WO2010132054A1 - Compositions et procédés pour antigènes immunodominants de mycobacterium tuberculosis - Google Patents

Compositions et procédés pour antigènes immunodominants de mycobacterium tuberculosis Download PDF

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WO2010132054A1
WO2010132054A1 PCT/US2009/043822 US2009043822W WO2010132054A1 WO 2010132054 A1 WO2010132054 A1 WO 2010132054A1 US 2009043822 W US2009043822 W US 2009043822W WO 2010132054 A1 WO2010132054 A1 WO 2010132054A1
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seq
idno
antigens
seq idno
antigen
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PCT/US2009/043822
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Philip Felgner
Maria Laura Gennaro
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Immport Therapeutics, Inc.
Liang, Xiaowu
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Priority to MX2011012120A priority Critical patent/MX344052B/es
Priority to MX2015001830A priority patent/MX359550B/es
Priority to PCT/US2009/043822 priority patent/WO2010132054A1/fr
Priority to EP09789675A priority patent/EP2429575A1/fr
Priority to CN200980160460.8A priority patent/CN102573896B/zh
Publication of WO2010132054A1 publication Critical patent/WO2010132054A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria

Definitions

  • the field of the invention is compositions and methods related to selected antigens from Mycobacterium tuberculosis, especially as they relate to their use in diagnostic and therapeutic compositions and methods.
  • Antigens for vaccination and/or diagnostic purposes are typically single antigens from a pathogen, or complex mixtures of multiple known antigens from a pathogen, or of multiple known and unknown antigens of a pathogen such as live, attenuated or inactivated bacteria or viruses.
  • single antigens may provide a quantifiable signal in immunodiagnostic tests (antibody or cellular responses).
  • immunodiagnostic tests antibody or cellular responses.
  • single antigen tests are often not sufficient to obtain useful diagnostic information with useful specificity and sensitivity.
  • TST tuberculin skin test
  • IGRAs interferon gamma release assays
  • Candidate antigens are typically tested for diagnostic utility in ELISAs and Western blots using TB sera and sera from healthy controls. CFPs are more widely studied because of the convenience of working with soluble proteins. Of the > 100 M. tuberculosis proteins in culture filtrates (representing about 2.5% of the M. tuberculosis proteome), roughly two dozen are recognized by sera from TB patients, most of which have been previously identified. Yet despite these efforts, there remains no effective serological test with the sensitivity and specificity required to accurately diagnose TB, particularly in the early stage of infection.
  • none of the heretofore known antigens is generally applicable to differentiate among stages (e.g., active disease versus non-active), secondary infections, etc., as the signal is either impossible to deconvolute (e.g., compound signal from inactivated pathogen) or only provides a single data point.
  • the present invention is directed to immunodominant antigens from Mycobacterium tuberculosis wherein the antigens are known to react, that is, have known reactivities (and particularly known relative reactivities) to serum of a population of patients infected with the pathogen.
  • the antigens presented herein will have a statistically high probability to elicit an immune response in a relatively large group of patients.
  • the antigens may also have a known association with a disease parameter.
  • an antigen composition comprises a plurality of antigens of M. tuberculosis encoded by nucleic acids selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:586 (or any subgroup of such sequences), or fragments thereof, wherein at least two of the antigens elicit an immune response.
  • an antigen composition comprises two or more immunodominant antigens of a pathogenic organism and are associated with a carrier, wherein the antigens have quantified and known relative reactivities with respect to sera of a population infected with the organism, and wherein the antigens have a known association with a disease parameter.
  • immunodominant antigens are polypeptides and are encoded by nucleic acids having a sequence according to SEQ ID NO: 1 to SEQ ID NO:586 (or comprise fragments thereof).
  • the known reactivities may be characterized by a variety of factors, however, it is particularly preferred that the known reactivities are characterized by strength of immunogenicity and/or time course of the infection. It is generally preferred that the parameter is activity state of the disease, a previous exposure to the pathogen, the duration of exposure to the pathogen, a chronic infection, past disease, active infection, inactive infection, at least partial immunity to infection with the pathogen, and/or outcome upon treatment.
  • the carrier is a pharmaceutically acceptable carrier, and the composition is formulated as a vaccine.
  • the vaccine comprises multiple (e.g., at least two, four, or six) antigens. It is still further contemplated that the antigens or fragments thereof are at least partially purified and/or recombinant.
  • the carrier may also be a solid carrier, and the plurality of antigens is disposed on the carrier either as a mixture or as an array.
  • the antigens have at least two distinct known reactivities and/or parameters.
  • the antigens or fragments thereof may be in crude expression extracts, in partially purified form (e.g., purity of less than 60%), or in highly purified form (e.g., purity of at least 95%).
  • the antigens in such arrays may be recombinant or native.
  • solid phases need not be limited to planar arrays, but may also include beads, columns, dipstick-type formats, etc.
  • aspects of this invention include diagnostic assay utilizing at least two immunodominant antigens of M. tuberculosis.
  • Antibody assays comprise contacting a sample of bodily fluid that contains antibodies against M. tuberculosis, for example, serum, with at least two immunodominant antigens of this invention and detecting antigen-antibody binding by any satisfactory method, preferably by formation of a color or generation of a fluorescent signal.
  • antigens immobilized on a solid surface either individually in discrete areas or in a mixture, may be used to immobilize antibodies from the sample, and an anti-antibody linked directly or indirectly to a color-forming enzyme may then be added for signal generation in the standard ELISA format.
  • fluorescence signals may be generated by methods such as linking (directly or indirectly) an anti-antibody to a fluorescence-emitting substance.
  • aspects of this invention also include use of at least two immunodominant antigens free in solution rather than immobilized on a surface.
  • a sample of peripheral blood, a bodily fluid containing T-lymphocytes may be contacted with such antigens in vitro. Reactions between T-lymphocytes and antigen (on an antigen-presenting cell) are, like antigen-antibody reactions, epitope-specific even though T- lymphocytes and antibodies may recognize different epitopes.
  • kits of reagents for performing assays include at least two immunodominant antigens according to this invention.
  • Figures IA and IB depict exemplary microarrays probed with sera from TB-positive and LTBI-negative (i.e., not infected with M. tuberculosis) individuals, respectively.
  • Figures 2 A and 2B are graphs depicting exemplary statistical representations in differences in signals for TB-positive and LTBI-negative results.
  • Figure 2C is a table listing selected TB antigens using one preferred exemplary ranking algorithm.
  • Figures 3 A and 3B depict exemplary fluorimetric and colorimetric visualization of exemplary microarrays and respective scatter plots indicating the correlation between the visualizations.
  • Figures 4A and 4B depict the proteins ranking in random forests, CERNO, and random forests with CERNO-pref ⁇ ltered data.
  • Figure 5 depicts a protein which exhibits a tail distribution of higher intensity signals in TB samples that is not seen in non-TB samples.
  • immunodominant antigens from M. tuberculosis that are suitable for diagnostic and therapeutic purposes.
  • Particularly preferred immunodominant antigens are those encoded by nucleic acids having a sequence according to SEQ ID NO:1 to SEQ ID NO:586, and it is generally contemplated that such antigens can be used as single antigens, or in combination (optionally also in combination with antigens from another pathogen) in the manufacture of various diagnostic devices, therapeutic compositions, and vaccines.
  • the immunodominant antigens suitable for diagnostic and therapeutic purposes are encoded by the sequences designated RvO798c (SEQ ID NO: 121), Rvl886c (SEQ ID NO:270), Rv2031c (SEQ ID NO:284), Rv3616c (SEQ ID NO:509), Rv3804c (SEQ ID NO:534), Rv3874 (SEQ ID NO:547), Rv0302 (SEQ ID NO:52), RvO379 (SEQ ID NO:65), RvO394c (SEQ ID NO:66), RvO456c (SEQ ID NO:74), RvO632c (SEQ ID NO:103), RvO944 (SEQ ID NO:142), RvO984(SEQ ID NO:146), Rvl030 (SEQ ID NO:153), RvI196 (SEQ ID NO:174), Rvl242 (SEQ ID NO:180), Rvl284 (SEQ ID NO:187), R
  • RvO856 SEQ ID NO:561), RvI 191 (SEQ ID NO:564), Rv2803 (SEQ ID NO:397), RvO783c (SEQ ID NO:118), RvlO54 (SEQ ID NO:563), Rvl689 (SEQ ID NO:240), Rv2539c (SEQ ID NO:572), Rv2859c (SEQ ID NO:573), Rv3777 (SEQ ID NO:528), and fragments thereof.
  • the immunodominant antigens are encoded by sequences designated RvO798c (SEQ ID NO:121); Rvl886c (SEQ ID NO:270); Rv2031c (SEQ ID NO:284); Rv3616c (SEQ ID NO:509); Rv3804c (SEQ ID NO:534); and Rv3874 (SEQ ID NO:547).
  • the term "immunodominant antigen” refers to an antigen that elicits in at least one stage of the infection production of one or more types of antibodies (e.g., IgG, IgA, IgE, IgM, etc.) in at least 20%, more typically at least 40%, and most typically at least 70% of a population exposed to the antigen, or wherein, when compared to other antigens of the same pathogen, the average binding affinity and/or average quantity of the antibodies produced in the patient in at least one stage of the disease is at least in the upper half, more typically upper tertile, and most typically upper quartile.
  • antibodies e.g., IgG, IgA, IgE, IgM, etc.
  • the average binding affinity and/or average quantity of the antibodies is reflected in the signal intensity and signal intensity can therefore be used as a surrogate marker for average binding affinity and/or average quantity of the antibodies.
  • preferred immunodominant antigens are also characterized by a response in the test group that is considered statistically significant when compared with control signal intensity, wherein the significance level p is preferably equal or less than 0.1, more preferably equal or less than 0.05, and most preferably equal or less than 0.01.
  • immunodominant antigens are identified from a proteome screen against sera of a population that has been previously exposed to the pathogen. Most preferably, the population is subdivided in several sub-populations to reflect various disease parameters (e.g., active disease, bacillary burden of disease, latent infection, presence of co-infection with HIV, absence of infection, etc.), which can then be correlated with antibody responses to the so identified antigens. It is still further preferred that the screening also provides data on relative reactivities with respect to the antigens and sera of the populations/sub-populations.
  • disease parameters e.g., active disease, bacillary burden of disease, latent infection, presence of co-infection with HIV, absence of infection, etc.
  • At least part of the pathogen's genome is obtained and all potential open reading frames and portions thereof are determined in silico.
  • suitable primers are determined to provide amplicons of the entire Open Reading Frames (ORFs),or, less preferably, portions thereof, wherein the primers are preferably designed to allow facile subcloning into an expression system.
  • ORFs Open Reading Frames
  • the subcloning uses recombinase-based subcloning using unpurified PCR mixtures to avoid cloning bias, and the so obtained recombinant plasmids are polyclonally multiplied, which enables unbiased presentation of the amplicons.
  • the plasmid preparations are then subjected to an in vitro transcription/translation reaction to thereby provide the recombinant ORF peptide, which is then spotted or otherwise immobilized onto a suitable addressable carrier (e.g., membrane, bead, etc.).
  • a suitable addressable carrier e.g., membrane, bead, etc.
  • proteomes can then be exposed to serum of a population of control individuals and/or population of individuals that are known to have current or previous exposure to the above pathogen from which the ORFs were prepared. Antibodies of the serum that bind to one or more of the ORFs are then detected using well known methods (e.g., use of secondary antibodies). In this manner, the entire proteome of the pathogen can be rapidly assessed for immunogenicity and potential binding with antibodies in serum.
  • Various preferred aspects, compositions, and methods of proteome preparation are disclosed in International patent publication number WO 06/088492, which is incorporated by reference herein.
  • contemplated compositions and methods presented herein will allow for preparation of vaccines and diagnostic compositions comprising a plurality of antigens with known and predetermined affinity to target ORFs of a pathogen.
  • methods and compositions contemplated herein will allow statistically supported antigen identification to identify immunodominant antigens in a population of patient. Consequently, multiple targets can be used to elicit an immune response and/or detect a prior exposure, even where one or more of the targets may be evasive for detection or provide only a weak response.
  • sequences need not be complete ORFs, but that suitable sequences may also be partial sequences (e.g., synthetic, recombinant or isolated) that typically comprise at least part of an antigenic epitope.
  • suitable sequences may also be partial sequences (e.g., synthetic, recombinant or isolated) that typically comprise at least part of an antigenic epitope.
  • contemplated DNA sequences include those that will hybridize under stringent hybridization conditions to respective sequences listed in the sequence listing.
  • sequences contemplated herein may be identified as DNA sequences encoding the antigenic peptide (partial or entire ORF), or may be identified as peptide sequence (or homologs thereof).
  • chemically modified antigens, and/or orthologs of the polypeptides presented herein are also deemed suitable for use herein.
  • proteome screening will provide a plurality of antigens as potentially useful molecules for diagnosis, vaccination, and/or therapy, such an approach only provides a raw cut of (a plurality) of individual responses. Therefore, as most individual immune reactions towards the same pathogen elicit a significantly distinct profile of antibodies (e.g., depending on disease stage, previous exposure, and/or inter-individual variability), results obtained from such screening are typically inhomogeneous.
  • immunodominant antigens are identified by selecting for an antigen (preferably within a well-defined sub-population) that (a) produces in at least 40-50% of a population a measurable signal, and (b) has a signal strength of at least 40% of the overall average signal intensity.
  • the signal strength will be at least above average of the overall average signal intensity, and even more preferably in the upper tertile (quartile, or even quintile) of signal intensities in the assay. Therefore, and viewed from another perspective, immunodominant antigens will preferably be selected in a comparison of at least two series of tests, wherein one series of tests is typically the sub-population (e.g., primary infection, active disease, latent infection, recovering, previously diseased, chronic, etc.) and the other series of tests is the control group (e.g., other sub-population or control group). Still further, it is generally preferred that the series of tests also include a negative control against which the potential immunodominant antigens are compared.
  • one series of tests is typically the sub-population (e.g., primary infection, active disease, latent infection, recovering, previously diseased, chronic, etc.) and the other series of tests is the control group (e.g., other sub-population or control group).
  • the series of tests also include a negative control
  • compositions comprising one or more selected immunodominant antigens can be prepared that will have a statistically high probability to elicit or have elicited an immune response in a relatively large group of patients.
  • the antigens are determined from selected sub-populations (e.g., active disease, severity of disease, latent infection, previously diseased patients, primary infection, etc.)
  • the antigens also have a known association with a disease parameter and thus allow staging of the disease and/or prediction of therapeutic efficacy.
  • vaccine compositions can be prepared with known or predictable immunogenicity.
  • antigens from M. tuberculosis encoded by the nucleic acids of SEQ ID NO: 1 to SEQ ID NO:586 were identified as immunodominant (see examples below).
  • the first base in the sequences is either the first base of the start codon or the first base in the first codon of the polypeptide that was identified with the methods and compositions provided herein.
  • the last three bases denote the stop codon, or the last base of the last codon of the polypeptide that was identified with the methods and compositions provided herein.
  • each of the antigens was characterized, inter alia, with regard to their individual and relative reactivities for the pathogen. Most typically, reactivity was measured as strength of immunogenicity (e.g., such that average binding affinity and/or average quantity of the antibodies produced a predetermined signal intensity (e.g., in the upper half, upper tertile, or even upper quartile)). Viewed from a different perspective, each one of the identified antigens has a known signal strength (reflecting the quantity of antibodies formed in the patient) in the assay as described below relative to another one of the identified antigens. Some proteins, such as the one depicted in Figure 5, exhibit a tail distribution of higher intensity signals in TB samples that is not seen in non-TB samples.
  • each of the identified antigens was also characterized by association with at least one parameter.
  • the disease parameter was active disease after infection, and in further cases, the disease parameter was number of tubercle bacilli in sputum or radiographic extent of disease, and in further cases, history of past disease in the non- diseased population. Therefore, it should be especially appreciated that identification of immunodominant antigens will not only allow for identification of statistically meaningful antigens for diagnosis, vaccine development, and treatment, but also allow to develop a stage specific tool to identify candidate molecules to fine-tune diagnosis and/or treatment.
  • suitable diagnostic devices especially include those comprising one or more of the immunodominant antigens, fragments, or analogs thereof that are encoded by nucleic acids according to SEQ ID NO:1 to SEQ ID NO:586, preferably RvO798c (SEQ ID NO: 121), Rvl886c (SEQ ID NO:270), Rv2031c (SEQ ID NO:284), Rv3616c (SEQ ID NO:509), Rv3804c (SEQ ID NO:534), Rv3874 (SEQ ID NO:547), Rv0302 (SEQ ID NO:52), RvO379 (SEQ ID NO:65),Rv0394c (SEQ ID NO:66), RvO456c (SEQ ID NO:74), RvO632c (SEQ ID NO: 103), RvO944 (SEQ ID NO: 142), RvO984(SEQ ID NO: 146), Rvl030 (SEQ ID NO:153), RvO798c (
  • RvO831c (SEQ ID NO:125), Rv2032 (SEQ IDNO:285), Rv3127 (SEQ ID NO:446), Rv3272 (SEQ ID NO:464), Rv3323c (SEQ IDNO:470), Rv3508 (SEQ ID NO:494), Rv3628 (SEQ IDNO:513), RvI173 (SEQ ID NO:167), Rv2623 (SEQ ID NO:376), RvO527 (SEQ ID NO:85), Rvl620c (SEQ ID NO:229), Rvl901 (SEQ ID NO:272), Rv2151c (SEQ ID NO:308),Rv0362 (SEQ ID NO:60), Rv3129 (SEQ ID NO:447), Rv3140 (SEQ ID NO:449), Rv0340 (SEQ ID NO:56), Rv2792c (SEQ ID NO:395), Rv3003c (SEQ ID NO:426), Rv30
  • the device may have only a single immunodominant antigen, fragment, or analog that may be used for detection of binding of antibodies from blood, plasma or serum or other bodily fluids containing antibody in an automated manner or by visual observation.
  • suitable devices may be in the format of a dipstick or competitive
  • suitable devices may be in the format of an array that can be read in an automated device (e.g., via scanner) or visual manner (e.g., dye-forming colorimetric reaction). Most typically, in such devices, the plurality of antigens is deposited in a spatially addressable manner (e.g., x-y matrix or beads with color association or microtiter plate).
  • diagnostic devices contemplated herein may be based on numerous well known manners of detection, including ELISA (sandwich or non-sandwich), competitive ELISA, anti-idiotypic antibodies, etc., wherein all known colorimetric and photometric (e.g., fluorescence, luminescence, etc.) or radiometric reactions are deemed suitable for use.
  • compositions can be employed to identify and/or characterize an immune response of an individual against selected antigens, and optionally assess the kind of immune response (e.g., identification of latent or chronic infection), as well as disease progression, efficacy of therapy, etc.
  • the plurality of antigens will include between 5 to 10 antigens, but significantly higher amounts of antigens are also contemplated, including at least 25%, more typically at least 50%, even more typically at least 75%, and most typically at least 90% of the proteome of the pathogen. Similarly, less than 5 antigens (1-4) are also deemed suitable.
  • contemplated arrays are most preferably processed in a micro fluidic device. For example, an array of antigens in such devices may be printed on a membrane or other material (e.g., nitrocellulose-coated carrier of less than 1 cm2 area) that is then placed in a micro fluidic device having sample/reagent inlet and outlet ports.
  • signals may be acquired using optical methods (e.g., CCD chip, flat bed scanner, etc.), electrical methods (e.g., voltametric or amperometric), or other methods well known in the art.
  • optical methods e.g., CCD chip, flat bed scanner, etc.
  • electrical methods e.g., voltametric or amperometric
  • visual detection or detection using a regular flat bed scanner at 1200 dpi resolution and/or fluorescence detection is also deemed suitable.
  • immunodominant antigens according to the inventive subject matter may also be employed to generate an antibody preparation that can be used as passive vaccination for therapeutic treatment of tuberculosis.
  • vaccines are subunit vaccines or attenuated live recombinant vaccines.
  • the immunodominant antigens presented herein may be employed in the manufacture of a vaccine that comprises at least one, and more typically at least two of the immunodominant antigens encoded by nucleic acids according to SEQ ID NO:1 to SEQ ID NO:586.
  • contemplated vaccines will include between two and five, or at least six, and even more antigens, of which at least one of the antigens is an immunodominant antigen.
  • Such vaccine compositions may be directed to elicit immunity against single or multiple subtypes and may thus comprise distinct immunodominant antigens, optionally from multiple and distinct subtypes.
  • vaccines may be produced that predominantly, or even exclusively, comprise immunodominant antigens of a single parameter.
  • a vaccine may comprise immunodominant antigens that are characteristic for a population that has a latent infection.
  • sequences according to SEQ ID NO:1 to SEQ ID NO:586 may also be employed as DNA vaccines, or be part of an in vivo expression system that triggers an immune response against the in vivo produced recombinant antigen or fragment thereof.
  • antigens identified herein may also be employed to generate (monoclonal or polyclonal) antibodies or fragments thereof (e.g., Fab, scFv, etc.) that can then be employed in a diagnostic test that directly detects the presence of the antigen in blood, blood derivatives or other body fluid of a patient where the antigen is circulating in the patient.
  • the antigen may circulate in association with the pathogen, in association with components of the pathogen, in free form, or bound to a molecule or cell of the patient.
  • the antigens are immunodominant and/or serodiagnostic antigens as presented herein.
  • suitable tests include those in which one or more labeled antibodies are used to detect presence of the antigen in bodily fluid where the antigen may be captured (specifically or in bulk with other proteins) on a surface.
  • the antigen may be captured (specifically or in bulk with other proteins) on a surface.
  • antigen detection methods known in the art and all of the known formats are deemed suitable for use herein.
  • the diagnostic tools of the present invention involve the recognition of the immunodominant antigens described herein in an in vitro cellular assay determining the release of cytokines, such as interferon gamma, from lymphocytes withdrawn from a subject currently or previously infected with a virulent mycobacterium.
  • cytokines such as interferon gamma
  • suitable vaccines may be formulated as injectable solutions, or suspensions, intranasal formulations, transdermal or oral formulations.
  • compositions, vaccines, diagnostic tests, etc., described herein may be used for both human and veterinary use.
  • M. tuberculosis proteome microarray chip fabrication and probing methods Proteome microarrays were fabricated as described previously (Proc Natl Acad Sci U S A 102(3): 547- 552; Proteomics 7(10): 1678-1686; Proteomics 7(13): 2172-2183) with modifications. This and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
  • High-throughput construction of the M. tuberculosis ORFeome Using the available M. tuberculosis sequence data primer pairs were designed for all ORFs encoded in the genome. Quality control was performed using gel electrophoresis of PCR products. After three rounds of PCR, the final tally was 97.3% successfully amplified. For cloning, PCR products were mixed with a linearized pXT7-based expression vector as described previously and transformed into supercompetent DH5 ⁇ cells. The transformed cells were cultured at 37 0 C with vigorous aeration and were checked for turbidity the following day.
  • Custom PCR primers comprising 20bp of gene-specific sequence with 33bp of "adapter" sequences were used in PCRs with genomic DNA as template. For genes >3kb, additional primer pairs were designed to amplify overlapping fragments of 3kb each. All primer pairs used for this work are published at the UCI Institute for Genomics and Bioinformatics (IGB) web portal at http://contactl4.ics.uci. edu/virus/tuber_index.php.
  • the adapter sequences which become incorporated into the termini flanking the amplified gene, are homologous to the cloning site of the linearized T7 expression vector pXT7 and allow the PCR products to be cloned by in vivo homologous recombination in competent DH5 ⁇ cells.
  • the resulting protein incorporates an ATG translation start codon, a 5 ' polyhistidine epitope, a 3 ' influenza hemagglutinin epitope and a T7 terminator.
  • Array fabrication Antibodies against the N-terminal poly-His and the C-terminal HA tags engineered into each protein were used to monitor the expression in each spot. Positive and negative controls were built into every array and remaining spots on the array were in vitro transcription/translation reactions expressing 4109 different M. tuberculosis clones representing the entire cloned ORFeome. Once RTS reactions were printed onto nitrocellulose and dried, no appreciable degradation was observed after 6 months of storage in a desiccator at 18 0 C (data not shown). However, a reduction in signal and a reduced signal-to-noise ratio associated with prolonged delay in printing after the end of the 5h in vitro protein expression was observed.
  • in vitro expression reactions were staggered through the print run.
  • the reactions are no more than 5, more preferably no more than 3, and most preferably no more than 1 hour old before printing. Remaining variance in signals was normalized using positive and negative controls as described below.
  • Microarray chips were fabricated in batches of 50-100 2-pad slides (i.e., 100-200 arrays). Two standard deviations above the average of the negative control intensity was used as a cut-off for detection of the tags. Overall, 3854 (96.4%) of the expressed proteins were positive for the HIS tag, 3730 (93.3%) were positive for the HA tag, 3538 (91%) were positive for both tags, and only 56 (1.4%) were negative for both tags, which means 98.6% of the expressed proteins were positive for at least 1 of the tags.
  • purified minipreps of DNA of 4109 clones (3998 full length ORFs plus 111 segments of ORFs > 3kb) were expressed in the E. coli based in vitro transcription/translation expression system from Roche (RTS-100).
  • 10 ⁇ l in vitro reactions were set up in sealed 384 well plates and incubated for 5h at 30 0 C in a platform microshaker at 300 RPM.
  • a mixture of Tween-20 to a final concentration of 0.05% and a protease inhibitor cocktail (Complete, Roche) were then added. To minimize delay in printing after completion of protein expression, the initiation of the RTS reactions was staggered.
  • the RTS reactions were printed in singlicate without further purification onto 2-pad nitrocellulose- coated FAST slides (Whatman) using an Omni Grid 100 microarray printer (Genomic Solutions) in 4 x 4 sub-array format, with each subarray comprising 17 x 17 spots.
  • Each sub- array included 6 negative control spots comprising 'mock' RTS reactions lacking DNA template.
  • Each sub-array also included positive control spots of 5 serial dilutions of mouse, rat and human whole IgG. Together these positive and negative controls are used to normalize the data from different arrays (infra).
  • Epstein-Barr virus nuclear antigen- 1 (EBNA-I) which is recognized by the majority of humans and which serves as a useful guide to serum quality.
  • EBNA-I Epstein-Barr virus nuclear antigen- 1
  • 38KDa RvO934
  • CFP-10 Rv3874
  • ESAT-6 Rv3875
  • VEGF vascular endothelial growth factor
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the arrays were rehydrated in blocking buffer (Candor Bioscience) for 30 min and probed with the pretreated sera overnight at 4 0 C with constant agitation.
  • the slides were then washed 5x in tris(hydroxymethyl)aminomethane (Tris)-buffered saline containing 0.05% (v/v) Tween 20, (T-TBS) and incubated in biotinylated anti-human IgG-Fc (Jackson Immuno Research) diluted 1/400 in dilution buffer. After washing the slides three times each in T- TBS, bound antibodies were visualized by incubation with streptavidin-conjugated SureLight® P-3 (Columbia Biosciences).
  • BCG vaccination status was not always known, but cohorts were all from countries that implement universal BCG vaccination at birth.
  • active TB cases were subdivided in smear positive TB (presence of M. tuberculosis in sputum) and smear negative active TB. Cases in the TB and non-TB disease group were also subdivided based on HIV comorbidity.
  • FIG. IA Screening the proteome for serodiagnostic antigens: Representative arrays probed with serum from each group are shown in Figures IA and IB.
  • panel (A) shows culture- confirmed TB-positive individual
  • panel (B) shows latent TB infected-negative control individual.
  • Each array contained positive and negative control spots.
  • the IgG control spots, which control for secondary antibody, were positive in both arrays.
  • Neither individuals reacted with the negative ('no DNA') control reactions.
  • Both groups of individuals reacted to EBNA-I indicating prior exposure to EBV, and the group of acute infected individuals, had a robust antibody response to several M. tuberculosis antigens.
  • Random Forests a classification method based on multiple classification trees. Random forests queries (comparisons of TB to non-TB disease classes) were performed with data collected from sera from endemic countries, and with data stratified for HIV status and for smear status of TB patients.
  • Antigens were ranked from most informative to least informative based on mean decrease accuracy output of a particular query (highest mean decrease accuracy corresponds to highest rank).
  • the random forests analysis was conducted with and without a pre-filtering step using the CERNO statistical calculation.
  • CERNO p-values provide an association of high relative intensities with active tuberculosis diagnosis. Antigens were ranked by decreasing p-value.
  • a p-value on the normalized data was prepared by comparing signals between the confirmed TB-positive and LTBI- negative control groups using a Bayes-regularized t-test adapted from Cyber-T for use with protein arrays (Bioinformatics 17(6): 509-519; J Biol Chem 276(23): 19937-19944; Bioinformatics 22(14): 1760-1766; Bioinformatics 23(13): 1508-518).
  • Benjamini Hochberg p-value adjustments were calculated. Reactive antigens were defined as serodiagnostic or cross-reactive by having a Benjamini Hochberg corrected p-value ⁇ 0.05 or >0.05, respectively, and an average signal intensity >2 std.
  • a total of 250 antigens were selected by combining top 50 ranks from Random Forests (RF) and CERNO for queries on all TB and non-TB disease patients, all HIV negative TB and non-TB disease patients (with and without stratification by smear), plus top 10 ranks for HIV-positive TB and non-TB disease query, plus reactivity calls of >3 in TB category, plus Benjamini Hochberg adjusted Cyber T p value ⁇ 0.05. Seven sets of antigens were prioritized based on agreement by the methods, with the antigens of the first set being the most preferred.
  • RvO281 SEQ ID NO:51
  • Rv2711 SEQ ID NO:383
  • Rv2744c SEQ ID NO:390
  • Rv3803c SEQ ID NO:533
  • Rvl239c SEQ ID NO:179
  • Rv2147c SEQ ID NO:307
  • Rv2253 SEQ ID NO:324
  • Rv0308 SEQ ID NO:53
  • RvO587 SEQ ID NO:95
  • Rvl564c SEQ ID NO:224
  • Rv2185c SEQ ID NO:313).
  • a ROC curve is a parametric plot of the false positive rate (1 - specificity) vs. the true positive rate (sensitivity) of a classifier as the underlying discrimination threshold is varied.
  • the area under the curve (AUC) summarizes the results.
  • An AUC of 1.0 indicates a perfect classifier, while an AUC of 0.51 (95% confidence interval, 0.43 to 0.59) is the expected value for a classifier that works by chance for the data set, as inferred by the method of Truchon and Bayly (J Chem Inf Model 47(2): 488-508).
  • kernel methods and support vector machines were used (Bioinformatics: the Machine Learning Approach, Second Edition edn.: MIT Press) to build linear and nonlinear classifiers.
  • the highest-ranking 1, 2, 5, 10, and 30, ORFs were used on the basis of either p-value or single antigen AUC and the results were validated with 10 runs of threefold cross-validation.
  • the results showed that increasing the antigen number from 1 to 5, and from 5 to 10 produced an incremental improvement in the classifier. Increasing numbers beyond this did not improve the algorithm's ability to discriminate the two populations. Contingency tables built on these data showed that using 10 antigens at an optimal threshold provides an accuracy of 94% of the true positives and 78% of the true negatives.
  • antigen selection was also performed as follows: Raw data were distributed into the two groups of the query and subsets were normalized using vsn on the control spots. A CyberT test was run on the normalized data, and SVM classifier was built with a subset of the top antigens. Duplicates were removed. Each sample included meta data that was used to build each of the 10 queries, and each group was a disjoint subset of the original data, and each query only had two groups. Data were normalized using arsinh normalization (Bioinformatics, 18 Suppl 1, 2002), which compensates for variance dependence on mean.
  • SVM Single-chain predictive model
  • Classifiers were built using a number of the top antigens to provide an estimate of what classification accuracy we could be obtained for each query. This allows for determination of the optimal number of antigens to be included in the final classifier. 3-fold cross-validation was used and ROC plots were generated to visualize the results. A list of exemplary results using this analysis is provided in the list of serodiagnostic antigens shown in Figure 2C.
  • Enrichment analysis To determine the features of proteins that were enriched in the immunodominant antigen set, proteins were classified into one of 11 functional categories according to the TubercuList genome database (http://genolist.pasteur.fr/TubercuList/). The number of 'hits' for each category was determined in the immunodominant antigen set. 7 immunodominant antigens were considered serodiagnostic, of which 4 (57.1%) were proteins with proline-glutamic (PE/PPE) motifs. Since the whole proteome contains 168 (4.2%) PE/PPE motif proteins, this represents a significant 13.6-fold enrichment relative to the whole proteome.

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Abstract

Les compositions, dispositifs, et procédés de la présente invention concernent divers antigènes issus du pathogène M. tuberculosis et leur utilisation comme vaccins, agents thérapeutiques, et divers tests diagnostiques. Dans des aspects particulièrement préférés, les antigènes sont immunodominants et ont des réactivités relatives quantifiées et connues par rapport aux sérums d'une population infectée par le pathogène, et/ou ont une association connue avec un paramètre de la maladie.
PCT/US2009/043822 2009-05-13 2009-05-13 Compositions et procédés pour antigènes immunodominants de mycobacterium tuberculosis WO2010132054A1 (fr)

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WO2016014240A3 (fr) * 2014-07-24 2016-03-17 Abbott Molecular Inc. Compositions et procédés pour la détection et l'analyse de mycobacterium tuberculosis
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