKMAMITLTLLAASLPTHAEESKIKNATITVTATGESQATPDMAIINLAIVTHDKTAQKALAANNKSMNDIVNAFKNNGIQANDLQTSGLSIYQSSPEKHREKKNTEIL YQVSNSLTVRIRDLSNAGKIFDQAMALGVNSVNGITFTNANTKPFYQEARKKAVTAAIEKAETIAQAANLKLGKIITINENDDNSYSRPHLMSRAAHASYADTTFSSGELNYNVSVTVVFAID

[0046] SEQ ID NO: 29 - Amino acid sequence of Bartonella vinsonii 26 kDa recombinant protein MKKTTFQPLNHYSVRIAMVALALITSSLPIHAEESKMKNATITVTATGESQAAPDMAIINLAVVTEDKTAQKALADNNKSMNDIVNTFKDNGIQANDLQTSGLSIYQSSPDKPHDKKNHEKL YHVSNSLTVRIRDLSNAGKIFDQAMALGVNSVNGITFTNADTKPLYQEARKKAITEAIEKAETIAQAANLKLGKIIEINENNDNYYPKPRLMSSAAHASYADTNFSGGELGYNVSVTVFAID

[0047] SEQ ID NO: 30 - Amino acid sequence of Bartonella elizabethoe 26 kDa recombinant protein IQPLKSSLVKIAILTFTLLTTSLAVVHAYAEESKRDATITVTAIGESQATPDMAIINLAVVTHDKTAQKALASNNQSINEIIKAFKNNGIQANDLQTSGLSIYQSSHEKKNNEKLYQVS NSLTVRIRDLSNAGKIFDQAMALGINSVNGITFTNADTKPFYQEARKKAITEAIEKAQTIAQAADLKLGKIIEINEKDDYYRPTPRLMSRAADASYADTNFAGGELNYNVSVTVVFAID

[0048] SEQ ID NO: 31 - Amino acid sequence of Bartonella baciliformis 26 kDa recombinant protein MTKAISQLFNIYWTKITILTILALLTSSLSVYAEKSTIKNSTITVTATGESQATPDMAIMNLAVITHNKTAQKALEANNKSINNIIDSFKKEGIQEKDIQTSNLFIHYINSDKHQEQRGDENL YQVSHSLTVRIRDLLNAGMILDQAMALGINSVRDITFTNTDTKPFYTAARKQAIAEAIEKAETLAHAASVKLGKIIEINESNDPPHLMPRLISRAQTASYADTHFSSGELNYSVTVTVTFAID

[0049] SEQ ID NO: 32 - Amino acid sequence of Bartonella claridiae 26 kDa recombinant protein MTKIIFQLLNTYRIKLSILTVLSLFATSLFINAEETIKDAPTITVTATGENQATPDMAIINLAVVTHDTTAQKALAANNKFMNDIVNTFKNNGIQANDLQTSGLSIYQTNSDKKREKKNNEII YHVSNSLTVNIRDLANAGKIFDQAMALGVNSVNGITFTNADTKPLYKEARKKAIAEAIEKAETLAQAANVKLGKIITISENNNGHYPTPRLMSRAQHARSYTDTNFSGGELNYNVSVTITFAID

[0050] SEQ ID NO: 33 - Amino acid sequence of Bartonella grahami 26 kDa recombinant protein VILAFTLLATSLTVRAYAEENKMKNATITVTAIGESQAAPDMAIINLAVVTYDKTAQKALASNNQSINDIIKAFKNDGIQANDLQTSGLSIYQSTSDKHHEKKNNEKLYHVSNSL TVRIRDLNNAGKIFDQAMTLGVNSVNGITFTNADTKPFYQEARKKAITEAIEKAQTIAQAADLKLGKIIEINERDDYYHPTPRLMSRAADASYENTNFAGGELTYNVSVTVVFAID

[0051] SEQ ID NO: 34 - Amino acid sequence of Bartonella coelerae 26 kDa recombinant protein QKARADNKSMDIVNAFKNNGIQENDLQTSGLSIQSNQGKHHEKNNNGGIVIHVSLTVRIRDSNAGKIFDQAMALGVNSVHGITFTNADTKPFYQEAKQAITAIEKAKTAEAAAANLKLGKIIKINDDDISRPHLMSKTVNASYTTAFSSGLNVSVTVFAIIN

[0052] SEQ ID NO: 35 - Amino acid sequence of Bartonella rosalimera 26 kDa recombinant protein MTKMIFQLKVYRCLSILTVLSLLTTSCFVHAETIKNAPTVTGATPDMAINLAVTTTTTAQKARANKFMNDTFKNNGIQANLTTGLSIYQTNSDKQQEKKNNNEITHVSNSLTVNIRNANAGKIFDQAMALGVNSVNGITTTNADTKPYKAKAAETLAQAANKLGKITNNNYYPPRPLMSRAQSASYADTNFSGGGERNHVSVTTFAID

[0053] SEQ ID NO: 36 - Amino acid sequence of Bartonella washoeensis 26 kDa recombinant protein QEALAANKSMNDIVNAFKSNGIQANDLQTSNLLYQSSPDNHEKKNKEILYRVSLTVRIRDRANAGKIFDQAMALGINSVNGITFTNADMKPFYQDARKKAIAIEKAETIAQKAAHLKLGKIIEENSSDGYHPSPRLMSRAANASYADTNFSGGELNYSVSVTVFTID

[0054] SEQ ID NO: 37 - Amino acid sequence of Bartonella henselae 17 kDa recombinant protein MKKISLVTLSLFCISAKTAPLTDEYYKKKALETKLDVAKSQTAQSIYSATQTAKIKDINQLKADTKPQLQALQESLQAQLQAKTKEREQTQKHEDLQKKLKKEKLQSDVRLL

[0055] SEQ ID NO: 38 - Amino acid sequence of Bartonella quintana 17 kDa recombinant protein MAAYISPKERKSMKYGLVTLLSLSCVSHTIAETALLADEYKRALENTQKLDAAKESTAESIYASANEITNKIKEINEKLRKAQAAEKTKPEFQALQIELSLLQAQLQADTLKIQFLSMIQQAKENTKTKEDIREQTQKKKKLKDDLQKLKEGLENSDVRL

[0056] SEQ ID NO: 39 - Amino acid sequence of Bartonella vinsonii 17 kDa recombinant protein MKKISLVTLSSFFISAVNSKDVDEYYKQARESTQKLNVKSETIFDHATQTAKKIESQQQQSQPDEKLQTLQVESVLQAKLQAEAKLQSLDMIQQAKDSRTKEIREIQQHEKIVQKLKKLKKLKSNVPSTLGKLND

[0057] SEQ ID NO: 40 - Amino acid sequence of Bartonella elizabethoe 17 kDa recombinant protein MKKYGLMTLLSFSFISHAFSQMSPDADEYYKQALENTQKLDTAKSETAETIYKTATETAKKIKEINGQLEQLKSQTETTATETTSDADKNSNLEELQKRQALRQELQVKVSLLQADLQANSLKLQALNMIQARDTKTKEELREEKEQQKHKTLQAQLKEKEEKLKEKLESTGADVRL

[0058] SEQ ID NO: 41 - Amino acid sequence of Bartonella coelerae 17 kDa recombinant protein MKKYSLVTLLSLFCISHAKAQIAPLPDEYYKKALENTQKLDVAKSQTAESIYESATQTANKIKDINDQLKTLKADTKTKPEQLQALQIELTLLQAQLQADTLKIQSLSMIQAKDTKTKEELREEQTQNHKKIEEKLKEKLEKSDVRL DETAILED DESCRIPTION OF THE INVENTION

[0059] This disclosure provides diagnostic panels and methods for diagnosing and treating bartonellosis caused by infection with multiple Bartonella species. To assess the impact of testing limitations and identify exposure to Bartonella, a modified Western blot procedure was developed and employed. This procedure, sometimes referred to herein as an "immunoblot," uses recombinant antigens from multiple Bartonella species for the serological diagnosis of Bartonella infection. Testing was performed on humans with suspected bartonellosis, as discussed in more detail elsewhere herein. Positive immunoblots were further characterized at the species level for Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethae.

[0060] The present disclosure provides diagnostic panels and methods for rapid, simple, and accurate detection of Bartonella antibodies in biological samples from subjects suspected of having Bartonellosis, thereby fulfilling the need for such testing. Because multiple Bartonella species are pathogenic for Bartonella disease, it can be beneficial for a test to be comprehensive. That is, the test can simultaneously detect antibodies to multiple Bartonella species. The present disclosure provides antigenic amino acid sequences specific to various Bartonella species. The disclosed amino acid sequences have high specificity and / or sensitivity for the indicated species. The inclusion of antigenic peptides that exhibit cross-reactivity across Bartonella species boundaries is also important for the development of comprehensive serological or other immunological-based assays, where the goal is to detect infection without necessarily identifying the specific species causing the infection. For example, the present disclosure includes immunoassays capable of detecting multiple Bartonella species in the context of a single test screening.

[0061] The present disclosure also provides diagnostic panels and methods for diagnosing infection with one or more species of the genus Bartonella. The present disclosure is based, in part, on the discovery of species-specific amino acid sequences encoding antigenic peptides, which may also be referred to in the art as peptide antigens or antigens, as described herein. Embodiments of the present disclosure provide antigen-specific amino acid sequences for Bartonella species, including Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethae. These amino acid sequences can be used in assays to identify infection with one or more species of the Bartonella genus, including, but not limited to, Bartonella henselae, Bartonella quintana, Bartonella vinsonii, Bartonella elizabethae, Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella grahamii, Bartonella hoehlerae, Bartonella rochalimae, and Bartonella washoensis, in a sample from a subject suspected of having bartonellosis. The amino acid sequences of the present disclosure allow for the identification of Bartonella infection in a subject sample with speed, sensitivity, and specificity at least equal to or greater than other current methods.

[0062] The amino acid sequences of the present disclosure can be used in diagnostic and scientific assays. Non-limiting examples of suitable assays include immunoblots, line immunoblots, enzyme-linked immunosorbent assays (ELISAs), and the like. The amino acid sequences of the present disclosure can be used, for example, to detect Bartonella-specific T cells using the IgXSPOT test (IGeneX, Milpitas, CA).

[0063] (Bartonella spp.) The genus Bartonella consists of fastidious, Gram-negative, slow-growing, facultative intracellular bacteria belonging to the α-2 subgroup of the Proteobacteria class and the Rhizobia order. These organisms are most commonly transmitted to humans through animal bites or scratches (e.g., cats, dogs, and other animals) or through the ingestion of infected flea or louse feces into the skin. Transmission can also occur through the bites of other vectors, such as ants, biting flies, pubic lice, mites, spiders, and ticks. Prior to the HIV epidemic in the early 1990s, Bartonella was not known to infect animals or humans in North America. Currently, at least 40 Bartonella species or subspecies have been characterized, of which 17 are involved in human zoonotic diseases. Table 1 lists known Bartonella species, their hosts, and vectors.

[0064] [Table 1]

[0065] (Production of amino acid sequences) The amino acid sequences of the present disclosure can be naturally occurring and isolated from natural sources. Furthermore, the amino acid sequences of the present disclosure can be non-naturally occurring synthetic sequences, such as sequences produced by recombinant technology or sequences synthesized by a protein synthesis apparatus. Thus, the amino acid sequences of the present disclosure can be isolated or can be prepared as described, for example, in Short Protocols in Molecular Biology, Second Edition, F.M. Ausubel, Ed., all John Wiley & Sons, NY, edition as of 2008, and Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001, and as known to those skilled in the art.

[0066] In some embodiments, the amino acid sequences of the present disclosure can be recombinantly produced in a vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another operably linked nucleic acid between different genetic environments. In addition to containing a nucleic acid sequence encoding an amino acid sequence of the present disclosure (e.g., SEQ ID NOS: 1-41) or a variant thereof that retains the immunological binding profile of the corresponding non-variant, the vector of the present disclosure can also contain a heterologous nucleic acid sequence. As used herein, a "heterologous nucleic acid sequence" is one that does not naturally occur in the organism from which the amino acid sequence of the present disclosure is derived.

[0067] The term "vector" can also refer to a virus or organism capable of transporting nucleic acid molecules. One type of vector is a plasmid, which is a small, circular, double-stranded, extrachromosomal deoxyribonucleic acid (DNA) molecule that is physically separate from and capable of autonomous replication independently of chromosomal DNA. Some useful vectors are capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0068] Vectors capable of directing the expression of an operably linked nucleic acid are referred to herein as "expression vectors." Other useful vectors include, but are not limited to, bacterial plasmids, bacterial artificial chromosomes (BACs), cosmids, and viruses, such as lentiviruses, retroviruses, adenoviruses, and phages.

[0069] The vector useful in the method of the present disclosure can contain additional sequences, including but not limited to one or more signal sequences, one or more promoter sequences, or a combination thereof.The promoter that can be used in the method and vector of the present disclosure includes, but is not limited to, cell-specific promoters and general promoters.Non-limiting examples of promoters that can be used in the vector of the present disclosure include ubiquitous promoters, including but not limited to CMV, CAG, CBA, and EF1a promoters.The method of selecting and using suitable promoters is within the knowledge of those skilled in the art.

[0070] Vectors useful in the methods of the present disclosure can be used to intracellularly express fusion proteins comprising the sequences of the present disclosure (e.g., SEQ ID NOS: 1-41) and / or variants thereof that retain the immunological binding profile of the corresponding non-variant. Expression vectors and methods for their preparation and use are within the knowledge of those of ordinary skill in the art. In some embodiments of the present disclosure, the nucleic acid sequence of the expression vector encodes a fusion protein comprising an amino acid sequence of the present disclosure or a variant thereof that retains the immunological binding profile of the corresponding non-variant. Methods for preparing and utilizing fusion proteins comprising the polypeptide sequences are within the knowledge of those of ordinary skill in the art.

[0071] In some embodiments, fusion proteins comprised of the amino acid sequences of the present disclosure, or variants thereof that retain the immunological binding profile of the corresponding non-variant, may also include a tag, as described elsewhere. In some embodiments, fusion proteins comprised of the amino acid sequences of the present disclosure, or variants thereof that retain the immunological binding profile of the corresponding non-variant, may include a label, as described elsewhere herein.

[0072] In some embodiments, the amino acid sequences of the present disclosure (eg, SEQ ID NOs: 1-41), or variants thereof that retain the immunological binding profile of the corresponding non-variant, can be produced recombinantly in E. coli.

[0073] (Amino acid sequence homology and variants) As used herein, a non-variant amino acid sequence is an amino acid sequence having 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41.

[0074] As used herein, an amino acid sequence variant that retains the immunological binding profile of the corresponding non-mutated amino acid sequence is an amino acid sequence having at least 90%, 95%, 98%, 99%, 99.9% sequence identity to SEQ ID NO:40, SEQ ID NO:40, or SEQ ID NO:41, and is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41.

[0075] Variants of the amino acid sequences SEQ ID NOS: 1-41 that retain the immunological binding profile of the corresponding non-variant may have conservative amino acid substitutions in conserved or non-conserved regions. Variants can have any modification of the designated amino acid sequence (e.g., SEQ ID NOS: 1-41) that retains the immunological binding profile of the corresponding non-variant. Such modifications can include insertions and deletions (e.g., internally or from the N- or C-terminus, or both). Those skilled in the art can, with no more than routine experimentation, design and generate antigenic amino acid sequences with conservative amino acid substitutions in non-conserved regions or non-conserved amino acid positions as identified by alignment comparison.

[0076] As used herein, the term "immunological binding profile" refers to the ability of a label, tag, or attached amino acid sequence to bind to antibodies present in a biological sample. Non-limiting examples of immunological binding profiles are shown in Figures 1-2.

[0077] Sequences with less than 100% homology may be modified with one or more substitutions, deletions, insertions, or other modifications relative to the amino acid sequences provided herein. Exemplary modifications include, but are not limited to, conservative amino acid substitutions, which produce molecules with similar functional properties to the molecule in which they are made. Conservative amino acid substitutions are those that do not significantly alter the activity or tertiary structure of a selected polypeptide or protein. Such substitutions typically involve replacing a selected amino acid residue with a different residue that has similar physicochemical properties. For example, substituting Glu for Asp is considered a conservative substitution because both amino acids have similar negative charges. Groupings of amino acids by physicochemical properties are known to those skilled in the art. Each of the following groups contains amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). One of skill in the art can determine whether sequences with less than 100% homology can bind to naturally occurring or non-naturally occurring Bartonella-associated antibodies, as well as the sensitivity and specificity of the antibodies to modified sequences. Given the teachings of this disclosure, one of skill in the art will be able to identify sequences with significant homology to SEQ ID NOs: 1-41 of the present disclosure that provide acceptable or equivalent responses in the methods of the present disclosure without undue experimentation.

[0078] Nucleic acid sequences, including polynucleotides and oligonucleotides, encoding the amino acid sequences of the present disclosure, and portions thereof, can be expressed in cultured cells to provide isolable amounts of peptides that exhibit the biological (e.g., immunological) properties of the antigenic amino acid sequences of the present disclosure. Due to the redundancy of the genetic code, multiple nucleic acid sequences may be suitable for producing the amino acid sequences of the present disclosure. Those skilled in the art will be able to determine one or more nucleic acid sequences for producing the amino acid sequences of the present disclosure. The nucleic acid sequences encoding the amino acid sequences of the present disclosure can be labeled with any suitable label known to those skilled in the art.

[0079] In this regard, nucleic acid sequences suitable for producing the amino acid sequences of the present disclosure may be substantially homologous to naturally occurring sequences. As used herein, substantial homology of a nucleic acid sequence means: (a) greater than 65%, 75%, 85%, 95%, 98%, or 99% homology with a naturally occurring sequence; or (b) a homologous nucleic acid sequence hybridizes to the sequence being compared or its complementary strand under stringent conditions of temperature and salt concentration. These stringent conditions generally include a temperature of about 22°C or higher, usually about 30°C or higher, more usually about 45°C or higher, and a salt concentration of generally less than about 1M, usually less than about 500mM, more usually less than about 200mM. The combination of temperature and salt concentration is more important in defining stringency than either temperature or salt concentration alone. Other conditions that affect stringency include the GC content of the sequences being compared, the degree of sequence complementarity, the length of the sequences involved in hybridization, and the composition of the buffer solution used in the hybridization mixture.These and other factors that affect stringency are well described in scientific and patent literature.Those skilled in the art will be able to determine the appropriate conditions for determining the homology of the nucleic acid sequence encoding the antigenic amino acid sequence of the present disclosure.

[0080] Homologous nucleic acid sequence can be determined based on the nature of nucleotide substitution in nucleic acid sequence.For example, synonymous nucleotide substitution, that is, the nucleotide change in nucleic acid sequence that does not change the encoded amino acid sequence, is more tolerant than non-synonymous nucleotide substitution, and therefore can be more abundant in certain nucleic acid sequence.Those skilled in the art can determine the appropriate number and position of substitution that can be tolerated in the nucleic acid sequence that codes the amino acid sequence of the present disclosure without adversely affecting the antigenicity of the encoded antigenic amino acid sequence without undue experimentation.

[0081] (binding amino acid sequence) In some embodiments, the amino acid sequences of the present disclosure (e.g., SEQ ID NOs: 1-41), and / or variants thereof that retain the immunological binding profile of the corresponding non-variant, can bind to a substrate(s).

[0082] As used herein, a "bound" amino acid sequence is an amino acid sequence that has been immobilized on a substrate to enable the use of the amino acid sequence in a biological test, such as, for example, an immunoassay. The bound amino acid sequence may be covalently or non-covalently bound to the substrate. The bound amino acid sequence may be directly or indirectly bound to the substrate. Furthermore, the "bound" amino acid sequences of the present disclosure may be bound, directly or indirectly, to a natural surface or material to which the amino acid sequence is not naturally associated.

[0083] As used herein, "substrate" refers to a non-native surface or material, or a native surface or material not naturally associated with an amino acid sequence of the present disclosure (e.g., SEQ ID NOS: 1-41), or a variant thereof that retains the immunological binding profile of the corresponding non-variant. Non-limiting examples of substrates include, but are not limited to, nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose (e.g., beads), plastic, and metal.

[0084] In a particular diagnostic panel of the present disclosure, the number of substrates used may depend on the number of amino acid sequences in the diagnostic panel. For example, if the diagnostic panel is a line immunoblot, multiple nitrocellulose sheets may be used as part of the diagnostic panel. For example, a first portion of the amino acid sequence may be applied to a first nitrocellulose sheet, a second portion of the amino acid sequence may be applied to a second nitrocellulose sheet, etc. Nevertheless, multiple nitrocellulose sheets may be analyzed together to determine the results of the line immunoblot.

[0085] Linking agents known to those of skill in the art can be used to assist and / or enhance the binding of the amino acid sequences of the present disclosure (e.g., SEQ ID NOS: 1-41), and / or variants thereof that retain the immunological binding profile of the corresponding non-variant, to a substrate(s).

[0086] (tagged amino acid sequence) In some embodiments, the amino acid sequences of the present disclosure (e.g., SEQ ID NOS: 1-41), and variants thereof that retain the immunological binding profile of the corresponding non-variant, can be tagged. As used herein, a "tagged" amino acid sequence is an amino acid sequence that includes a tag. As used herein, a "tag" refers to a peptide sequence that is a physical part of an amino acid sequence of the present disclosure or that is chemically attached (e.g., covalently, or via hydrogen or ionic bonds) to an amino acid sequence of the present disclosure, where the peptide sequence can be detected by microscopy or other detection means to enable visualization and / or localization of the corresponding amino acid sequence.

[0087] Numerous tags are well known in the art, and routine methods can be used to include such tags in the amino acid sequences and methods of the present disclosure. A non-limiting list of tags includes, but is not limited to, affinity tags, chromatography tags, epitope tags, enzyme-conjugated tags, and fluorescent tags.

[0088] Affinity tags allow the conjugated moiety to be purified from biological samples using affinity techniques. Examples of affinity tags include, but are not limited to, chitin-binding protein (CBP), maltose-binding protein (MBP), strepto-tag, poly-histidine tag (His), glutathione-S-transferase (GST), etc.

[0089] Chromatographic tags alter one or more chromatographic properties of the tagged amino acid sequence, thereby enabling separation of the tagged amino acid sequence over a particular separation technique. Examples of chromatographic tags include, but are not limited to, polyanionic amino acids such as FLAG tags and polyglutamic acid tags.

[0090] Epitope tags are peptide sequences that have high affinity for one or more antibodies that can be used for antibody purification. Examples of epitope tags include, but are not limited to, ALFA tags, V5 tags, Myc tags, human influenza hemagglutinin (HA) tags, Spot tags, T7 tags, FLAG tags, and NE tags.

[0091] Enzyme-conjugated tags are enzymes that can be used to conjugate antibodies, streptavidin, and other proteins via various mechanisms. Enzyme conjugates typically require the addition of a substrate to produce a suitable colorimetric, chemiluminescent, or fluorescent signal. Examples of enzyme-conjugated tags include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase.

[0092] Fluorescent tags allow the tagged amino acid to be visually read. An example of a fluorescent tag is green fluorescent protein (GFP).

[0093] Because the tagged amino acid sequences of the present disclosure must retain immunological specificity for the antibody, the particular tag must be carefully selected to avoid, for example, steric hindrance to such immunological specificity. One of skill in the art can select a tag with no more than routine experimentation.

[0094] (labeled amino acid sequence) In some embodiments, the amino acid sequences of the present disclosure (e.g., SEQ ID NOS: 1-41), and variants thereof that retain the immunological binding profile of the corresponding non-variant, can be labeled. As used herein, a "labeled" amino acid sequence is an amino acid sequence that includes a label. As used herein, "label" refers to a detectable moiety that is a physical part of the amino acid sequence of the present disclosure or that is chemically attached (e.g., covalently or via hydrogen or ionic bonds) to the amino acid sequence of the present disclosure, where the detectable moiety can be detected by microscopy or other detection means to allow visualization and / or localization of the corresponding amino acid sequence.

[0095] Numerous labels are well known in the art and can be incorporated into the disclosed amino acid sequences and methods using routine methods. A non-limiting list of labels includes, but is not limited to, stable isotopes, mass tags, and fluorescent dyes.

[0096] Stable isotopes allow for the measurement and identification of the corresponding labeled amino acid sequences using mass spectrometry. Examples of stable isotopes include, but are not limited to, (2)H, (3)H, (13)C, (14)C, (15)N, (32)P, (35)S, and 125I.

[0097] Mass tags allow for the measurement and identification of corresponding labeled amino acid sequences using mass spectrometry. Mass tags include tandem mass tags, which are chemical labels that facilitate sample multiplexing for mass spectrometry-based quantification and identification of biopolymers. Tandem mass tags belong to a class of reagents called isobaric mass tags, which are groups of molecules with the same mass but generate reporter ions of different masses after fragmentation. The relative ratio of the measured reporter ions represents the relative abundance of the tagged molecules. Examples of tandem mass tags include, but are not limited to, TMTzero, TMTduplex, TMTsixplex, TMT 10-plex, TMTpro, and TMTpro Zero.

[0098] Fluorescent dyes are compounds that re-emit light upon excitation and are detected by a fluorescent imager. Examples of fluorescent dyes that can be used in accordance with the present disclosure include organic dyes such as fluorescein, rhodamine, and aminomethylcoumarin acetate. Such small molecules, when used as labels, are less likely to present steric hindrance with respect to antibody binding.

[0099] (Diagnostic Panel) In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, including SEQ ID NOS: 1-41, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infections with Bartonella at the genus level, and optionally, with the specific species Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethae.

[0100] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or linked amino acid sequences, including SEQ ID NOS: 1-12, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella at the genus level.

[0101] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences consisting of SEQ ID NOS: 13-20 and 27-30, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infections with the specific species Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethoe.

[0102] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences comprise SEQ ID NOs: 13, 17, and 27, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella henselae.

[0103] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences comprise SEQ ID NOs: 14, 18, and 28, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella quintana.

[0104] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, wherein the amino acid sequences comprise SEQ ID NOs: 15, 19, and 29, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella vinsonii.

[0105] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences comprise SEQ ID NOs: 16, 20, and 30, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella elizabethoe.

[0106] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences include SEQ ID NOS: 1-41 and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infections with Bartonella at the genus level, and optionally, also for the specific species Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethae.

[0107] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences comprise SEQ ID NOS: 1-12, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infection with Bartonella at the genus level.

[0108] In some embodiments, diagnostic panels of the present disclosure comprise individually identifiable labeled, tagged, or conjugated amino acid sequences, where the amino acid sequences include SEQ ID NOS: 13-20 and 27-30, and / or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. Such diagnostic panels can be used to detect infections by specific species of Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and Bartonella elizabethae.

[0109] As used herein, when referring to the amino acid sequence of a diagnostic panel, the terms "consist of," "consists of," and "consisting of" refer to a diagnostic panel having an amino acid sequence limited to the specified amino acid sequence. That is, a diagnostic panel may have components other than the specific amino acid sequence, but the amino acid sequence included in the diagnostic panel is limited to the specified amino acid sequence. In other words, although the amino acid sequence is limited to a specific one, this does not prevent the diagnostic panel from having other non-amino acid sequence components.

[0110] Assays and Detection Methods The amino acid sequences of the present disclosure exhibit antigenic activity with various antibodies derived from Bartonella, as demonstrated by the examples provided herein below. In other words, antibodies derived from Bartonella exhibit specificity for the amino acid sequences of the present disclosure. Specificity for the amino acid sequence (i.e., antibody specificity) is a property of an antibody that allows it to react preferentially with certain antigenic determinants and not with other antigenic determinants. Specificity depends on the chemical composition, physical strength, and molecular structure of the binding site. Sensitivity refers to how strongly an antibody binds to an antigenic determinant. Those skilled in the art can easily determine the specificity and sensitivity of an antibody for a particular amino acid sequence using standard affinity assays such as immunoblotting, Ouchterlony assays, and titer assays.

[0111] Bartonella antibodies can be detected, for example, by immunoblot, ELISPOT, ELISA, Western blotting, lateral flow assay, or other suitable immunoassay methods known to those skilled in the art, all of which may be individually referred to herein as diagnostic panels. These techniques are known to those skilled in the art, and their procedures are described in the general technical literature. Although similar, these techniques each have their own advantages and disadvantages. Other suitable techniques are known to those skilled in the art and are incorporated herein.

[0112] To assess the impact of testing limitations and determine levels of exposure to Bartonella spp., a modified Western blot procedure, line immunoblot, was developed and employed, as described in the Examples provided herein below. According to the present disclosure, line immunoblot uses recombinant antigens (i.e., amino acid sequences of the present disclosure) from multiple Bartonella species for the serological identification and diagnosis of Bartonella infection in biological samples (e.g., serum) from subjects suspected of having a Bartonella infection. Infection with more than one species of Bartonella spp. is possible and may occasionally be observed.

[0113] In Western blotting, proteins are separated by electrophoresis and transferred to nitrocellulose or other solid media (such as polyvinylidene fluoride, PVDF, or nylon membranes).

[0114] In immunoblotting, proteins can be applied manually or mechanically to a solid medium. Preferably, the proteins are applied in lines or spots, dried, and bound to a solid support medium (e.g., nitrocellulose). The bound proteins are then exposed to a biological sample or samples suspected of having antibodies specific to the target protein. This procedure allows the use of known antibodies to determine the presence of the protein in a sample, such as when proteins from lysed cells are separated by electrophoresis and transferred to a solid medium. Western blotting allows proteins to be identified not only by their specificity for a particular antibody but also by their size.

[0115] In immunoblotting, a known protein is bound to a solid medium and a biological sample, such as a sample from a subject suspected of Bartonella infection, is contacted with the bound protein to test for the presence of a specific antibody in the sample. The antibody that binds to the target protein is typically referred to as the primary antibody. A secondary antibody (e.g., rabbit anti-human IgG antibody) specific for a conserved region of the primary antibody can be used to detect the bound primary antibody. The secondary antibody is typically labeled with a detectable moiety for visualization. Non-limiting examples of suitable labels include, for example, chromophores such as biotin, radioactive sites, and enzymes such as alkaline phosphatase. The use of these and other materials for antibody visualization is well known to those skilled in the art.

[0116] The ELISPOT method can detect human T cells responding to Bartonella-specific antigens in vitro. In an ELISPOT assay, the surface of a PVDF membrane in a 96-well microtiter plate is coated with a capture antibody that binds, for example, anti-interferon-γ (IFNγ) or other cytokine-specific antibodies. Between cell incubation and stimulation steps, T cells isolated from a subject's whole blood are seeded into the wells of the plate with the amino acid sequences of the present disclosure, forming a substantial monolayer on the membrane surface of the wells. Upon stimulation of the antigen-specific cells with one or more amino acid sequences of the present disclosure, the antigen-specific cells are activated and release IFNγ, which is directly captured on the membrane surface by the immobilized antibody. IFNγ is thus "trapped" in the area immediately surrounding the secreting cells, before it has a chance to diffuse into the culture medium, be degraded by proteases, or be bound by receptors on bystander cells. In a subsequent detection step, the immobilized IFNγ is visualized as an immunospot.

[0117] In a specific example of an ELISPOT test, each well of a plate is coated with a purified cytokine-specific antibody specific to the test or cell to be detected. T cells from a subject (i.e., a subject suspected of Bartonella infection) are isolated, cultured in each well, and stimulated with recombinant antigens of one or more sequences of the present disclosure. Bartonella-positive cells secrete cytokines in response to the stimulation, which are captured by the antibody coated on the well and then detected by ELISA.

[0118] ELISA assays are also used to detect antigens. ELISA assays allow for the quantification of specific proteins in a protein mixture (e.g., a lysate) or can determine whether a peptide is present in a sample. Similarly, ELISA assays can be used to determine the presence of a specific antibody by using a specific antigen as a target. As used herein, the target amino acid sequence is attached to a surface. If present in the biological sample being tested, a reactive antibody can bind to the antigen. A secondary antibody linked to an enzyme is added, and in a final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

[0119] Lateral flow assays, also referred to by various other names including, but not limited to, lateral flow tests, lateral flow devices, lateral flow immunoassays, lateral flow immunochromatographic assays, and rapid tests, are further examples of diagnostic panels of the present disclosure. Lateral flow assays are simple, versatile, paper-based platforms for detecting and / or quantifying the presence of one or more analytes, such as antigens, in a mixture, such as a liquid sample. Lateral flow assays can be qualitative or quantitative. In a lateral flow assay, a sample containing one or more analytes is applied to an absorbent sample pad and is drawn by capillary action through various zones of a polymeric test strip, each containing molecules that can interact with the analytes. The pad contains molecules that specifically bind to the analytes and are bound to fluorescent, colored, or otherwise detectable particles. Finally, the sample containing the bound analytes migrates to a detection zone, where biological components, such as antibodies or antigens, are immobilized within a porous membrane and react with the detectable particles. Lateral flow assays typically have a control line to verify the flow of sample through the strip and one or more test lines to detect the presence of the analyte of interest. Results can be read visually or by a machine that can read and interpret the results. Lateral flow assays can be designed as direct or "sandwich" assays, where the presence of a colored line at the test line indicates a positive result, or as competitive assays, where the absence of a colored line indicates a positive result. Direct and competitive assays can be multiplexed.

[0120] (Methods for detecting infection with one or more Bartonella species) The present disclosure provides methods for rapidly and accurately detecting infection with one or more Bartonella species. In other words, the present disclosure provides methods for detecting Bartonella antibodies having specificity for the amino acid sequences of the present disclosure in a biological sample from a subject suspected of infection with one or more Bartonella species.

[0121] The methods of the disclosure can include providing or obtaining a biological sample (eg, blood or saliva) obtained from a subject suspected of being infected with one or more Bartonella species.

[0122] The disclosed methods may further include contacting a biological sample with a diagnostic panel of the present disclosure. For example, the diagnostic panel may include amino acid sequences set forth in SEQ ID NOS: 1-41, or one or more variants thereof that retain the immunological binding profile of the corresponding non-mutant, or may consist of SEQ ID NOS: 1-41, or one or more variants thereof that retain the immunological binding profile of the corresponding non-mutant. As a further example, the diagnostic panel may include amino acid sequences set forth in SEQ ID NOS: 1-12, or one or more variants thereof that retain the immunological binding profile of the corresponding non-mutant, or may consist of SEQ ID NOS: 1-12, or one or more variants thereof that retain the immunological binding profile of the corresponding non-mutant.

[0123] The disclosed methods may further include determining that the biological sample is positive for Bartonella infection after contacting the biological sample with the diagnostic panel. Determining that the biological sample is positive for Bartonella infection may include determining a positive immunobinding reaction indicative of the presence of one or more Bartonella antibodies in the biological sample.

[0124] In the methods of the present disclosure, any primary antibody bound to the amino acid sequence of the present disclosure can be detected with an anti-human antibody, such as an IgM class antibody, an IgG class antibody, or an IgA class antibody, used as a secondary antibody, which can be linked, bound, or otherwise associated with a detectable moiety (e.g., a chromophore, a radioactive moiety, an enzyme, or other detectable moiety known to those skilled in the art). In some embodiments, the detectable moiety comprises alkaline phosphatase. In some embodiments, the detectable moiety comprises biotin.

[0125] In some embodiments, when the diagnostic panel comprises or consists of an amino acid sequence comprising SEQ ID NOs: 1 to 41, if a positive immune binding reaction by a secondary antibody of the IgM class, IgG class, or IgA class is detected for at least one of SEQ ID NOs: 1 to 12 and at least one of SEQ ID NOs: 13 to 41, the biological sample can be determined to be positive for Bartonella infection.

[0126] In some embodiments, when the diagnostic panel comprises or consists of an amino acid sequence comprising SEQ ID NOs: 1-41, a biological sample may be determined to be positive for Bartonella infection if a positive immune binding reaction with a secondary antibody of the IgM class, IgG class, or IgA class (i.e., bound, conjugated, or otherwise attached to a detectable moiety) is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NO: 9 and SEQ ID NO: 12.

[0127] In some embodiments, when the diagnostic panel comprises or consists of an amino acid sequence comprising SEQ ID NOs: 1-12, a biological sample may be determined to be positive for Bartonella infection if a positive immune binding reaction with a secondary antibody of the IgM class, IgG class, or IgA class (i.e., bound, conjugated, or otherwise attached to a detectable moiety) is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9 and 12.

[0128] Non-limiting examples of combinations of SEQ ID NOs: 1-12 that can be included in a diagnostic panel of the present invention are shown in Table 2. In some embodiments, a diagnostic panel of the present invention comprises a combination of sequences shown in Table 2.

[0129] [Table 2]

[0130] In embodiments in which a diagnostic panel includes an amino acid sequence comprising or consisting of SEQ ID NOS: 1-41 and a sample is determined to be positive for Bartonella infection, one or more specific species causing Bartonella infection can be identified. For example, if a positive immune binding reaction is detected with at least one of SEQ ID NOS: 13, 17, and 27, with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety), the biological sample can be determined to be positive for Bartonella henselae infection. As a further example, if a positive immune binding reaction is detected with at least one of SEQ ID NOS: 14, 18, and 28, with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety), the biological sample can be determined to be positive for Bartonella quintana infection. As another example, a biological sample may be determined to be positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 15, 19, and 29. Further, for example, a biological sample may be determined to be positive for Bartonella elizabethae infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 16, 20, and 30.

[0131] (Methods for detecting infection with one or more specific Bartonella species) The present disclosure also provides methods for rapidly and accurately detecting infection with one or more specific Bartonella species. Such methods of the present disclosure may include providing or obtaining a biological sample (e.g., blood or saliva) obtained from a subject known to have a Bartonella infection.

[0132] The disclosed methods may further include contacting a biological sample with a diagnostic panel of the present disclosure. For example, the diagnostic panel may include amino acid sequences including or consisting of SEQ ID NOS: 13-20 and 27-30, or one or more variants thereof that retain the corresponding non-mutant immunological binding profile. As a further example, the diagnostic panel may include amino acid sequences including or consisting of SEQ ID NOS: 13, 17, and 27, or one or more variants thereof that retain the corresponding non-mutant immunological binding profile. As another example, the diagnostic panel may include amino acid sequences including or consisting of SEQ ID NOS: 14, 18, and 28, or one or more variants thereof that retain the corresponding non-mutant immunological binding profile. As a further example, the diagnostic panel may include amino acid sequences including or consisting of SEQ ID NOS: 15, 19, and 29, or one or more variants thereof that retain the corresponding non-mutant immunological binding profile. As another example, a diagnostic panel may include amino acid sequences comprising or consisting of SEQ ID NOs: 16, 20, and 30, or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant. As a further example, a diagnostic panel may include amino acid sequences comprising or consisting of SEQ ID NOs: 13, 17, and 27, or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant.

[0133] The disclosed methods may further include, after contacting the biological sample with the diagnostic panel, determining that the biological sample is positive for at least one of a Bartonella henselae infection, a Bartonella quintana infection, a Bartonella vinsonii infection, and a Bartonella elisabethoe infection. Determining that the biological sample is positive for at least one of a Bartonella henselae infection, a Bartonella quintana infection, a Bartonella vinsonii infection, and a Bartonella elisabethoe infection may include determining a positive immune binding reaction indicative of the presence of at least one of a Bartonella henselae antibody, a Bartonella quintana antibody, a Bartonella vinsonii antibody, and a Bartonella elisabethoe antibody in the biological sample.

[0134] For example, a biological sample may be determined to be positive for Bartonella henselae infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., bound, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 13, 17, and 27. As a further example, a biological sample may be determined to be positive for Bartonella quintana infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., bound, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 14, 18, and 28. As another example, a biological sample may be determined to be positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 15, 19, and 29. As a further example, a biological sample may be determined to be positive for Bartonella elizabethoeae infection if a positive immune binding reaction with an IgM-class, IgG-class, or IgA-class secondary antibody (i.e., linked, conjugated, or otherwise associated with a detectable moiety) is detected for at least one of SEQ ID NOs: 16, 20, and 30.

[0135] (biological samples) As used herein, the term "biological sample" includes, but is not limited to, whole blood, serum, plasma, synovial fluid, cerebrospinal fluid (CSF), urine, saliva, and mucus.

[0136] (subject) As used herein, a subject can be an animal, such as a mammal or a non-mammal. Non-limiting examples of mammalian subjects include primates (including but not limited to humans), rodents (including but not limited to mice, rats, squirrels, chipmunks, and prairie dogs), rabbits, deer, canines (including but not limited to dogs, foxes, coyotes, and wolves), felines (including but not limited to house cats, ocelots, cougars, and other wild cats), bears, horses, cows, sheep, goats, and pigs. Non-limiting examples of non-mammalian subjects include birds, amphibians, lizards, insects, arthropods, etc.

[0137] (Treatment of Bartonella infections) In some embodiments, the present disclosure includes treating a Bartonella infection in a subject. As used herein, the terms "therapy," "treatment," and "treating" refer to reversing, alleviating, or inhibiting the progression of a Bartonella infection.

[0138] Treatment of a Bartonella infection can include administration of one or more compounds, or one or more pharmaceutical compositions thereof. As used herein, the terms "administer," "administering," and "administration" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing one or more compounds, or one or more pharmaceutical compositions thereof, for treating a Bartonella infection.

[0139] The dosage of a compound or pharmaceutical composition thereof for treating a Bartonella infection is within the knowledge of, or can be determined by, one of ordinary skill in the art using routine experimentation. The compound or pharmaceutical composition thereof for treating a Bartonella infection can be administered to a subject in a pharmaceutically effective dose.

[0140] A variety of administration routes are available. The particular delivery mode selected will depend on the particular condition being treated and the dosage required for therapeutic efficacy. The methods of the present disclosure can generally be practiced using any medically acceptable mode of administration, i.e., any mode that provides an effective level of therapy without causing clinically unacceptable side effects. In some embodiments, a compound for treating a Bartonella infection, or a pharmaceutical composition thereof, can be administered via oral, enteral, mucosal, transdermal, and / or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, and intrathoracic injections, as well as infusion techniques. Other routes include, but are not limited to, intranasal (e.g., via a nasogastric tube), transdermal, vaginal, rectal, and sublingual. Delivery routes of the present disclosure can include intrathecal, intraventricular, or intracranial. In some embodiments, a compound for treating a Bartonella infection, or a pharmaceutical composition thereof, can be placed in a sustained-release matrix and administered by placing the matrix in a subject.

[0141] The compounds for treating Bartonella infection, or pharmaceutical compositions thereof, may be administered in a formulation, which may be administered in a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0142] According to the method of the present disclosure, Bartonella infection can be treated using a pharmaceutical composition. Generally, a pharmaceutical composition is a composition containing a pharmaceutically active ingredient and one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are well known to those skilled in the art and can be selected and utilized using routine methods. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic substance that does not inhibit the effectiveness of the biological activity of the pharmaceutically active ingredient. Pharmaceutically acceptable carriers can include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

[0143] In some embodiments, the methods of the disclosure may include treating a Bartonella infection in a subject with one or more commercially available agents.

[0144] In situations where one or more species causing a Bartonella infection are specifically identified (e.g., B. henselae, B. quintana, B. vinsonii, and B. elisabethoe), treatment(s) specific for one or more identified species can be administered.

[0145] (Example) Example 1. Detection of Bartonella infection in subject samples

[0146] (method) Bartonella recombinant proteins obtained from a total of 41 species of Bartonella, namely, B. hensel, B. quintana, B. elisabethae, B. vinsonii, B. bacillus formis, B. clarigiae, B. grahamii, B. corellae, B. locallimae, and B. washoensis (listed in Table 3), were used to prepare antigen strips for Bartonella immunoblots as described by Liu S et al.: “Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease,” Healthcare (Basel) 2018 Aug 14;6(3):99. doi: 10.3390 / healthcare6030099 is incorporated herein by reference in its entirety. Protein sequences were obtained from published data. Recombinant antigens were prepared by cloning hybrid gene constructs or portions of selected genes into pET vectors and then expressing the proteins in E. coli (GenScript, Piscataway, NJ, USA). Recombinant Bartonella proteins produced in E. coli were purified using metal affinity chromatography and gel filtration. All recombinant proteins showed a purity of 90% or higher by Coomassie blue staining after SDS-PAGE.

[0147] [Table 3]

[0148] The 61 remaining sequenced human sera (including 31 samples previously tested with the Bartonella Western blot IgM and IgG panel and 30 serum samples with antibody positive samples to other tick-borne pathogens and controls) were tested by Bartonella IgM and IgG immunoblot using the protocol described by Liu S. et al., “Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease,” Healthcare (Basel) 2018 Aug 14;6(3):99. doi: 10.3390 / healthcare6030099.

[0149] Bartonella antigen strips were incubated with human serum at room temperature for 1 hour and washed three times to remove unbound serum. The washed strips were then incubated with alkaline phosphatase-conjugated goat anti-human IgG at a dilution of 1:10,000 or IgM at a dilution of 1:6,000 for 1 hour. After three washes, bands were visualized by reaction with 5-bromo-4-chloro-3-indolylphosphatenitroblue tetrazolium. The strips were dried and read manually.

[0150] (Interpretation and Reporting) The Bartonella IgG and IgM immunoblot tests are qualitative tests that detect Bartonella-specific IgG and IgM antibodies, respectively, in human serum. Recombinant Bartonella antigens were sprayed onto specific locations on nitrocellulose membranes and cut into strips. These strips were used to detect Bartonella-specific antibodies in human serum. If samples tested positive for Bartonella antibodies, they were speciated into the following Bartonella species: Bartonella henselae, Bartonella quintana, Bartonella vinsonii, Bartonella querzabethoe, and Bartonella sp.

[0151] Human sera were identified as positive for Bartonella antibodies if at least one band from each of the following two groups was detected using IgM-class or IgG-class secondary antibodies: Group 1: SucB, NIpD, HTRA Group 2: P35, P31, P26, and P17 were present, or at least one band from each of the following groups was detected: SucB, NIpD, and HTRA were present.

[0152] If only two bands in group 1 or only one band in group 2 were detected using the IgM-class or IgG-class secondary antibody, the test failed.

[0153] Alternatively, human serum was judged to be positive for Bartonella antibodies if at least one band from each of SucB, N1pD, and HTRA was detected using IgM-class or IgG-class secondary antibodies.

[0154] Profiles that did not meet the above-mentioned criteria for positive or negative results were considered negative.

[0155] If the Bartonema rhizoblot was positive, speciation was analyzed as follows: If at least one Bartonella henselae band was detected from at least one of P35, P31, and P26 using an IgM-class or IgG-class secondary antibody, the result was positive for Bartonella henselae; Bartonella quintana positive if at least one Bartonella quintana band was detected using an IgM-class or IgG-class secondary antibody from at least one of P35, P31, and P26; If at least one B. vinsonii band was detected using an IgM-class or IgG-class secondary antibody from at least one of P35, P31, and P26, the sample was considered B. vinsonii-positive; and If there is at least one B. elizabetzea band detected using an IgM-class or IgG-class secondary antibody from at least one of P35, P31, and P26, the sample is B. elizabetzea-positive.

[0156] If a human serum tested positive for Bartonella , the human serum was identified as positive for Bartonella spp., but the aforementioned speciation was not determined due to the presence of bands from multiple species.

[0157] (Restrictions) A positive result suggests exposure to Bartonella. For diagnostic purposes, immunoblot test results should be used in conjunction with clinical symptoms and other evidence available to the diagnostician. If the test result is inconclusive, it is recommended to test by an alternative method or repeat the test in 6 to 8 weeks.

[0158] (Test results) Figure 1 is an image showing the results of Bartonella immunoblotting of IgM and IgG classes used to test nine human specimens suspected of Bartonella infection.

[0159] Figures 2A-B show comparative images of a Bartonella Western blot (Figure 2A) and a Bartonella immunoblot (Figure 2B).

[0160] The results are summarized in Tables 4 and 5. As shown in Table 4 below, of the 17 samples that were positive by Western blot, 16 were also positive by the immunoblot described above. Of the 6 samples that were indeterminate by Western blot, 5 were also indeterminate by the immunoblot described above. All 8 samples that were negative by Western blot were also negative by immunoblot. As shown in Table 5 below, of the 30 samples tested for specificity, none were positive by the Bartonella immunoblot. From this data, the sensitivity of the immunoblot compared to the Bartonella Western blot is 94% and the specificity is 100%.

[0161] [Table 4]

[0162] [Table 5]

[0163] [Table 6]

[0164] (Discussion and Conclusion) In Western blots, antigens are separated by size. Therefore, multiple proteins may be present at any given location on the blot. This can result in false positives. In immunoblots, individual antigens are deposited (e.g., sprayed) at specific locations, leaving no ambiguity in reading the blot. This data demonstrates that Bartonella immunoblots have the specificity and sensitivity to be used clinically.

[0165] (equivalent) While several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art can readily envision various other means and / or structures for performing and / or obtaining the functions and / or results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular use or application for which the teachings of the present disclosure are / will be used. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. In addition, combinations of two or more such features, systems, articles, materials, and / or methods are included within the scope of the present disclosure, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0166] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0167] The indefinite articles "a" and "an," as used herein and in the claims, should be understood to mean "at least one" unless clearly indicated to the contrary. The term "and / or," as used herein and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, whether relevant or not, other than the elements specifically identified by the "and / or" clause may optionally be present unless clearly indicated to the contrary.

[0168] All references, patents and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety.

Claims

1. A diagnostic panel comprising individually identifiable labeled, tagged, or conjugated amino acid sequences comprising SEQ ID NOs: 1-41, and variants thereof that retain the immunological binding profile of the corresponding non-variant.

2. 2. The diagnostic panel of claim 1, wherein the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

3. 10. The diagnostic panel of claim 1, wherein the amino acid sequence comprises one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

4. 2. The diagnostic panel of claim 1, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

5. 1. A method for detecting infection with one or more Bartonella species, comprising: contacting a biological sample obtained from a subject suspected of having a Bartonella infection with a diagnostic panel comprising an amino acid sequence comprising SEQ ID NOs: 1-41, or one or more variants thereof that retain the immunological binding profile of the corresponding non-variant, wherein the biological sample comprises at least one of IgM class antibodies, IgG class antibodies, and IgA class antibodies; If a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 1 to 12 and at least one of SEQ ID NOs: 13 to 41, or If a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9-12, determining that the biological sample is positive for Bartonella infection; A method comprising:

6. 6. The method of claim 5, wherein the amino acid sequence is an unlabeled, untagged, and unlinked sequence.

7. 6. The method of claim 5, wherein the amino acid sequence comprises one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

8. 6. The method of claim 5, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

9. The method of claim 5 , wherein the amino acid sequence is attached to one or more substrates.

10. 10. The method of claim 9, wherein the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

11. 6. The method of claim 5, wherein the immunological binding of IgM class antibodies is detected by using an anti-human IgM antibody linked to a detectable moiety.

12. 6. The method of claim 5, wherein the immune binding of IgG class antibodies is detected by using an anti-human IgG antibody linked to a detectable moiety.

13. 6. The method of claim 5, wherein the immune binding of IgA class antibodies is detected by using an anti-human IgA antibody linked to a detectable moiety.

14. The method of any one of claims 11 to 13, wherein the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme.

15. The method of claim 14 , wherein the detectable moiety comprises alkaline phosphatase.

16. The method of claim 14 , wherein the detectable moiety comprises biotin.

17. 6. The method of claim 5, further comprising treating the subject for a Bartonella infection in response to the biological sample testing positive for a Bartonella infection.

18. determining that the biological sample is positive for Bartonella henselae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 13, 17, and 27; Determining that the biological sample is positive for Bartonella quintana infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 14, 18, and 28; Determining that the biological sample is positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 15, 19, and 29; Determining that the biological sample is positive for Bartonella elizabesae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 16, 20, and 30; The method of claim 5 further comprising:

19. 20. The method of claim 18, further comprising treating the subject for the Bartonella henselae infection in response to determining that the biological sample is positive for the Bartonella henselae infection.

20. 20. The method of claim 18, further comprising treating the subject for the Bartonella quintana infection in response to determining that the biological sample is positive for the Bartonella quintana infection.

21. 20. The method of claim 18, further comprising treating the subject for the Bartonella vinsonii infection in response to determining that the biological sample is positive for the Bartonella vinsonii infection.

22. 20. The method of claim 18, further comprising treating the subject for the Bartonella elizabethoeae infection in response to determining that the biological sample is positive for the Bartonella elizabethoeae infection.

23. A diagnostic panel comprising individually identifiable labeled, tagged or conjugated amino acid sequences comprising one, two, three or four of SEQ ID NOs: 1-4, one, two, three or four of SEQ ID NOs: 5-8, and one, two, three or four of SEQ ID NOs: 9-12, and variants thereof that retain the immunological binding profile of the corresponding non-variant.

24. 24. The diagnostic panel of claim 23, comprising individually identifiable labeled, tagged, or conjugated amino acid sequences comprising SEQ ID NOs: 1-12 and variants thereof that retain the immunological binding profile of the corresponding non-variant.

25. 24. The diagnostic panel of claim 23, wherein the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

26. 24. The diagnostic panel of claim 23, wherein the amino acid sequence comprises one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

27. 24. The diagnostic panel of claim 23, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

28. 1. A method for detecting infection with one or more Bartonella species, comprising:

1. A method for detecting Bartonella infection comprising contacting a biological sample obtained from a subject suspected of having a Bartonella infection with a diagnostic panel comprising amino acid sequences comprising one, two, three, or four of SEQ ID NOs: 1-4; one, two, three, or four of SEQ ID NOs: 5-8, and one, two, three, or four of SEQ ID NOs: 9-12; or one, two, three, or four of SEQ ID NOs: 9-12, and variants thereof that retain the immunological binding profile of the corresponding non-variants; wherein the biological sample is determined to be positive for Bartonella infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9-12.

29. 29. The method of claim 28, wherein the diagnostic panel comprises the amino acid sequences of SEQ ID NOs: 1-12, and the biological sample is determined to be positive for Bartonella infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected for at least one of SEQ ID NOs: 1-4, at least one of SEQ ID NOs: 5-8, and at least one of SEQ ID NOs: 9-12.

30. 29. The method of claim 28, wherein the amino acid sequence is an unlabeled, untagged, unlinked sequence.

31. 29. The method of claim 28, wherein the amino acid sequence comprises one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

32. 29. The method of claim 28, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

33. 29. The method of claim 28, wherein the amino acid sequence is attached to one or more substrates.

34. 30. The method of claim 29, wherein the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

35. 30. The method of claim 29, wherein the immune binding of IgM class antibodies is detected by the use of an anti-human IgM antibody linked to a detectable moiety.

36. 30. The method of claim 29, wherein the immune binding of IgG class antibodies is detected by the use of an anti-human IgG antibody linked to a detectable moiety.

37. 30. The method of claim 29, wherein the immune binding of IgA class antibodies is detected by the use of an anti-human IgA antibody linked to a detectable moiety.

38. 38. The method of any one of claims 35 to 37, wherein the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme.

39. 39. The method of claim 38, wherein the detectable moiety comprises alkaline phosphatase.

40. 39. The method of claim 38, wherein the detectable moiety comprises biotin.

41. 30. The method of claim 28, further comprising treating the subject for the Bartonella infection in response to the biological sample determining that the biological sample is positive for the Bartonella infection.

42. A diagnostic panel comprising individually identifiable labeled, tagged, or conjugated amino acid sequences comprising SEQ ID NOs: 13-20 and 27-30, and variants thereof that retain the immunological binding profile of the corresponding non-variant.

43. 43. The diagnostic panel of claim 42, wherein the amino acid sequences are bound to one or more substrates selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

44. 43. The diagnostic panel of claim 42, wherein the amino acid sequences comprise one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

45. 43. The diagnostic panel of claim 42, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

46. 1. A method for detecting species-specific infection with Bartonella henselae, Bartonella quintana, Bartonella vinsonii, and / or Bartonella elizabesae, comprising: contacting a biological sample obtained from a subject determined to have a Bartonella infection with a diagnostic panel comprising amino acid sequences comprising SEQ ID NOS: 13-20 and 27-30, or one or more variants that retain the immunological binding profile of the corresponding non-variant, wherein the biological sample comprises at least one of IgM class antibodies, IgG class antibodies, and IgA class antibodies; determining that the biological sample is positive for Bartonella henselae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 13, 17, and 27; Determining that the biological sample is positive for Bartonella quintana infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 14, 18, and 28; Determining that the biological sample is positive for Bartonella vinsonii infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 15, 19, and 29; Determining that the biological sample is positive for Bartonella elizabesae infection if a positive immune binding reaction with an IgM class antibody, an IgG class antibody, or an IgA class antibody is detected against at least one of SEQ ID NOs: 16, 20, and 30; A method comprising:

47. 47. The method of claim 46, wherein the amino acid sequence is an unlabeled, untagged, unlinked sequence.

48. 47. The method of claim 46, wherein the amino acid sequence comprises one or more labels selected from the group consisting of stable isotopes, mass tags, and fluorescent dyes.

49. 47. The method of claim 46, wherein the amino acid sequence comprises one or more peptide sequence tags selected from the group consisting of affinity tags, chromatography tags, epitope tags, enzyme conjugate tags, fluorescent tags, and combinations thereof.

50. 47. The method of claim 46, wherein the amino acid sequence is attached to one or more substrates.

51. 51. The method of claim 50, wherein the one or more substrates are selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), magnetic beads, agarose, plastic, and metal.

52. 47. The method of claim 46, wherein the immune binding of IgM class antibodies is detected by the use of an anti-human IgM antibody linked to a detectable moiety.

53. 47. The method of claim 46, wherein the immunological binding of IgG class antibodies is detected by the use of an anti-human IgG antibody linked to a detectable moiety.

54. 47. The method of claim 46, wherein the immune binding of IgA class antibodies is detected by the use of an anti-human IgA antibody linked to a detectable moiety.

55. 55. The method of any one of claims 52 to 54, wherein the detectable moiety is selected from the group consisting of a chromophore, a radioactive moiety, and an enzyme.

56. 56. The method of claim 55, wherein the detectable moiety comprises alkaline phosphatase.

57. 56. The method of claim 55, wherein the detectable moiety comprises biotin.

58. 56. The method of claim 55, further comprising treating the subject for a Bartonella henselae infection in response to determining that the biological sample is positive for a Bartonella henselae infection.

59. 47. The method of claim 46, further comprising treating the subject for Bartonella quintana infection in response to determining that the biological sample is positive for Bartonella quintana infection.

60. 47. The method of claim 46, further comprising treating the subject for a Bartonella vinsonii infection in response to determining that the biological sample is positive for a Bartonella vinsonii infection.

61. 47. The method of claim 46, further comprising treating the subject for a Bartonella elizabesae infection in response to determining that the biological sample is positive for a Bartonella elizabesae infection.

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