[go: up one dir, main page]

WO2006110367A2 - Procedes et compositions pour toxines mycoplasma - Google Patents

Procedes et compositions pour toxines mycoplasma Download PDF

Info

Publication number
WO2006110367A2
WO2006110367A2 PCT/US2006/012266 US2006012266W WO2006110367A2 WO 2006110367 A2 WO2006110367 A2 WO 2006110367A2 US 2006012266 W US2006012266 W US 2006012266W WO 2006110367 A2 WO2006110367 A2 WO 2006110367A2
Authority
WO
WIPO (PCT)
Prior art keywords
toxin
subject
seq
penetrans
cards
Prior art date
Application number
PCT/US2006/012266
Other languages
English (en)
Other versions
WO2006110367A3 (fr
Inventor
Joel Barry Baseman
Thirumalai Rengasamy Kannan
Peter Dube
Original Assignee
Board Of Regents, University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Board Of Regents, University Of Texas System filed Critical Board Of Regents, University Of Texas System
Priority to US11/887,995 priority Critical patent/US20090104185A1/en
Publication of WO2006110367A2 publication Critical patent/WO2006110367A2/fr
Publication of WO2006110367A3 publication Critical patent/WO2006110367A3/fr

Links

Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • the present invention relates to Mycoplasma pneumoniae and Mycoplasma penetrans toxins, antibodies thereto, and their use in diagnostic and therapeutic methods.
  • M. pneumoniae is the etiologic agent of primary atypical pneumonia and is also responsible for many respiratory tract infections, such as tracheobronchitis, bronchiolitis, pharyngitis and croup, especially in older children and young adults and in elderly populations. It accounts for 20-30% of all pneumonias and also is linked to asthma and chronic obstructive pulmonary disease. M.
  • M. pneumoniae is among the smallest self-replicating cells (816 kb genome) and is spread by direct contact or aerosol among individuals, usually young children and adolescents, although the age spectrum for susceptibility includes adults and the elderly.
  • Large, sustained outbreaks of M. pneumoniae have occurred in closed and semi-closed populations, such as child care centers, college dormitories, hospitals, psychiatric institutions, military and religious communities, and prisons.
  • M. pneumoniae can disseminate to other organ sites and cause gastrointestinal, hematologic, neurologic, dermatologic, musculoskeletal and cardiovascular pathologies. This secondary involvement by M. pneumoniae leads to a spectrum of complicated extrapulmonary sequelae, including arthritis, pericarditis and central nervous system disorders, which attests to the significance of M.
  • M. pneumoniae in human disease Definitive diagnosis and therapeutic decisions concerning M. pneumoniae are often delayed or lacking because of the relatively long incubation period (average 1-2 weeks) before clinical symptoms can be observed and before M. pneumoniae broth or colony growth can be visualized. Although antibiotic therapy appears to be relatively effective in controlling mycoplasma pneumonia, the bacteria continue to persist. At present, no known virulence determinants of M. pneumoniae have been functionally identified and linked to the wide range of pathologies associated with M. pneumoniae mediated diseases. Furthermore, there are no specific and standardized diagnostic tests available for reliable and rapid detection of M pneumoniae infection, or effective vaccines to control M. pneumoniae infection.
  • Mycoplasma penetrans a member of the Mollicute (includes mycoplasmas) family, was first isolated in 1992 from the urine of HIV-infected homosexual males and is associated with the progression of AIDS. For example, 40% of HIV-infected individuals with AIDS exhibit high antibody titers against M. penetrans, while 20% of HIV-infected individuals who have not progressed to AIDS exhibit high-titered antibodies to M. penetrans, hi contrast, antibodies against M. penetrans are rarely found in heterosexual and non-HIV infected groups (Giron et al. 1996.
  • M. penetrans is implicated in the deterioration of the immune system in AIDS patients and thought to be involved in the rapid decline of CD4+ lymphocytes.
  • M. penetrans is mainly considered a urogenital tract pathogen, it has been infrequently isolated from non-HIV-infected patients with urethritis and respiratory disease manifestations.
  • the present invention overcomes previous shortcomings in the art by providing a Mycoplasma pneumoniae polypeptide and biologically active fragments thereof, known as community acquired respiratory distress syndrome (CARDS) toxin, as well as nucleic acids encoding this polypeptide and its fragments and antibodies specific thereto. Also provided is a Mycoplasma penetrans polypeptide and biologically active fragments thereof, as well as nucleic acids encoding this polypeptide and antibodies specific thereto. These compositions are used, for example, in methods of diagnosing, treating and preventing infection by M. pneumoniae and M. penetrans.
  • CARDS community acquired respiratory distress syndrome
  • rCARDS TX (rMPN372) yielded two different distinct and stable domains.
  • the amino acid (aa) sequence from aa 1-307 is ⁇ 37 kDa and represents the N -terminal part of the toxin domain; this sequence encodes the ADP ribosylation region and likely represents the catalytic activity of the toxin.
  • the remaining aa sequence from 308-591 is -34 kDa and represents the C-terminal part. Attached is the CARDS TX sequence with identified trypsin cleavage site labeled in red.
  • C-terminal domain (308-591) probably represents the "B" domain of toxins like diphtheria toxin, which is likely involved in binding of CARDS TX to specific host targets. Because it I trypsin-resistant, it would be a prime candidate for diagnostic, vaccine and anti-drug targets.
  • RJLl (clinical strain) amino acid sequence: (SEQ ID N0:4)
  • Reference nucleotide sequence M129/B9 (contains tga codons that need to be changed to tgg before expression in E. coli) (SEQ ID NO:7)
  • RJLl nucleotide sequence with each tga changed to tgg for expression in E. coli SEQ ID NOrIl atgccaaatc ctgttagatt tgtttaccgt gttgatttga gaagccctga agaaattttt 60 gaacatggct tttcaacttt aggtgatgtg agaaatttct ttgaacacat tctctccact 120 aattttggta gaagctatttt tattccact tcagaaacac ccacagcagc tattcgcttc 180 ttggtagct ggttacggga atatgtacca gagcacccca gaagggctta cttatatgaa 240 attcgtgccg accaacactt tacaatgccc 180
  • Tryptophans 10, 247, 491, 493, 572, 587 and 620 are encoded by TGA and they were changed to TGG to express recombinant M. penetrans toxin in E. coli. 0
  • Figure 1 shows an immunoblot that demonstrates both production of the CARDS toxin and anti-CARDS antibodies in three patients during infection with Mycoplasma pneumoniae.
  • Figure 2 shows ADP-ribosylation of G-like proteins in HEp-2 cells following incubation with CARDS protein. Lane 1 : HEp-2 cells in medium alone followed by preparation of cell free extract and addition of CARDS protein. Lane 2: HEp-2 cells pretreated with CARDS protein, followed by preparation of cell free extract and addition of CARDS protein. The marked reduction in ADP-ribosylation of specific proteins in the CARDS protein-pretreated cells is indicated by arrows. Also, ADP- ribosylation of other Hep-2 cell proteins is diminished (lane 2).
  • Figure 3 shows an ELISA and an immunoblot employing rDl as antigen that demonstrates production of anti-CARDS antibodies in sequential serum samples of two patients infected with Mycoplasma pneumoniae.
  • Figures 4A-B Vacuolating effect of CARDS toxin on monolayers of CHO cells and HeLa cells.
  • B HeLa cells: 10 ⁇ g/ml, panels start top, left to right; control non-intoxicated cells; then CARDS toxin-treated cells at 16 hours, 36 hours, 54 hours and 72 hours after exposure.
  • CARDS toxin delays time to death when mice are challenged with a high lethal dose of Yersinia pestis. Mice were treated with 50 ⁇ g of CARDS toxin intranasally (IN) or 50 ⁇ g of CARDS toxin heated to 6O 0 C for 30 minutes (HK TOX). Four days later, animals were challenged with 40,000 CFU of Y pestis IN. There was a delayed time to death in 50% of the toxin-treated animals at day 2. AU animals died at day 3. Time is on the X axis, % survival is on the Y axis and treatment is on the Z axis.
  • CARDS toxin delays time to death when mice in a dose dependent manner when are challenged with low lethal dose of Yersinia pestis. Mice were treated with the indicated amount of CARDS toxin IN. Four days later, animals were challenged with 800 CFU of Y. pestis. There was a delayed time to death in animals treated with 1 or 10 ⁇ g of toxin. The surviving animals appeared clinically ill and then recovered and remained alive. Time is on the X axis, % survival is on the Y axis and treatment is on the Z axis.
  • FIG. 7 CARDS TX treatment 2-4 days prioro to infection with lethal dose of Y. pestis provides protection. Mice were treated with 1 ⁇ g of CARDS TX IN at the indicated time prior to infection. Mice pretreated for two days were 20% protected but 3-4 days pretreatment provided 60% protection. Time is on the X axis, % survival on the Y axis and treatment on the Z axis.
  • FIG. 8 Growth of Y. pestis strain KIM 5 was evaluated in the presence of varying concentrations of CARDS TX.
  • the bacteria were grown overnight in heart infusion broth with 0.2% xylose at 37 0 C.
  • the culture was diluted to approximately 1000 cfu/ml and then the indicated amount of toxin or control treatment was added. Cultures were then grown an additional 1.5 hours at 37 0 C and the actual number of colony forming units per ml was determined. Results are the average of duplicate experiments.
  • the present invention provides Mycoplasma pneumoniae toxin (CARDS toxin) from subjects infected with Mycoplasma pneumoniae.
  • CARDS toxin Mycoplasma pneumoniae toxin
  • the present invention provides a polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:2 (Sl isolate), a polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO: 3 (JL isolate), a polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:4 (RJLl isolate), a polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO: 5 (L2 isolate), a polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:1 (reference sequence), and/or a polypeptide comprising, consisting essentially
  • the present invention further provides biologically active fragments of the polypeptides of this invention, as well as antibodies that specifically bind the polypeptides and/or fragments of the polypeptides of this invention.
  • nucleotide sequences that encode the polypeptides and fragments of this invention.
  • the present invention provides an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO: 8 (Sl isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO: 10 (JL isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO: 11 (RJLl isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotides sequence of SEQ ID NO: 9 (L2 isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotides sequence of SEQ ID NO:7 (reference sequence), and/or an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the
  • nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO:2 (Sl isolate), a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO: 3 (JL isolate), a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO:4 (RJLl isolate), a nucleic acid comprising, consisting essentially
  • probes and primers for the detection and/or amplification of the nucleic acids of this invention including
  • M. pneumoniae and/or M. penetrans in a subject comprising contacting a biological sample from the subject with a polypeptide or antibody of this invention under conditions whereby an antigen/antibody complex can form; and detecting formation of an antigen/antibody complex, thereby diagnosing infection by M. pneumoniae and/or
  • Methods are also provided herein for diagnosing infection by M. pneumoniae and/or M. penetrans in a subject comprising contacting a biological sample from the subject with a nucleic acid of this invention under conditions whereby hybridization of nucleic acid molecules can occur; and detecting hybridization, thereby diagnosing infection by M. pneumoniae and/or M. penetrans in the subject.
  • the present invention provides a method of detecting the presence of the CARDS toxin of this invention in a sample (e.g., a biological sample from a subject or a food or water sample or other sample that could contain CARDS toxin) and/or a subject and/or diagnosing infection by M. pneumoniae in a subject, comprising contacting the sample with surfactant protein A (SP-A) under conditions whereby a toxin/SP-A complex can form; and detecting formation of the toxin/SP-A complex, thereby detecting the presence of CARDS toxin in a sample and/or diagnosing infection by M. pneumoniae in a subject.
  • a sample e.g., a biological sample from a subject or a food or water sample or other sample that could contain CARDS toxin
  • SP-A surfactant protein A
  • the present invention provides methods of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a polypeptide and/or biologically active fragment of a polypeptide of this invention and/or by administering to a subject an effective amount of a nucleic acid comprising a nucleotide sequence encoding a polypeptide and/or biologically active fragment of a polypeptide of this invention.
  • the present invention additionally provides methods of providing passive immunity to a subject, comprising administering to the subject an effective amount of an antibody of this invention.
  • the present invention provides methods of treating and/or preventing infection by M. pneumoniae and/or M. penetrans in a subject, comprising administering to the subject an effective amount of a polypeptide of this invention and/or an effective amount of a biologically active fragment of a polypeptide of this invention and/or an effective amount of a nucleic acid comprising a nucleotide sequence encoding a polypeptide of this invention and/or an effective amount of a nucleic acid comprising a nucleotide sequence encoding a biologically active fragment of a polypeptide of this invention.
  • Also provided are methods of treating and/or preventing infection by M. pneumoniae and/or M. penetrans in a subject, comprising administering to the subject an effective amount of an antibody of this invention.
  • the present invention provides methods of identifying substances having the ability to inhibit or enhance various activities of the polypeptides and/or biologically active fragments of this invention, including but not limited to, binding activity, translocating activity, immunogenic activity, ADP- ribosylating activity, cytopathology inducing activity and/or toxin activity. These methods are carried out by contacting the polypeptides and/or biologically active fragments of this invention and/or the nucleic acids of this invention, with the substance to be tested for inhibitory or enhancing activity, under conditions whereby the inhibition or enhancement of activity can be detected, as described herein.
  • Various other objectives and advantages of the present invention will become apparent from the following detailed description.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • ⁇ as used herein when referring to a measurable value such as an amount of virus ⁇ e.g., titer), dose (e.g., an amount of a non-viral vector), time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • the present invention is based on the discovery of polypeptides of Mycoplasma pneumoniae and M. penetrans having the respective amino acid sequence described herein and encoded by the nucleic acids described herein and the identification of activities of these polypeptides and various fragments or "domains" of these polypeptides.
  • CARDS community acquired respiratory distress syndrome
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:2 (Sl isolate), an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:3 (JL isolate), an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:4 (RJLl isolate), an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO: 5 (L2 isolate), an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:1 (reference sequence), and/or an isolated polypeptide comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:6 (composite sequence), either individually or in any combination.
  • Sl isolate an isolated polypeptide comprising, consisting essentially of, and/
  • the present invention further provides biologically active fragments of the polypeptides of this invention, as well as antibodies that specifically bind the polypeptides and/or fragments of the polypeptides of this invention.
  • nucleotide sequences that encode the polypeptides and fragments of this invention.
  • the present invention provides an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO: 8 (Sl isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO: 10 (JL isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotide sequence of SEQ ID NO:11 (RJLl isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotides sequence of SEQ ID NO: 9 (L2 isolate), an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the nucleotides sequence of SEQ ID NO: 7 (reference sequence), and/or an isolated nucleic acid comprising, consisting essentially of, and/or consisting of the
  • nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO:2 (Sl isolate), a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO: 3 (JL isolate), a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence that encodes an amino acid sequence comprising, consisting essentially of, and/or consisting of the amino acid sequence or a biologically active fragment of the amino acid sequence of SEQ ID NO:4 (RJLl isolate), a nucleic acid comprising, consisting essentially
  • probes and primers for the detection of the nucleic acids of this invention including TTTTTAC ATATGCCAAATCCTGTT (SEQ ID NO:12; Primer 1), CGTTAAAGGATCCTCGCTAAAAGCGATC (SEQ ID N0:13; Primer 2), CTAGCCAAGCACTACGGACATTAGC (SEQ ID NO: 14; Primer 3), CGTAGTGCTTGGCTAGTAGATGCTGTT (SEQ ID NO:15; Primer 4), CCTGGTGTTGGCAACCATGGTTG (SEQ IDN0:16; Primer 5), GATCAACCATGGTTGCCAACACC (SEQ ID NO:17; Primer 6), AAGGTGGACTCCAATCAGGGCACG (SEQ ID NO: 18; Primer 7), CGTGCCCTGATTGGAGTCCACCTT (SEQ ID NO:19; Primer 8), GCGGTGTCATTTTCCACTTTTGG (SEQ ID NO:20; Primer 9), CCAAAAGTGGAAAA
  • a Mycoplasma penetrans toxin is provided.
  • a polypeptide of this invention as described herein include
  • the present invention provides a composition comprising an isolated polypeptide comprising, consisting essentially of and/or consisting of the amino acid sequence of SEQ ID NO: 79 (M penetrans toxin) or a biologically active fragment thereof and a pharmaceutically acceptable carrier.
  • a composition comprising, consisting essentially of and/or consisting of an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:77 (M penetrans coding sequence) and a pharmaceutically acceptable carrier.
  • an isolated nucleic acid comprising, consisting essentially of and/or consisting of the nucleotide sequence of SEQ ID NO:78 (UGG modified coding sequence of M. penetrans toxin).
  • nucleic acid can also be present in a composition comprising a pharmaceutically acceptable carrier.
  • the present invention provides a composition comprising, consisting essentially of and/or consisting of an antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 79 or an antigenic fragment thereof in a pharmaceutically acceptable carrier.
  • the present invention provides a method of diagnosing infection by Mycoplasma penetrans in a subject, comprising contacting a biological sample from the subject with a polypeptide having the amino acid sequence of SEQ ID NO:79 (M penetrans toxin) or an antigenic fragment thereof under conditions whereby an antigen/antibody complex can form and detecting formation of an antigen/antibody complex, thereby diagnosing infection by Mycoplasma penetrans in the subject.
  • a method of diagnosing infection by Mycoplasma penetrans in a subject comprising contacting a biological sample from the subject with an antibody that specifically binds a polypeptide having the amino acid sequence of SEQ ID NO: 79 under conditions whereby an antigen/antibody complex can form and detecting formation of an antigen/antibody complex, thereby diagnosing infection by Mycoplasma penetrans in the subject.
  • the present invention provides a method of diagnosing infection by Mycoplasma penetrans in a subject, comprising contacting a biological sample from the subject with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77 (M penetrans WT coding sequence) and/or a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 78 (M penetrans coding sequence with UGG modifications) under conditions whereby hybridization of nucleic acid molecules can occur to form a hybridization complex and detecting the hybridization complex, thereby diagnosing infection by Mycoplasma penetrans in the subject.
  • a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77 (M penetrans WT coding sequence) and/or a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 78 (M penetrans coding sequence with UGG modifications) under conditions whereby hybridization of nucleic acid molecules can occur to form a hybridization complex and detecting
  • kits for detecting M penetrans in a sample and/or for diagnosing an infection by Mycoplasma penetrans in a subject, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 79 or an antigenic fragment thereof, an antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 79, a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77 (WT), a nucleic acid comprising the nucleotide sequence of SEQ ID NO:78 (UGG modified) and any combination thereof.
  • Also provided herein is a method of detecting an M penetrans toxin or an antigenic fragment thereof in a sample, comprising: a) contacting the sample with an antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO:79 under conditions whereby an antigen/antibody complex can form; and b) detecting formation of the antigen/antibody complex, thereby detecting M. penetrans toxin or an antigenic fragment in the sample.
  • a method is further provided of detecting an antibody to M. penetrans toxin in a sample, comprising: a) contacting the sample with a polypeptide comprising the amino acid sequence of SEQ ID NO: 79 or an antigenic fragment thereof under conditions whereby an antigen/antibody complex can form; and b) detecting formation of the antigen/antibody complex, thereby detecting an antibody to M. penetrans toxin in the sample.
  • the present invention provides a method of detecting a nucleic acid comprising a nucleotide sequence encoding M. penetrans toxin in a sample, comprising: a) contacting the sample with an oligonucleotide comprising a nucleotide sequence that is complementary to the nucleotide sequence of SEQ ID NO:77 or SEQ ID NO:78, under conditions whereby nucleic acid hybridization can occur; and b) detecting nucleic acid hybridization, thereby detecting nucleic acid comprising the nucleotide sequence encoding M. penetrans toxin in the sample.
  • the present invention provides a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO: 79 or an immunogenic fragment thereof. Also provided is a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77 (WT) and/or a nucleic acid comprising the nucleotide sequence of SEQ ID NO:78 (UGG modified).
  • the present invention further provides a method of providing passive immunity to a subject, comprising administering to the subject an effective amount of an antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO:79.
  • the present invention provides a method of treating or preventing infection by Mycoplasma penetrans in a subject, comprising administering to the subject an effective amount of a polypeptide comprising the amino acid of SEQ ID NO: 79 or an immunogenic fragment thereof.
  • a method of treating or preventing infection by Mycoplasma penetrans in a subject comprising administering to the subject an effective amount of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77 (WT) and/or a nucleic acid comprising the nucleotide sequence of SEQ ID NO:78 (UGG modified). Further provided is a method of treating or preventing infection by Mycoplasma penetrans in a subject, comprising administering to the subject an effective amount of an antibody that specifically binds a polypeptide comprising the amino acid sequence of SEQ ID NO:79.
  • the present invention also provides a method of treating or preventing a detrimental immune response in a subject, comprising administering to the subject an effective amount of CARDS toxin and/or M. penetrans toxin or an active fragment thereof.
  • a method of enhancing an immunomodulating effect of a substance comprising combining the substance with CARDS toxin or an active fragment thereof.
  • Also provided herein is a method of treating and/or preventing Yersinia pestis infection in a subject, comprising administering to the subject an effective amount of CARDS toxin and/or M. penetrans toxin or an active fragment thereof and/or a nucleotide sequence encoding CARDS toxin and/or M. penetrans toxin or an active fragment thereof.
  • the methods of this invention can be used to treat an infection caused by any pathogen that elicits a ThI immune response, as described herein.
  • the CARDS TX described herein can elicit a predictable pattern of chemokines and cytokines that essentially holds 'in check' specific pathogens (e.g., Y. pestis). During this initial protective period, the CARDS TX-dependent response recruits cells of the immune system.
  • the present invention provides a method of treating or preventing a detrimental immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid encoding CARDS toxin or an active fragment thereof.
  • a detrimental immune response as described herein can be, for example, a detrimental immune response produced by a pathogenic agent that acts to inhibit the host's natural immune response in order to establish infection and/or cause disease.
  • pathogenic agents include Yersinia pestis, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Francisella tularensis and any other pathogens now known or later identified to elicit a ThI immune response.
  • pathogenic agents include Yersinia pestis, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Francisella tularensis and any other pathogens now known or later identified to elicit a ThI immune response.
  • a detrimental immune response include but are not limited to allergic reaction or allergy, immune-mediated inflammation of the organs (e.g., heart; central nervous system; kidney; liver), pathogen-induced immunopathology, autoimmune diseases and disorders (e.g., diabetes, SLE, MS), etc.
  • Also provided herein is a method of treating or preventing a detrimental immune response in a subject wherein the detrimental immune response is caused by CARDS toxin activity, comprising administering to the subject an effective amount of a substance that inhibits CARDS toxin activity.
  • a substance that inhibits CARDS toxin activity can be, but is not limited to a ligand (e.g., an antibody or antibody fragment) that specifically binds a CARDS toxin or active fragment thereof and/or a nucleic acid that inhibits transcription or translation of nucleic acid encoding a CARDS toxin or active fragment thereof (e.g., an antisense nucleic acid that binds a coding sequence of the A35R protein, an interfering RNA that inhibits or suppresses transcription and/or translation of the A35R protein, a ribozyme, etc.)
  • small molecules and other compounds and substances that inhibit the activity of CARDS toxin could be used in the methods of this invention.
  • the present invention provides a method of enhancing an immunomodulating effect of a substance, comprising combining the substance with a nucleic acid encoding CARDS toxin or an active fragment thereof.
  • a substance that has an immunomodulating effect can be but is not limited to steroids, immunosuppressive drugs, interferons, corticosteroids, azathioprine, cyclophosphamide, prednisone, methotrexate, rituximab, etc., as well as any other immunomodulating agent now known or later identified.
  • an enhancement of an immunomodulating effect of a substance would be 5 identified by comparison of an immunomodulating effect of the substance in a subject with and without the presence of the CARDS toxin or active fragment thereof or nucleic acid encoding the CARDS toxin or active fragment thereof.
  • an "immunomodulating effect” is any action or activity of a cell or tissue of the immune system. Such an immunomodulating effect can be positive or negative, an
  • the present invention provides a method of treating or preventing an autoimmune disorder in a subject, comprising administering to the subject an effective amount of CARDS toxin or an active fragment thereof and/or a nucleic acid encoding a CARDS toxin or active fragment thereof.
  • Nonlimiting examples of autoimmune disorders that can be treated and/or prevented by the methods of this invention include acute and chronic arthritis (e.g., rheumatoid arthritis or RA), multiple sclerosis (MS), diabetes (e.g., insulin dependent diabetes mellitus or IDDM), systemic lupus erythematosus (SLE), myasthenia gravis, Crohns' disease, regional enteritis, vasculitis, ulcerative colitis, Sjogren's syndrome, 0 ankylosing spondylitis, polymyositis and any other autoimmune disorder now known or later identified.
  • the methods of the present invention can further be employed to treat allergies, allergic reactions, and any other disease or disorder associated with an aberrant and/or undesirable immune response or reaction.
  • a method of reducing the likelihood of transplant 5 rejection (or increasing the likelihood of successful transplantation) in a transplant recipient comprising administering to the transplant recipient an effective amount of CARDS toxin or an active fragment thereof and/or administering to the transplant recipient an effective amount of a nucleic acid encoding a CARDS toxin or active fragment thereof.
  • the reduction in the likelihood of transplant rejection or increase in the likelihood of successful transplantation is in comparison to the likelihood of transplant rejection or likelihood of successful transplantation in a transplant recipient that did not receive a CARDS toxin or active fragment thereof or a nucleic acid encoding a CARDS toxin or active fragment thereof, as such likelihoods would be know and/or determined according to art-known standards.
  • the protein, active fragment and/or nucleic acid of these methods can be administered to the transplant recipient at any time relative to the transplantation (i.e., before, after and/or simultaneously, in any combination.
  • Also provided herein is a method of modulating (e.g., enhancing or inhibiting) an immune response in a subject, comprising administering to the subject an effective amount of CARDS toxin or an active fragment thereof and/or a nucleic acid encoding CARDS toxin or an active fragment thereof.
  • an enhancement is identified by comparison with an immune response in a subject that did not receive the protein, active fragment and/or nucleic acid of this invention.
  • methods provided herein for inhibiting an immune response such an inhibition is identified by comparison with an immune response in a subject that did not receive the protein, active fragment and/or nucleic acid of this invention.
  • Such comparative studies can be carried out according to well known protocols in the art for detecting and/or measuring any number of immune responses.
  • Nonlimiting examples of an immune response that can be enhanced by the methods of this invention include antibody response (e.g., protective antibody response; neutralizing antibody response), cytotoxic T cell response, T helper response, interleukin-2 (IL-2) production; and vaccine efficacy.
  • Additional embodiments of this invention provide a method of treating and/or preventing an obstructive airway disorder in a subject, comprising administering to the subject an effective amount of an inhibitor of CARDS toxin activity and/or an inhibitor of M. penetrans toxin activity.
  • An obstructive airway disease that can be treated and/or prevented according to the present invention can include, but is not limited to, asthma, allergy, pneumonia, tracheobronchitis, pharyngitis, croup, chronic obstructive pulmonary disease (COPD), sarcoidosis, interstitial pneumonia, interstitial pulmonary fibrosis, hypersensitivity pneumonitis, antiphospholipid syndrome, bronchiolitis and other chronic lung and allergic diseases and disorders, in any combination.
  • the obstructive airway disorder is asthma.
  • the present invention also contemplates the treatment or prevention of allergies and/or allergic reaction, caused by various allergens, which can include, but are not limited to, environmental allergens such as dust mite allergens; plant allergens such as pollen, including ragweed pollen; insect allergens such as bee and ant venom; and animal allergens such as cat dander, dog dander and animal saliva allergens.
  • environmental allergens such as dust mite allergens
  • plant allergens such as pollen, including ragweed pollen
  • insect allergens such as bee and ant venom
  • animal allergens such as cat dander, dog dander and animal saliva allergens.
  • An inhibitor of CARDS toxin activity can be an antibody or other ligand that Specifically binds CARDS toxin and inhibits CARDS toxin activity.
  • An inhibitor of CARDS toxin activity can also be a nucleic acid that inhibits the expression of a gene encoding the CARDS toxin and/or that inhibits transcription of CARDS toxin niRNA and/or that inhibits translation of the CARDS toxin from mRNA . (e.g., interfering RNA, antisense nucleic acid sequences, ribozymes, etc.).
  • An inhibitor of M. penetrans toxin activity can be an antibody or other ligand that specifically binds M. penetrans toxin and inhibits M. penetrans toxin activity.
  • An inhibitor of M. penetrans toxin activity can also be a nucleic acid that inhibits the expression of a gene encoding the M. penetrans toxin and/or that inhibits transcription of M. penetrans toxin mRNA and/or that inhibits translation of the M. penetrans toxin from mRNA (e.g., interfering RNA, antisense nucleic acid sequences, ribozymes, etc.).
  • the term "inhibit" or “inhibits” includes complete or partial reduction in an activity of the toxin described herein, as determined according to protocols known in the art and as described herein.
  • isolated as used herein means the nucleic acid or polypeptide of this invention is sufficiently free of contaminants or cell components with which nucleic acids or polypeptides normally occur. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the nucleic acid or polypeptide in a form in which it can be used therapeutically.
  • Epitope or "antigenic epitope” or “antigenic peptide” or “antigenic fragment” or “immunogenic fragment” as used herein means a specific amino acid sequence of limited length which, when present in the proper conformation, provides a reactive site for an antibody or T cell receptor.
  • the identification of epitopes on antigens can be carried out by immunology protocols that are well known in the art.
  • polypeptide or "protein” is used to describe a chain of amino acids that correspond to those encoded by a nucleic acid.
  • a polypeptide of this invention can be a peptide, which usually describes a chain of amino acids of from two to about 30 amino acids.
  • polypeptide as used herein also describes a chain of amino acids having more than 30 amino acids and can be a fragment or domain of a protein or a full length protein.
  • polypeptide can refer to a linear chain of amino acids or it can refer to a chain of amino acids that has been processed and folded into a functional protein.
  • polypeptides of the present invention are obtained by isolation and purification of the polypeptides from cells where they are produced naturally, by enzymatic (e.g., proteolytic) cleavage, and/or recombinantly by expression of nucleic acid encoding the polypeptides or fragments of this invention.
  • the polypeptides and/or fragments of this invention can also be obtained by chemical synthesis or other known protocols for producing polypeptides and fragments.
  • the amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right.
  • nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right. However, it is intended that the nucleic acids of this invention can be either single or double stranded (i.e., including the complementary nucleic acid).
  • a nucleic acid of this invention can be the complement of a nucleic acid described herein.
  • a "biologically active fragment" includes a polypeptide of this invention that comprises a sufficient number of amino acids to have one or more of the biological activities of the polypeptides of this invention.
  • Such biological activities can include, but are not limited to, in any combination, binding activity, translocating activity, immunogenic activity, ADP-ribosylating activity, and/or cytopathology inducing activity, as well as any other activity now known or later identified for the polypeptides and/or fragments of this invention.
  • a fragment of a polypeptide of this invention can be produced by methods well known and routine in the art. Fragments of this invention can be produced, for example, by enzymatic or other cleavage of naturally occurring peptides or polypeptides or by synthetic protocols that are well known.
  • Such fragments can be tested for one or more of the biological activities of this invention according to the methods described herein, which are routine methods for testing activities of polypeptides, and/or according to any art-known and routine methods for identifying such activities.
  • Such production and testing to identify biologically active fragments of the polypeptides described herein would be well within the scope of one of ordinary skill in the art and would be routine.
  • Fragments of the polypeptides of this invention are preferably at least about ten amino acids in length and retain one or more of the biological activities and/or the immunological activities of the CARDS toxin.
  • Examples of the fragments of this invention include, but are not intended to be limited to, the following fragments identified by the amino acid number as shown in the Sequence Listing for each of the isolates of SEQ ID NO:2 (SI isolate), SEQ ID NO:3 (JL isolate), SEQ ID NO:4 (RJLl isolate), SEQ ID NO:5 ( L2 isolate), SEQ ID NO:6 (composite sequence) and SEQ ID NO:1 (reference sequence): Amino acids 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60- 70, 70-80, 80-90, 90-100, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230,
  • fragment of this invention can be any amino acid sequence containing any combination of contiguous amino acids that are numbered in the Sequence Listing as amino acids 1 through 591 even if that combination is not specifically recited as an example herein. It is also understood that these fragments can be combined in any order or amount. For example, fragment 1-10 can be combined with fragment 10-20 to produce a fragment of amino acids 1-20. Also fragments can be present in multiple numbers and in any combination in a fragment of this invention. Thus, for example, fragment 1-150 can be combined with a second fragment 1-150 and/or combined with fragment 400-500 to produce a fragment of this invention.
  • Other exemplary fragments of this invention include the domains of the CARDS toxin described herein [e.g., domain 1 (N terminal 249 amino acids), domain 2 (256 amino acids) and domain 3 (247 amino acids at carboxy terminus)].
  • homology refers to a degree of similarity between two or more sequences. There may be partial homology or complete homology (i.e., identity).
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence can be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of low stringency, as this term is known in the art.
  • low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding can be tested by the use of a second target sequence that lacks even a partial degree of complementarity (e.g., less than about 30% identity). In the absence of non-specific binding, the probe will nothybridize to the second non-complementary target sequence.
  • hybridization refers to any process by which a first strand of nucleic acid binds with a second strand of nucleic acid through base pairing.
  • Nucleic acids encoding the polypeptides and/or fragments of this invention can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, primers and/or fragments of polynucleotides encoding the polypeptides and/or fragments of this invention and/or designed to detect and/or amplify the nucleic acids of this invention.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., C o t or R o t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells and/or nucleic acids have been fixed).
  • nucleotide sequence refers to a heteropolymer of nucleotides or the sequence of these nucleotides.
  • nucleic acid refers to a heteropolymer of nucleotides or the sequence of these nucleotides.
  • oligonucleotide refers to a heteropolymer of nucleotides or the sequence of these nucleotides.
  • nucleic acid segments are also used interchangeably herein to refer to a heteropolymer of nucleotides.
  • nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
  • Nucleic acids of this invention can comprise a nucleotide sequence that can be identical in sequence to the sequence which is naturally occurring or, due to the well-characterized degeneracy of the nucleic acid code, can include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids of this invention can comprise nucleotide sequences that can include codons which represent conservative substitutions of amino acids as are well known in the art, such that the biological activity of the resulting polypeptide and/or fragment is retained.
  • modifications of the toxins of this invention leading to specific amino acid substitutions can alter the toxin's ADP ribosylation, vacuolating and immunomodulatory activities and make the toxins better candidates as vaccine and/or immunomodulatory molecules.
  • Other amino acid substitutions as would be known to one of skill in the art can alter other structural and/or functional properties of the CARDS TX and M. penetrans toxin to improve their therapeutic capabilities.
  • the predictive adjuvant properties of CARDS TX and/or the M. penetrans toxin of this invention can be used as an adjuvant and/or in combination with other antigens (viral, bacterial, protozoal, fungal) to enhance the immunogenic properties of such antigens, e.g., for vaccine purposes. This would be most beneficial when the other antigen (i.e., not toxin) is poorly antigenic.
  • the toxin should be able to force the shifting of a Th2 response that is elicited by many environmental stresses, including specific infectious agents and allergens, to a ThI response that would benefit the well-being of the host (human and animal) by reducing inflammation and other clinical signs and symptoms.
  • probe or “primer” includes naturally occurring or recombinant or chemically synthesized single- and/or double-stranded nucleic acids. They can be labeled for detection by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes and primers of the present invention, their preparation and/or labeling are described in Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al. 1989. Current Protocols in Molecular Biology, John Wiley & Sons, New York N. Y., both of which are incorporated herein by reference in their entirety for these teachings.
  • stringent refers to hybridization conditions that are commonly understood in the art to define the conditions of the hybridization procedure. Stringency conditions can be low, high or medium, as those terms are commonly know in the art and well recognized by one of ordinary skill, hi various embodiments, stringent conditions can include, for example, highly stringent (i.e., high stringency) conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 0 C, and washing in O.lxSSC/0.1% SDS at 68°C), and/or moderately stringent (i.e., medium stringency) conditions (e.g., washing in 0.2xSSC/0.1% SDS at 42°C).
  • highly stringent i.e., high stringency
  • SDS sodium dodecyl sulfate
  • moderately stringent i.e., medium stringency
  • Amplification includes the production of multiple copies of a nucleic acid molecule and is generally carried out using polymerase chain reaction (PCR) and/or other amplification technologies as are well known in the art
  • PCR polymerase chain reaction
  • antibody includes intact immunoglobulin molecules as well as fragments thereof, such as Fab, F(ab')2, and Fc 5 which are capable of binding the epitopic determinant of an antigen (i.e., antigenic determinant).
  • Antibodies that bind the polypeptides of this invention are prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or fragment used to immunize an animal can be derived from enzymatic cleavage, recombinant expression, isolation from biological materials, synthesis, etc., and can be conjugated to a carrier protein, if desired.
  • Commonly used carriers that are chemically coupled to peptides and proteins for the production of antibody include, but are not limited to, bovine serum albumin, thyroglobulin and keyhole limpet hemocyanin.
  • the coupled peptide or protein is then used to immunize the animal (e.g., a mouse, rat, or rabbit).
  • the polypeptide or peptide antigens can also be administered with an adjuvant, as described herein and as otherwise known in the art.
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibody can be monoclonal or polyclonal and can be of any species of origin, including, for example, mouse, rat, rabbit, horse, goat, sheep or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403-11 (1989).
  • the antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Patent No. 4,474,893 or U.S. Patent No. 4,816,567.
  • the antibodies can also be chemically constructed according to the method disclosed in U.S. Patent No. 4,676,980.
  • the antibody can further be a single chain antibody or bispecific antibody.
  • Antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, and the corresponding fragments obtained from antibodies other than IgG.
  • Such fragments can be produced by known techniques.
  • F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse etal., (1989) Science 254:1275-1281).
  • Monoclonal antibodies can be produced in a hybridoma cell line according to the technique of Kohler and Milstein, (1975) Nature 265:495-97.
  • a solution containing the appropriate antigen can be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable medium and the supernatant screened for monoclonal antibodies having the desired specificity.
  • Monoclonal Fab fragments can be produced in bacterial cell such as E. coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, (1989) Science 246:1275-81.
  • Antibodies can also be obtained by phage display techniques known in the art or by immunizing a heterologous host with a cell containing an epitope of interest.
  • sample as used herein is used in its broadest sense.
  • a biological sample suspected of containing a polypeptide, fragment, antibody and/or nucleic acid of this invention can be any biological fluid, an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue print, and the like.
  • Effective amount refers to an amount of a compound or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic effect.
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art.
  • an "effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. ⁇ See, for example, Remington, The Science And Practice of Pharmacy (20th ed. 2000)).
  • a “pharmaceutically acceptable” component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue” when their risk outweighs the benefit provided by the composition.
  • Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.
  • a pharmaceutically acceptable carrier be a sterile carrier that is formulated for administration to or delivery into a subject of this invention.
  • Treating refers to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the condition, prevention or delay of the onset of the disorder, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art.
  • modulate “modulates” or “modulation” refers to enhancement
  • the term “enhancement,” “enhance,” “enhances,” or “enhancing” refers to an increase in the specified parameter (e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or more increase) and/or an increase in the specified parameter of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.
  • the specified parameter e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or more increase
  • an increase in the specified parameter of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99%
  • inhibitor refers to a decrease in the specified parameter (e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or more increase) and/or a decrease or reduction in the specified parameter of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.
  • the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or about 5%).
  • an "immunomodulatory molecule" of this invention can be, but is not limited to an immunostimulatory cytokine that can be, but is not limited to, GM/CSF, interleukin- 2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules.
  • an immunostimulatory cytokine can be, but is not limited to, GM/CSF, interleukin- 2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules.
  • an immunomodulatory molecule of this invention include the adjuvants of this invention, including, for example, SYNTEX adjuvant formulation 1 (SAF-I) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
  • SAF-I SYNTEX adjuvant formulation 1
  • Suitable adjuvants also include an aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.
  • aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin
  • alum aluminum hydroxide gel
  • aluminum phosphate aluminum phosphate
  • algannmulin algannmulin
  • Other adjuvants are well known in the art and include QS-21, Freund's adjuvant
  • N-acetyl-muraniyl-L-threonyl-D- isoglutamine thr-MDP
  • N-acetyl-normuramyl-L-alanyl-D-isoglutamine CGP 11637, referred to as nor-MDP
  • N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r- 2'-dipalmitoyl-sn -glycero-3-hydroxyphosphoryloxy)-ethylamine CGP 19835A, referred to as MTP-PE
  • RIBI which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80 emulsion.
  • Additional adjuvants can include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl. lipid A (3D- MPL) together with an aluminum salt.
  • An enhanced adjuvant system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in PCT publication number WO 94/00153 (the entire contents of which are incorporated herein by reference), or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in PCT publication number WO 96/33739 (the entire contents of which are incorporated herein by reference).
  • nucleic acid of this invention can include an adjuvant by comprising a nucleotide sequence encoding a A35R protein or active fragment thereof of this invention and a nucleotide sequence that provides an adjuvant function, such as CpG sequences.
  • CpG sequences, or motifs are well known in the art.
  • a subject of this invention includes any animal susceptible to infection by Mycoplasma pneumoniae and/or M. penetrans. Such a subject can be a mammal and in particular embodiments, is a human. As used herein, a "subject" or “subject in need thereof is a subject known to be, or suspected of being, infected with Mycoplasma pneumoniae and/or M. penetrans. A subject of this invention can also include a subject not previously known or suspected to be infected by Mycoplasma pneumoniae and/or M. penetrans or in need of treatment for Mycoplasma pneumoniae and/or M. penetrans infection.
  • a subject of this invention can be administered the compositions of this invention even if it is not known or suspected that the subject is infected with Mycoplasma pneumoniae and/or M. penetrans (e.g., prophylactically).
  • a subject of this invention is also a subject known or believed to be at risk of infection by Mycoplasma pneumoniae and/or M. penetrans.
  • the fragments and/or polypeptides of this invention can be fused with a "carrier" protein or peptide to produce a fusion protein.
  • the carrier protein or peptide can be fused to a polypeptide and/or fragment of this invention to increase the stability thereof (e.g., decrease the turnover rate) in the cell and/or subject.
  • exemplary carrier proteins include, but are not limited to, glutathione-S-transferase or maltose-binding protein.
  • the carrier protein or peptide can alternatively be a reporter protein.
  • the fusion protein can comprise a polypeptide and/or fragment of this invention and a reporter protein or peptide (e.g., Green Fluorescent Protein, ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, and the like) for easy detection of transformed cells and transgene expression.
  • a reporter protein or peptide e.g., Green Fluorescent Protein, ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, and the like
  • the fusion protein attached to the polypeptides and/or fragments and a carrier protein or peptide can be targeted to a subcellular compartment of interest, i.e., to affect the co-localization of the polypeptide and/or fragment.
  • Any suitable carrier protein as is well known in the art can be used to produce a fusion protein of this invention.
  • the polypeptides and/or fragments of the present invention can 1) be used in assays to determine the biological activity of other proteins or peptides; 2) be included in a panel of multiple proteins for high-throughput screening; 3) be used to raise antibodies or to elicit an immune response; 4) be used as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or receptor) in biological fluids; and 5) be used as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
  • EIA enzyme immunoassays
  • agglutination assays immunoblots (Western blot; dot/slot blot, etc.)
  • radioimmunoassays RIA
  • immunodiffusion assays chemiluminescence assays
  • antibody library screens expression arrays
  • enzyme-linked immunosorbent assays ELISA
  • radioimmunoassays RIA
  • immunoprecipitation Western blotting, competitive binding assays, immunofluorescence, immunohistochemical staining precipitation/flocculation assays and fluorescence-activated cell sorting (FACS).
  • nucleic acid sequences are well known in the art.
  • labeling and conjugation techniques are known in the art that are used in various nucleic acid detection and amplification assays.
  • Methods for producing labeled hybridization probes and/or PCR or other ligation primers for detecting and/or amplifying nucleic acid sequences can include, for example, oligolabeling, nick translation and end-labeling, as well as other well known methods.
  • nucleic acid sequences encoding the polypeptides of this invention, and/or any functional fragment thereof can be cloned into a plasmid or vector for detection and amplification.
  • reporter molecules or labels include, for example, radionuclides, enzymes, fluorescence agents, chemiluminescence agents and chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like as are well known in the art.
  • the present invention further includes isolated polypeptides, peptides, proteins, fragments, domains and/or nucleic acid molecules that are substantially equivalent to those described for this invention.
  • substantially equivalent can refer both to nucleic acid and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an undesirable adverse functional dissimilarity between reference and subject sequences.
  • this invention can include substantially equivalent sequences that have an adverse functional dissimilarity.
  • sequences having equivalent biological activity and equivalent expression characteristics are considered substantially equivalent.
  • the invention further provides homologs, as well as methods of obtaining homologs, of the polypeptides and/or fragments of this invention from other strains of Mycoplasma and/or other organisms.
  • an amino acid sequence or protein is defined as a homolog of a polypeptide or fragment of the present invention if it shares significant homology to one of the polypeptides and/or fragments of the present invention.
  • Significant homology means at least 75%, 80%, 85%, 90%, 95%, 98% and/or 100% homology with another amino acid sequence.
  • nucleic acids disclosed herein as a probe or as primers, and techniques such as PCR amplification and colony/plaque hybridization, one skilled in the art can identify homologs of the polypeptides and/or fragments of this invention in Mycoplasma and/or other organisms.
  • the present invention also provides an antibody that specifically binds the polypeptides and/or biologically active fragments of this invention, as well as a method of making an antibody specific for a polypeptide and/or fragment of this invention comprising: a) immunizing an animal with a polypeptide and/or fragment of this invention under conditions whereby the animal produces antibodies that specifically bind the polypeptide and/or fragment of this invention; and b) removing biological materials comprising the antibodies from the animal. Also provided herein is an antibody produced by the methods set forth herein.
  • Antibodies of this invention can be generated using methods that are well known in the art.
  • Such antibodies and immunoglobulin molecules of this invention can include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (e.g., scFv), Fab fragments, and fragments produced by a Fab expression library.
  • any animal known to produce antibodies can be immunized with a polypeptide, fragment and/or antigenic epitope of this invention.
  • Methods for immunization of animals to produce antibodies are well known in the art.
  • such methods can include subcutaneous or interperitoneal injection of the polypeptide, fragment and/or antigenic epitope of this invention.
  • the polypeptide, fragment or antigenic epitope that is used as an immunogen can be modified or administered in an adjuvant in order to increase antigenicity.
  • Methods of increasing the antigenicity of a protein or peptide include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ⁇ -galactosidase
  • various hosts including goats, rabbits, rats, mice, humans, and others, can be immunized by injection with the polypeptides and/or fragments of this invention, with or without a carrier protein.
  • various adjuvants may be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette- Guerin
  • Corynebacterium parvum are especially preferable.
  • Polypeptides, peptides and/or fragments of this invention used as antigens to produce the antibodies of this invention can have an amino acid sequence consisting of at least five amino acids and in certain embodiments, at least ten amino acids.
  • the antigen is identical to a portion of the amino acid sequence of the natural protein, and it can contain the entire amino acid sequence of a small, naturally-occurring molecule. Short stretches of the polypeptides and/or fragments of this invention can be fused with all or a fragment of another protein that acts as a carrier protein (e.g., keyhole limpet hemocyanin) and antibodies can be produced against the chimeric polypeptide or peptide.
  • a carrier protein e.g., keyhole limpet hemocyanin
  • Monoclonal antibodies to the polypeptides and/or fragments of this invention are prepared using any technique, which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. 1975. Nature 256:495-497; Kozbor et al. 1985. J Immunol. Methods 81:31- 42; Cote et al. 1983. Proc. Natl. Acad. Sci. 80:2026-2030; Cole et al. 1984. MoI. Cell Biol. 62:109-120).
  • spleen cells from the immunized animal are removed, fused with myeloma cells, and cultured in selective medium to become monoclonal antibody-producing hybridoma cells, according to techniques routine in the art. Any one of a number of methods well known in the art can be used to identify the hybridoma cell, which produces an antibody with the desired characteristics. These include screening the hybridomas by ELISA assay, Western blot analysis, or radioimmunoassay. Hybridomas secreting the desired antibodies are cloned and the class and subclass are identified using standard procedures known in the art.
  • antibody-containing serum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using any of the well known procedures as described herein.
  • the present invention further provides antibodies of this invention in detectably labeled form.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescence labels (such as FITC or rhodamine, etc.), paramagnetic atoms, gold beads, etc.
  • affinity labels such as biotin, avidin, etc.
  • enzymatic labels such as horseradish peroxidase, alkaline phosphatase, etc.
  • fluorescence labels such as FITC or rhodamine, etc.
  • paramagnetic atoms gold beads, etc.
  • the present invention further provides the above- described antibodies immobilized on a solid support (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene).
  • a solid support e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene.
  • solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., Handbook of Experimental Immunology 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986)).
  • Antibodies can likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S, 125 1, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescence labels (e.g., fluorescein) in accordance with known techniques. Determination of the formation of an antibody/antigen complex in the methods of this invention can be by detection of, for example, precipitation, agglutination, flocculation, radioactivity, color development or change, fluorescence, luminescence, etc., as is well know in the art.
  • radiolabels e.g., 35 S, 125 1, 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescence labels e.g., fluorescein
  • Antibody fragments that specifically bind the polypeptides and/or fragments of this invention can also be generated.
  • fragments include, but are not limited to, the F(ab ') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab ') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. 1989. Science 254:1275-1281).
  • immunoassays can be used for screening to identify antibodies having the desired specificity for the proteins and peptides of this invention.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between an antigen and its specific antibody (e.g., antigen/antibody complex formation).
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on the proteins or peptides of this invention can be used, as well as a competitive binding assay.
  • kits for detection of the polypeptides and/or fragments of this invention in a sample can comprise one or more antibodies of this invention, along with suitable buffers, wash solutions and/or other reagents for the detection of antibody/antigen complex formation.
  • a kit of this invention can comprise a polypeptide, an antigenic peptide of the polypeptide of this invention, a fragment of this invention and/or an antigenic peptide of a fragment of this invention, along with suitable buffers, wash solutions and/or other reagents for the detection of antibody/antigen complex formation.
  • the present invention further provides a kit for the detection of nucleic acid encoding the polypeptides and/or fragments of this invention.
  • the kit can comprise one or more nucleic acids of this invention, along with suitable buffers, wash solutions and/or other reagents for the detection of hybridization complex formation.
  • kits of this invention can comprise one or more containers and/or receptacles to hold the reagents (e.g., antibodies, antigens, nucleic acids) of the kit, along with appropriate buffers and/or wash solutions and directions for using the kit, as would be well known in the art.
  • reagents e.g., antibodies, antigens, nucleic acids
  • kits can further comprise adjuvants and/or other irnmunostimulatory or immunomodulating agents, as are well known in the art.
  • the nucleic acids encoding the polypeptides and/or fragments of this invention can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations.
  • the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding a polypeptide and/or biologically active fragment of this invention.
  • the present invention further provides a vector comprising a nucleic acid encoding a polypeptide and/or fragment of this invention.
  • the vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art.
  • the expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols.
  • the expression vector can comprise viral nucleic acid including, but not limited to, vaccinia virus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid.
  • the nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle, which can be taken up by a cell via receptor-mediated or other type of endocytosis.
  • the nucleic acid of this invention can be in a cell, which can be a cell expressing the nucleic acid whereby a polypeptide and/or biologically active fragment of this invention is produced in the cell.
  • the vector of this invention can be in a cell, which can be a cell expressing the nucleic acid of the vector whereby a polypeptide and/or biologically active fragment of this invention is produced in the cell.
  • the nucleic acids and/or vectors of this invention can be present in a host animal (e.g., a transgenic animal), which expresses the nucleic acids of this invention and produces the polypeptides and/or fragments of this invention.
  • the nucleic acid encoding the polypeptide and/or fragment of this invention can be any nucleic acid that functionally encodes the polypeptides and/or fragments of this invention.
  • the nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc.
  • a nucleic acid encoding a selected polypeptide and/or fragment can readily be determined based upon the genetic code for the amino acid sequence of the selected polypeptide and/or fragment and many nucleic acids will encode any selected polypeptide and/or fragment. Modifications in the nucleic acid sequence encoding the polypeptide and/or fragment are also contemplated.
  • nucleic acid of this invention can be generated by means standard in the art, such as by recombinant nucleic acid techniques and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
  • the present invention provides a Dl domain of CARDS Toxin comprising, consisting essentially of and/or consisting of the amino acid sequence of SEQ ID NO:69 and/or SEQ ID NO:75, a D2 domain of CARDS Toxin comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 70, and/or a D3 domain of CARDS Toxin comprising, consisting essentially of, and/or consisting of the amino acid sequence of SEQ ID NO:71, in any combination.
  • an isolated nucleic acid encoding the amino acid sequence of the domains Dl, D2 and D3 of this invention can comprise, consist of and/or consist essentially of the nucleotide sequence of SEQ ID NO:74.
  • domain peptides can be used as antigens for the production of antibodies, which can be polyclonal and/or monoclonal, according to well known protocols.
  • the domain peptides and antibodies can be used in the methods described herein for the detection of M. pneumoniae antibodies and proteins and/or for diagnosis of M. pneumoniae infection, as well as in therapeutic methods to treat M. pneumoniae infection and related diseases as described herein.
  • the present invention further provides a method of producing a polypeptide and/or biologically active fragment according to the methods set forth in the Examples provided herein, and as are well known in the art for polypeptide synthesis.
  • a nucleic acid encoding the polypeptides and/or fragments of this invention can be synthesized according to standard nucleic acid synthesis protocols and the nucleic acid can be expressed according to methods well known for expression of nucleic acid.
  • the resulting polypeptide and/or fragment can then be removed from the expression system by standard isolation and purification procedures and tested for any of the various biological activities described herein according to methods as taught herein as well as methods routine in the art.
  • the present invention also provides a method for producing the polypeptides and/or biologically active fragments of this invention comprising producing the cells of this invention which contain the nucleic acids or vectors of this invention as exogenous nucleic acid; culturing the cells under conditions whereby the exogenous nucleic acid in the cell can be expressed and the encoded polypeptide and/or fragment can be produced; and isolating the polypeptide and/or fragment from the cell.
  • the polypeptides and/or fragments of this invention can be produced in quantity in vitro in either prokaryotic or eukaryotic expression systems as are well known in the art.
  • E. coli ⁇ Escherichia col ⁇ E. coli ⁇ Escherichia col ⁇ expression vectors known to one of ordinary skill in the art useful for the expression of nucleic acid that encodes polypeptides.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species.
  • These prokaryotic hosts can support expression vectors that will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters can be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences for example, for initiating and completing transcription and translation.
  • an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the polypeptide.
  • the carboxy-terminal extension of the polypeptide can be removed using standard oligonucleotide mutagenesis procedures.
  • the nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences.
  • selection markers e.g., tetracycline resistance or hygromycin resistance
  • yeast expression system can be used for eukaryotic system expression. There are several advantages to yeast expression systems. First, evidence exists that polypeptides produced in a yeast expression system exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast expression systems.
  • the Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFa-I gene) is routinely used to direct protein secretion from yeast.
  • the leader region of pre-pro-alpha-factor contains a signal peptide and a pro- segment, which includes a recognition sequence for a yeast protease encoded by the KEX2 gene. This enzyme cleaves the precursor protein on the carboxyl side of a Lys- Arg dipeptide cleavage-signal sequence.
  • the polypeptide coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region.
  • This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
  • the coding sequence is followed by a translation termination codon, which is followed by transcription termination signals.
  • the coding sequence of interest can be fused to a second polypeptide coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification of the resulting fusion polypeptide by affinity chromatography.
  • the insertion of protease cleavage sites to separate the components of the fusion polypeptide is applicable to constructs used for expression in yeast. Efficient post-translational glycosylation and expression of recombinant polypeptides can also be achieved in Baculovirus systems in insect cells, as are well known in the art.
  • the peptides, polypeptides and/or fragments of this invention can be expressed in mammalian cells.
  • Mammalian cells permit the expression of peptides and polypeptides in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein.
  • Vectors useful for the expression of peptides and polypeptides in mammalian cells are characterized by insertion of the coding sequence between a strong (e.g., viral) promoter and a polyadenylation signal.
  • the vectors can contain genes conferring either, e.g., gentamicin or methotrexate resistance, for use as selectable markers.
  • the coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector. Presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the polypeptide or fragment coding sequence can be confirmed by Southern and Northern blot analysis, respectively.
  • suitable host cell lines capable of producing exogenous polypeptides have been developed in the art and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells and the like. Expression vectors for these cells can include expression control sequences, as described above.
  • nucleic acids and/or vectors of this invention can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cell hosts.
  • polypeptides, fragments, nucleic acids, vectors and cells of this invention can be present in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected polypeptide, fragment, nucleic acid, vector or cell without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • compositions of this invention can comprise a pharmaceutically acceptable carrier and a suitable adjuvant.
  • suitable adjuvant describes an adjuvant capable of being combined with the polypeptide and/or fragment and/or nucleic acid of this invention to further enhance an immune response without deleterious effect on the subject or the cell of the subject.
  • a suitable adjuvant can be, but is not limited to, MONTANIDE ISA51 (Seppic, Inc., Fairfield, NJ), SYNTEX adjuvant formulation 1 (SAF-I), composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
  • SAF-I SYNTEX adjuvant formulation 1
  • Suitable adjuvants include QS-21, Freund's adjuvant (complete and incomplete), alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L- alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalen
  • compositions of the present invention can also include other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. It is contemplated that the above-described compositions of this invention can be administered to a subject or to a cell of a subject to impart a therapeutic benefit.
  • the present invention further provides a method of producing an immune response in a subject, comprising administering to the subject or to a cell of the subject an effective amount of a polypeptide and/or biologically active fragment of this invention and/or a nucleic acid comprising a nucleotide sequence encoding a polypeptide and/or biologically active fragment of this invention.
  • the cell of the subject can be in vivo or ex vivo and can be, but is not limited to a CD 8+ T lymphocyte (e.g., a cytotoxic T lymphocyte) or an MHC I-expressing antigen presenting cell, such as a dendritic cell, a macrophage and/or a monocyte.
  • Detection of an immune response in the subject or in the cells of the subject can be carried out according to methods standard in the art for detecting a humoral and/or cellular immune response.
  • the present invention provides a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a polypeptide and/of fragment of this invention. Also provided herein is a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid and/or vector of this invention.
  • the present invention provides a method of providing passive immunity to a subject, comprising administering to the subject an effective amount of an antibody of this invention to the subject.
  • compositions of this invention can also be employed as a therapeutic and/or prophylactic formulation and administered to a subject in need thereof.
  • the present invention provides a method of treating or preventing infection or intoxication by Mycoplasma pneumoniae and/or M. penetrans in a subject, comprising administering to the subject an effective amount of a polypeptide and/or fragment of this invention, a nucleic acid and/or vector of this invention, and/or an antibody of this invention.
  • the present invention provides a method of treating or preventing infection or intoxication caused by Mycoplasma pneumoniae and/or M. penetrans in a subject comprising contacting an immune cell of the subject with any of the polypeptides, fragments, nucleic acids, vectors and/or antibodies of this invention.
  • the cell can be in vivo or ex vivo and can be, for example, a CD8 + T cell which is contacted with the polypeptide and/or fragment of this invention in the presence of a class I MHC molecule, which can be a soluble molecule or it can be present on the surface of a cell which expresses class I MHC molecules.
  • the cell can also be an antigen presenting cell or other class I MHC-expressing cell which can be contacted with the nucleic acids and/or vectors of this invention under conditions whereby the nucleic acid or vector is introduced into the cell by standard methods for uptake of nucleic acid and vectors.
  • the nucleic acid encoding the polypeptide and/or fragment of this invention is then expressed and the polypeptide and/or fragment product is processed within the antigen presenting cell or other MHC I-expressing cell and presented on the cell surface as an MHC I/antigen complex.
  • the antigen presenting cell or other class I MHC-expressing cell is then contacted with an immune cell of the subject which binds the class I MHC /antigen complex and elicits an immune response which treats or prevents Mycoplasma pneumoniae and/or M. penetrans infection in the subject.
  • compositions of this invention are administered to a subject or to a cell of a subject
  • methods can further comprise the step of administering a suitable adjuvant to the subject or to a cell of the subject.
  • the adjuvant can be in the composition of this invention or the adjuvant can be in a separate composition comprising the suitable adjuvant and a pharmaceutically acceptable carrier.
  • the adjuvant can be administered prior to, simultaneous with, or after administration of the composition containing any of the polypeptides, fragments, nucleic acids and/or vectors of this invention.
  • QS-21 similar to alum, complete Freund's adjuvant, SAF, etc.
  • QS-21 can be administered within days/weeks/hours (before or after) of administration of the composition of this invention.
  • the effectiveness of an adjuvant can be determined by measuring the immune response directed against the polypeptide and/or fragment of this invention with and without the adjuvant, using standard procedures, as described in the Examples herein and as are well known in the art.
  • the subject of this invention can be any subject in need of the immune response of this invention and/or in need of treatment for or prevention from Mycoplasma pneumoniae and/or M. penetrans infection, as well as any subject in whom it is desirable to induce an immune response to Mycoplasma pneumoniae and/or M. penetrans.
  • Symptoms of Mycoplasma pneumoniae infection can include tracheobronchitis and pneumonia with extrapulmonary pathologies, such as neurologic, cardiac, gastrointestinal, dermatologic, renal and joint complications.
  • a range of serological (elevated IgM and IgG seroconversion) assays and PCR detection can be used for diagnosing M. pneumoniae infection.
  • Appropriate treatment can lead to resolution of respiratory symptoms such as decreased fever and cough, complete recovery of respiratory function including normal lung radiogram, and normal levels of tissue enzymes and CSF analysis. Also, decreased levels of M. pneumoniae and/or M penetrans cells, antigens and nucleic acids in blood, sputum, bronchial lavage should identify an effective treatment. Symptoms of M. penetrans infection can include AIDS progression (multiple tissue involvement and clinical manifestations), urethritis, acute and chronic kidney infections, respiratory disease, antiphospholipid syndrome, etc. as would be known in the art. Effective treatment would result in, for example, improved immune function, reduction or elimination of tissue-associated pathologies, reduction of fever and reduction of tissue inflammation.
  • M. pneumoniae infection can include infected individuals coughing, sneezing and transmitting aerosols containing M. pneumoniae.
  • the transmission rate is very high, which is why M. pneumoniae is such a common cause of community acquired pneumonia.
  • Highest targets of infection are children, especially 5-9 years old and adults between ages 25-40, although infection can occur among all 'healthy' individuals.
  • a subject for whom the methods of this invention would be indicated for preventing M. pneumoniae infection can, in some embodiments, be a child or young adult.
  • compositions of this invention can be administered to a cell of a subject or to a subject either in vivo or ex vivo.
  • the compositions of this invention can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, subcutaneous injection, transdermally, extracorporeally, topically or the like.
  • the compositions of this invention can be pulsed onto dendritic cells, which are isolated or grown from a subject's cells, according to methods well known in the art, or onto bulk peripheral blood mononuclear cells (PBMC) or various cell subfractions thereof from a subject.
  • PBMC peripheral blood mononuclear cells
  • composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition of this invention. However, effective amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • between about 50-1000 nM and more preferably, between about 100-500 nM of a polypeptide and/or biologically active fragment of this invention can be administered subcutaneously and can be in an adjuvant, at one to three hour/day/week intervals until an evaluation of the subject's clinical parameters indicate that the subject is not infected by M. pneumoniae and/or M. penetrans and/or the subject demonstrates the desired immunological response.
  • a polypeptide and/or fragment of this invention can be pulsed onto dendritic cells at a concentration of between about 10-100 ⁇ M and the dendritic cells can be administered to the subject intravenously at the same time intervals.
  • the treatment can be continued or resumed if the subject's clinical parameters indicate that M. pneumoniae and/or M. penetrans infection is present and can be maintained until the infection is no longer detected by these parameters and/or until the desired immunological response is achieved.
  • cells or tissues can be removed and maintained outside the subject's body according to standard protocols well known in the art.
  • polypeptides and/or biologically active fragments of this invention can be introduced into the cells via known mechanisms for uptake of polypeptides into cells (e.g., phagocytosis, pulsing onto class I MHC-expressing cells, liposomes, etc.).
  • the cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or transplanted 2006/012266
  • Standard methods are known for transplantation or infusion of various cells into a subject.
  • the nucleic acids and vectors of this invention can also be administered to a cell of the subject either in vivo or ex vivo.
  • the cell can be any cell that can take up and express exogenous nucleic acid and produce the polypeptides and/or fragments of this invention.
  • the polypeptides and/or fragments of this invention can be produced by a cell that secretes them, whereby the polypeptide and/or fragment is produced and secreted and then taken up and subsequently processed by an antigen presenting cell or other class I MHC-expressing cell and presented to the immune system for induction of an immune response.
  • nucleic acids and/or vectors of this invention can be directly introduced into an antigen presenting cell and/or other class I MHC-expressing cell in which the polypeptide and/or fragment is produced and processed directly and presented to the immune system on the cell surface.
  • nucleic acids and vectors of this invention can be administered orally, intranasally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, hi the methods described herein which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the polypeptides and/or fragments of this invention.
  • the vector can be a commercially available preparation or can be constructed in the laboratory according to methods well known in the art.
  • Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO- BRL 5 Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
  • vector delivery can be via a viral system, such as a retroviral vector system, which can package a recombinant retroviral genome.
  • the recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding the polypeptide and/or fragment of this invention.
  • the exact method of introducing the exogenous nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors.
  • adenoviral vectors alphaviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, pseudotyped retroviral vectors and vaccinia viral vectors, as well as any other viral vectors now known or developed in the future.
  • Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms. This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
  • the dosage for administration of adenovirus to humans can range from about 10 7 to 10 9 plaque forming units (pfu) per injection, but can be as high as 10 12 , 10 15 and/or 10 20 pfu per injection.
  • a subject will receive a single injection. If additional injections are necessary, they can be repeated at daily/weekly/monthly intervals for an indefinite period and/or until the efficacy of the treatment has been established.
  • the efficacy of treatment can be determined by evaluating the symptoms and clinical parameters described herein and/or by detecting a desired immunological response.
  • nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every nucleic acid or vector. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art.
  • the nucleic acids and vectors of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes.
  • the transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
  • Parenteral administration of the peptides, polypeptides, nucleic acids and/or vectors of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • parenteral administration includes intradermal, intranasal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes, as well as a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein in its entirety.
  • the efficacy of treating or preventing Mycoplasma pneumoniae and/or M. penetrans infection by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters, as would be well known to one of skill in the art.
  • compositions of the present invention can be used in diagnostic and therapeutic applications.
  • the present invention provides a method of detecting the presence of a polypeptide and/or fragment of this invention in a sample, comprising contacting the sample with an antibody of this invention under conditions whereby an antigen/antibody complex can form and detecting formation of an antigen/antibody complex, thereby detecting the presence of a Mycoplasma pneumoniae polypeptide and/or fragment and/or a Mycoplasma penetrans toxin of fragment thereof of this invention in the sample.
  • the present invention provides a method of detecting the presence of an antibody of this invention in a sample, comprising contacting the sample with a polypeptide and/or fragment of this invention under conditions whereby an antigen/antibody complex can form and detecting formation of an antigen/antibody complex, thereby detecting the presence of a Mycoplasma pneumoniae and/or M. penetrans antibody of this invention in the sample.
  • the present invention provides a method of detecting the presence of the CARDS toxin of this invention in a sample (e.g., a biological sample from a subject or a food or water sample or other sample that could contain CARDS toxin) and/or a subject and/or diagnosing infection by M. pneumoniae in a subject, comprising contacting the sample with surfactant protein A (SP-A) under conditions whereby a toxin/SP-A complex can form; and detecting formation of the toxin/SP-A complex, thereby detecting the presence of CARDS toxin in a sample and/or diagnosing infection by M. pneumoniae in a subject.
  • a sample e.g., a biological sample from a subject or a food or water sample or other sample that could contain CARDS toxin
  • SP-A surfactant protein A
  • the sample of this invention can be any sample in which Mycoplasma pneumoniae CARDS toxin and/or M. penetrans toxin can be present.
  • the sample can be a body fluid, cells or tissue that can contain Mycoplasma pneumoniae toxin and/or M.
  • penetrans toxin including but not limited to, blood, serum, plasma, saliva, sputum, bronchoalveolar lavage, urine, semen, joint fluid, cerebrospinal fluid and cells, fluids and/or tissue from all organs to which CARDS toxin can disseminate including lung, liver, heart, brain, kidney, spleen, muscle, etc.
  • a sample of this invention can also be a sample in which a polypeptide, nucleic acid and/or fragment of this invention can be detected, such as water, soil, effluent, food, feedstuff, air, swabs of surfaces, T/US2006/012266
  • the present invention provides a method of diagnosing Mycoplasma pneumoniae and/or M. penetrans infection in a subject comprising contacting a biological sample from the subject with a polypeptide and/or fragment of this invention under conditions whereby an antigen/antibody complex can form; and detecting formation of an antigen/antibody complex, thereby diagnosing Mycoplasma pneumoniae and/or M. penetrans infection in the subject.
  • a method of diagnosing Mycoplasma pneumoniae and/or M. penetrans infection in a subject comprising contacting a biological sample from the subject with an antibody of this invention under conditions whereby an antigen/antibody complex can form; and detecting formation of an antigen/antibody complex, thereby diagnosing Mycoplasma pneumoniae and/or M. penetrans infection in the subject.
  • the present invention provides a method of diagnosing infection by Mycoplasma pneumoniae and/or M. penetrans in a subject, comprising contacting a biological sample from the subject with the nucleic acid of this invention under conditions whereby hybridization of nucleic acid molecules can occur and detecting a hybridization complex, thereby diagnosing infection by Mycoplasma pneumoniae and/or M. penetrans in the subject.
  • the present invention provides a method of identifying a subject infected with Mycoplasma pneumoniae and/or M. penefrans as having a poor prognosis, comprising: a) establishing a correlation between the presence of and/or an amount of a polypeptide, fragment, nucleic acid and/or antibody of this invention in a sample of test subjects infected with Mycoplasma pneumoniae and/or M.
  • a biological sample from the subject the presence of and/or an amount of the polypeptide, fragment, nucleic acid and/or antibody of this invention correlated with a poor prognosis, thereby identifying the subject infected with Mycoplasma pneumoniae and/or M. penetrans as having a poor prognosis.
  • a correlation can be made between a level of antibodies to the CARDS toxin and/or antibodies to M. penetrans and a degree of respiratory and/or pulmonary dysfunction indicative of a poor prognosis.
  • the present invention also provides various screening assays that employ the polypeptides, fragments and/or nucleic acids of this invention.
  • a method of identifying a substance having the ability to inhibit or enhance the binding activity of a polypeptide and/or biologically active fragment of this invention comprising contacting the substance with the CARDS protein or a biologically active fragment thereof under conditions whereby binding can occur and detecting a decrease or increase in the amount of binding in the presence of the substance as compared to a control amount of binding in the absence of the substance, thereby identifying a substance having the ability to inhibit or enhance the binding activity of the CARDS toxin.
  • Inhibition or enhancement of binding activity can be detected by any of a variety of art-recognized methods for evaluating binding activity.
  • the substance to be tested and the CARDS polypeptide and/or fragment can be contacted in the presence of target cells or a target substrate (e.g., surfactant protein A; SP-A) known to bind the polypeptide or fragment.
  • target substrate e.g., surfactant protein A; SP-A
  • the amount of binding of polypeptide or fragment to the cells or the substrate in the presence of the substance and the amount of binding of polypeptide or fragment to the cells or the substrate in the absence of the substance is determined and a decrease or increase in the amount of binding in the presence of the substance identifies the substance as having the ability to inhibit or enhance binding.
  • binding of polypeptide and/or fragment to target cells or a target substrate can be measured by attaching a detectable moiety to the polypeptide or fragment (e.g., a fluorescence moiety, histochemically detectable moiety, radioactive moiety, etc.).
  • a detectable moiety e.g., a fluorescence moiety, histochemically detectable moiety, radioactive moiety, etc.
  • the amount of detectable moiety can be measured in the presence and absence of the substance to be tested and the amounts can be compared to determine inhibition or enhancement.
  • Binding activity can also be determined by comparing the amount of cytopathology observed in a monolayer of target cells in the presence and absence of the substance to be tested.
  • Target cells that can be used in such a binding assay include, but are not limited to, Chinese hamster ovary (CHO) cells, Hep2 cells, human lung and kidney epithelial and fibroblast cells, and any other mammalian cells that exhibit sensitivity to CARDS toxin now known or later identified.
  • CHO Chinese hamster ovary
  • Hep2 cells Hep2 cells
  • human lung and kidney epithelial and fibroblast cells include, but are not limited to, Chinese hamster ovary (CHO) cells, Hep2 cells, human lung and kidney epithelial and fibroblast cells, and any other mammalian cells that exhibit sensitivity to CARDS toxin now known or later identified.
  • the present invention provides a method of identifying a substance having the ability to inhibit or enhance the translocating activity of a polypeptide and/or a biologically active fragment of this invention, comprising contacting the substance with the polypeptide of this invention and/or a biologically active fragment thereof under conditions whereby translocation activity can occur and detecting a decrease or increase in the amount of translocation activity in the presence of the substance as compared to a control amount of translocation activity in the absence of the substance, thereby identifying a substance having the ability to inhibit or enhance the translocating activity of the CARDS toxin.
  • Inhibition or enhancement of translocating activity can be detected by any of a variety of art-recognized methods for evaluating translocating activity.
  • the substance to be tested and the CARDS polypeptide and/or fragment can be contacted in the presence of target cells known to translocate the CARDS toxin.
  • the amount of translocation of polypeptide or fragment into the cells in the presence of the substance and the amount of translocation of polypeptide or fragment into the cells in the absence of the substance is determined and a decrease or increase in the amount of translocation in the presence of the substance identifies the substance as having the ability to inhibit or enhance translocation of the CARDS toxin.
  • Translocation of polypeptide and/or fragment into target cells can be measured by attaching a detectable moiety to the polypeptide or fragment (e.g., a fluorescence moiety, histochemically detectable moiety, radioactive moiety, etc.).
  • the amount of translocated detectable moiety can be measured in the presence and absence of the substance to be tested and the amounts can be compared to determine inhibition or enhancement of translocation.
  • Translocation activity can also be determined by comparing the amount of cytopathology observed in a monolayer of target cells in the presence and absence of the substance to be tested.
  • Target cells that can be used in such a translocation assay include, but are not limited to, Chinese hamster ovary (CHO) cells, etc.
  • a method of identifying a substance having the ability to enhance or inhibit the immunogenic activity of the CARDS toxin of this invention and/or a biologically active fragment thereof comprising contacting the substance with the CARDS toxin or an immunogenic fragment thereof under conditions whereby a measurable immune response can be elicited and detecting an increase or decrease in the amount of immune response in the presence of the substance, as compared to a control amount of immune response in the absence of the substance, thereby identifying a substance having the ability to enhance or inhibit immunogenic activity of the CARDS toxin.
  • Assays to detect and measure immune responses are well known in the art and can be employed to detect either humoral or cellular immune responses.
  • the present invention provides a method of identifying a substance having the ability to inhibit or enhance the ADP-ribosylating activity of the CARDS toxin of this invention and/or biologically active fragments thereof, comprising contacting the substance with the CARDS toxin or biologically active fragment thereof under conditions whereby ADP ribosylation can occur and detecting a decrease or increase in the amount of ADP ribosylation in the presence of the substance as compared to a control amount of ADP ribosylation in the absence of the substance, thereby identifying a substance having the ability to inhibit or enhance the ADP ribosylating activity of the CARDS toxin.
  • Methods for detecting ADP ribosylating activity are well known in the art and are described, for example, in the Examples section provided herein.
  • a method of identifying a substance having the ability to inhibit or enhance the cytopathology-inducing activity of the CARDS toxin of this invention and/or a biologically active fragment thereof comprising contacting the substance with the CARDS toxin or biologically active fragment thereof under conditions whereby cytopathology (e.g., changes in cell morphology, monolayer characteristics, etc.) of target cells can be induced and detecting a decrease or increase in the amount of cytopathology in the presence of the substance, as compared to a control amount of cytopathology in the absence of the substance, thereby identifying a substance having the ability to inhibit or enhance the cytopathology-inducing activity of the CARDS toxin or biologically active fragment thereof.
  • cytopathology e.g., changes in cell morphology, monolayer characteristics, etc.
  • Methods of detecting cytopathology of cells are well known in the art and are described, for example, in the Examples section herein. Additionally provided is a method of identifying a substance having the ability to inhibit the binding activity of the M. penetrans toxin, comprising contacting the substance with the toxin or a biologically active fragment thereof under conditions whereby binding can occur and detecting a decrease in the amount of binding in the presence of the substance as compared to a control amount of binding in the absence of the substance, thereby identifying a substance having the ability to inhibit the binding activity of the M. penetrans toxin.
  • a method is also provided herein of identifying a substance having the ability to enhance the immunogenic activity of the M. penetrans toxin, comprising contacting the substance with the toxin or an immunogenic fragment thereof under conditions whereby a measurable immune response can be elicited and detecting in increase in the amount of immune response in the presence of the substance, as compared to a control amount of immune response in the absence of the substance, thereby identifying a substance having the ability to enhance immunogenic activity of the M. penetrans toxin. Also provided herein is a method of identifying a substance having the ability to inhibit the ADP-ribosylating activity of the M.
  • penetrans toxin comprising contacting the substance with the toxin or biologically active fragment thereof under conditions whereby ADP ribosylation can occur and detecting a decrease in the amount of ADP ribosylation in the presence of the substance as compared to a control amount of ADP ribosylation in the absence of the substance, thereby identifying a substance having the ability to inhibit the ADP ribosylating activity of the M. penetrans toxin.
  • the present invention provides a method of identifying a substance having the ability to inhibit the cytopathology-inducing activity of the M. penetrans toxin, comprising contacting the substance with the toxin or biologically active fragment thereof under conditions whereby cytopathology of target cells can be induced and detecting a decrease in the amount of cytopathology in the presence of the substance, as compared to a control amount of cytopathology in the absence of the substance, thereby identifying a substance having the ability to inhibit the cytopathology-inducing activity of the M. penetrans toxin or biologically active fragment thereof.
  • Substances identified in the screening assays of this invention to have the ability to inhibit or enhance various of the activities of the polypeptides and/or fragments of this invention can be employed in methods of diagnosing M. pneumoniae and/or M. penetrans infection, as well as in methods of treating and/or preventing M. pneumoniae and/or M. penetrans infection.
  • such substances can be present in a pharmaceutically acceptable carrier for administration to a subject and an effective amount of the substance can be administered to a subject to treat and/or prevent infection by Mycoplasma pneumoniae and/or M. penetrans. It is also contemplated that the present invention includes methods of screening
  • Mycoplasma pneumoniae and/or M. penetrans cultures for mutants defective in one or more of the biological activities of the CARDS toxin and/or M. penetrans toxin, for use in a vaccine preparation.
  • Such mutants can be identified as having a defect in any of the biological activities of the CARDS toxin and/or M. penetrans toxin according to the protocols described herein and as are known in the art.
  • Such mutants can be further tested for being attenuated in the ability to produce a clinical infection in a subject (i.e., for virulence potential) and then further evaluated for use as a vaccine according to known protocols.
  • CARDS toxin mutants of Mycoplasma pneumoniae e.g., having a mutation in the CARDS coding sequence or lacking the CARDS coding sequence
  • toxin mutants of M penetrans can be generated through such art-known techniques as gene disruption and their virulence potential determined by challenge studies in hamsters and by adherence and cytopathology assessments in hamster tracheal rings in organ culture and in cell culture, as is well known in the art.
  • complementation studies can be performed to restore the defective activity of the CARDS toxin and/or of the M. penetrans toxin, in order to characterize the mutant.
  • RJLl were grown to late logarithmic phase in SP-4 medium at 37 0 C for 72 h in 150- cm 2 tissue culture flasks.
  • Mycoplasmas were harvested by washing three times with PBS [150 mM NaCl, 10 niM sodium phosphate, pH 7.4] and pelleting at 12,500 g for 15 min at 4 0 C.
  • M. pneumoniae chromosomal DNA was isolated using Easy DNA kit according to the manufacturer's protocol (Invitrogen). Mycoplasma culture conditions for radiolabeling.
  • Wild-type Mycoplasma pneumoniae M129/B9 and clinical isolates were grown in SP-4 medium as above.
  • Mycoplasma monolayers in logarithmic growth phase were washed two times with 10 ml PBS (pH 7.4) and one time with Dulbecco Modified Eagle Medium (DMEM) without L-cysteine and L-methionine and resuspended in 10 ml Dulbecco Modified Eagle Medium (DMEM) without L-cysteine and L-methionine supplemented with 10 % heat-inactivated fetal bovine serum and 100 ⁇ Ci L- [ 3S S]methionine.
  • DMEM Dulbecco Modified Eagle Medium
  • Cell pellets were resuspended in 1 ml complete lysis buffer (CLB) prepared shortly before use (150 mM NaCl, 10 mM Tris, 20 ⁇ M EGTA, 0.5 M Triton-X 114, 1 mM CaCl 2 and protease inhibitors 1 ⁇ M pepstatin A, 200 ⁇ M PMSF, 1 mM iV- ⁇ -p-tosyl-L-lysine chloromethyl ketone (TLCK), and 10 ⁇ M leupeptin. Cell pellets in CLB were sheared through 25 gauge needles using 3 ml syringes to obtain clear lysis.
  • CLB complete lysis buffer
  • a 20 x 1.2 cm control glass column was packed with 3 ml uncoupled Sepharose, another identical (experimental) column was packed with 3 ml Sepharose coupled to SP-A.
  • Coupling of SP-A to Sepharose CL-4B was performed as follows: A total of 1.5 mg of SP- A was coupled to 2g of CNBr-activated Sepharose CL-4B according to the manufacture's instructions except the coupling buffer was 10 mM sodium bicarbonate, pH 8.3. SP-A coupled Sepharose was stored in 5 ml of 5 mM Tris pH 7.5, containing ImM NaN 3 . Columns were equilibrated with 50 ml CLB prior to addition of radiolabeled cell lysates.
  • Radiolabeled cell lysates were collected and reapplied to each column 3-4 times. After samples were added, columns were washed with 10 times volume of packed material to remove unbound proteins. M. pneumoniae SP-A-binding proteins were eluted using a NaCl gradient (0.2 to 3 MNaCl) containing 10 mM EDTA. Eluates were collected as 1 ml fractions, and 20 ⁇ l from each fraction was assayed for specific activity with a scintillation counter.
  • SDS-polyacrylamide gels containing M. pneumoniae SP-A binding proteins were stained with Coomassie brilliant blue and washed thoroughly in distilled water. Individual protein bands were excised from acrylamide gels and subjected to MALDI- TOF by the microsequencing facility at Baylor College of Medicine (Houston, TX).
  • Escherichia coli INVaF' [ ⁇ 'endAlreclhsdR.17supE44gyrA96lacZM15 (lacZYAargF)] (Invitrogen) and E. coli BL21(DE3) [F'ompT hsdS (r B ' m B " ) gal dcm ⁇ (DE3) pLysS] were grown in Luria Bertani (LB) broth and used to clone and express mycoplasma CARDS toxin genes.
  • LB Luria Bertani
  • This method is based on the principle that two overlapping complementary ends may prime on each other and be extended to yield a hybrid product, and a second PCR with two primers annealing at the non-overlapping ends will amplify this hybrid.
  • An example of a stepwise strategy for SOE-PCR is as follows. 1. 'a' and 'd' are primers for a gene and 'b' and 'c' are primers to mutagenize the UGA region. 2. Amplification carried out with primers 'a' and 'b' and using genomic DNA as template gives a DNA fragment "AB" of the gene. 3. Amplification carried out with primers 'c' and 'd' and using genomic DNA as template will give DNA fragment "CD" of the gene. 4.
  • Mycoplasma total proteins or purified recombinant CARDS protein were resolved on 4-12% SDS-polyacrylamide gels (NuPAGE 5 InVitrogen) (His-tag released, i.e., minus His tag) and transferred electrophoretically to nitrocellulose membranes (Towbin et ah, 1979). Membranes were blocked for two hours with 5% (wt/vol) blotto [nonfat dry milk in TBS containing 0.1% Tween-20 (TBST)], followed by three washes with TBST, and incubated with M. pneumoniae infected patient sera (1 :50 to 1 : 100 in 2% blotto) at RT for 2 h.
  • Figure 1 is an immunoblot of sera from three patients, RJ, 1970 and MJ, infected with Mycoplasma pneumoniae.
  • Purified M. pneumoniae recombinant CARDS toxin was resolved in 4-12% SDS-PAGE and transferred to nitrocellulose membranes.
  • Membranes were blocked for two hours with 5% blotto and treated with patients' sera for two hours at room temperature. Patients' sera were diluted as follows. RJ and MJ: 1:50, and 1970: 1 : 100 in 2% blotto. Membranes were washed and treated with alkaline phosphatase-conjugated goat anti-human antibodies diluted 1 :2000 in TBS-T and two hours and color developed.
  • M. pneumoniae total cell preparations of different clinical isolated (RJl, Jl, Sl and L2) and laboratory strain (B9) were dissolved in 150 ⁇ l SDS sample buffer, boiled for two minutes and separated by SDS-PAGE using 4- 12% NuPAGE SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Shleicher & Schull, Dassel, Germany) by electroblotting.
  • Membranes were blocked for one hour at room temperature with blocking buffer (20 mM Tris-base, 150 mM NaCl, 3% skim milk powder) and incubated with anti-CARDS Toxin mouse polyclonal antibodies diluted 1 :2000 in antibody buffer (20 mM Tris-base, 150 mM NaCl, 3% skim milk powder) for one hour at 37 0 C. Bound IgG was detected with alkaline phosphatase (AP)-conjugated goat-antimouse IgG diluted 1 :3000.
  • blocking buffer 20 mM Tris-base, 150 mM NaCl, 3% skim milk powder
  • Membranes were developed for 1-5 minutes with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidium (BCIP) solution. Results of the immunoblotting show a colored band of 68 kDa molecular weight on each membrane and thus demonstrate the presence of the CARDS toxin protein in each clinical isolate at concentrations that appear to vary among individuals.
  • NBT nitroblue tetrazolium
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidium
  • Proteins were transferred to nitrocellulose membranes (Schleicher & Schull, Dassel, Germany) by electroblotting and membranes were blocked for one hour at room temperature with blocking buffer (20 mM Tris-base, 150 mM NaCl, 3% skim milk powder). Membranes were cut into 3 mm strips and incubated with human serum samples diluted 1 :200 in buffer (20 mM Tris-base, 150 mM NaCl, 3% skim milk powder) for one hour at 37 0 C. Serum samples were from M. pneumonia-infected patients designated patients 1 and 2 and the first serum samples were collected during the acute phase of disease (designated 1-1 and 2-1, respectively). The second serum samples (1-2 and 2-2) and third serum samples (1-3 and 2-3) were obtained 14 and 28 days later, respectively.
  • Bound IgG was detected with alkaline phosphatase (AP)-conjugated goat- antihuman IgG diluted 1 :3000. Individual strips were developed for 1-5 minutes with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidium (BCIP) solution.
  • NBT nitroblue tetrazolium
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidium
  • Results of the immunoblotting showed a colored band of 68 IdDa molecular weight on each membrane containing rCARDs toxin and colored bands of 32 kDa and 28 kDa on each membrane containing the Dl domain, thus demonstrating seroconversion in these patients and detection of antibodies to the CARDS toxin, either as a recombinant protein or as the Dl domain.
  • the color intensity of each band appears to increase in the samples in a manner consistent with the time course of collection from the patient during the course of the disease (i.e., l-l ⁇ l-2 ⁇ l-3) ( Figure 3).
  • ELISAs were also carried out on the samples collected from patients 1 and 2 described above (i.e., samples 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3). In these assays, washing at each stage was performed at least three times with PBS and sera and antibodies were diluted in 1% BSA in PBS. Each well of Immulon 4 HBX Immunoplates (Dynox) was coated overnight at 4 0 C with 50 ⁇ l of rCARDS toxin/Dl (1 ⁇ g/well) diluted in carbonate/bicarbonate buffer (32 mM Na 2 COs, 64 mM NaHCOs).
  • PNPP p-nitrophenyl phosphate
  • phosphate buffered saline PBS
  • a cell suspension cells grown 2-3 days at 37 0 C in SP-4 medium; total cells ⁇ 1 X 10 9
  • the pellet was resuspended in 200 ⁇ l of water and incubated at 4 0 C for 20 minutes.
  • the sample was then boiled at 100 0 C for 15 minutes. 37-40 ⁇ l of this sample was used for PCR in a total reaction volume of 50 ⁇ l.
  • the samples were serially diluted 10 '1 to 10 "9 in PBS.
  • PCR conditions were 95 0 C for five minutes; 94 0 C for one minute; 55 0 C for one minute; 72 0 C for one minute and 72 0 C for 10 minutes, for 30 cycles.
  • the amplification primer set was Primer 12a forward: (nts. 1197-1220; 24 bp) 5 ? gcttgttctggaataccaagagtg 3' (SEQ ID NO:23) and Primer 15a reverse: (nts. 1541-1564; 24 bp) 5' ccattctacccaatcccagctgta 3' (SEQ ID NO:26).
  • the product size of the amplicon was 368 base pairs.
  • Detection was by ethidium bromide staining or autoradiography with a P- labeled probe.
  • the probe used to detect the amplicon by autoradiography was Primer 14a forward: (nts 1371-1429; 59 bp) 5' gctggtattggaggggttattactataccccacaattaagtggttggtcttatcagatg 3' (SEQ ID NO:25). Results of this study demonstrate that M. pneumoniae nucleic acid can be detected in the presence or absence of saliva and that one mycoplasma cell can be identified using this primer/probe set. Cloning and sequencing of CARDS
  • the entire cards sequence was amplified using forward primer 5'- tttttacatatgccaaatcctgtt-3' (primer I 5 SEQ ID NO: 12) and reverse primer 5'- gatcgcttttagcgaggatectttaacg -3' (primer 2, SEQ ID NO.64), which produces Ndel and BamEl (underlined) sites at 5' and 3' ends of the cards ORF, respectively. Both fragments were ligated into the pCR.2.1 vector and transformed into E. coli INVaF' cells for automated sequencing using Ml 3 forward and reverse primers.
  • CARDS Oligonucleotide sequences within selected (above) nucleotide sequence.
  • Pri 1-16 Modified oligonucleotide sequence* to amplify the cards sequence. * modified nucleotides are given in bold. Complementary oligonucleotide sequence are given underneath the reverse primers (2, 3, 5, 7, 9, 11, 13 and 15)
  • MPN372 23 tttttaaaaatgccaaatcctgtt 46 (SEQ ID NO:28)
  • Pri-3 20 aatgtccgtagtgcttggct 1 (SEQ ID NO:30) ttacaggcatcacgaaccga (SEQ ID NO:31)
  • MPN372 469 tgcttgactagtagatgctgtt 490 (SEQ ID NO: 32)
  • Pri-4 1 tIgMctItIgIgcItIaIgItIaIgIaItIgIcItIgItItI 22 (SEQ ID NO:33)
  • MPN372 613 atgattgccaacaccagg 630 (SEQ ID NO:34)
  • Pri-5 18 a ItIgIgtItMgcIcMaaMcaMccIaMggI 1 (SEQ ID NO:35) taccaacggttgtggtcc (SEQ ID NO: 36)
  • MPN372 610 accatgattgccaacacc 627 (SEQ ID NO:37) Pri-6 : 1 a MccIaItIgIgtItlgMccMaaMcaIclcI 18 (SEQ ID NO:38) MPN372: 722 cctgattgaagtccacctt 740 (SEQ ID NO:39)
  • Pri-7 19 cctgattggagtccacctt 1 (SEQ ID NO:40) ggactaacctcaggtggaa (SEQ ID NO: 41)
  • MPN372 717 cgtgccctgattgaagtc 734 (SEQ ID NO: 42)
  • MPN372 1115 caaaagtgaaaatgacacc 1134 (SEQ ID NO:47)
  • Pri-12 1 gcttgttctggaataccaagagt 23 (SEQ ID NO:52)
  • MPN372 1368 taggctggtattgaaggggt 1387 (SEQ ID NO-.53)
  • IMIMMMMI 1 gcttgttctggaataccaagagt 23 (SEQ ID NO:52)
  • MPN372 1368 taggctggtattgaaggggt 1387 (SEQ ID NO-.53)
  • MPN372 1374 ggtattgaaggggttattactataccccacaattaagtggttgatcttatcagatg 1429 IMMM MMMIMMMIMMMMMMIMM IMMIIMMI
  • MPN372 1541 tacagctgggattgagtagaa 1561 (SEQ ID NO: 58) MMMMMMM
  • MPN372 1541 tacagctgggattgagtagaa 1561 (SEQ ID NO:61) MIMMMMMI
  • Pri-16 1 tacagctgggattgggtagaa 21 (SEQ ID NO:62)
  • MPN372 1796 gatcgcttttagcgattaagctttaacg 1824 (SEQ ID NO:63)
  • MIMMMMMII I MMIIII Pri-2 28 gatcgcttttagcgaggatcctttaacg 1 (SEQ ID NO: 64) ctagcgaaaatcgctcctaggaaattgc (SEQ ID NO: 13)
  • the cards gene of M. pneumoniae reference strain M 129/ B9 and clinical isolates (Sl, L2, JL and RJLl) were cloned in a PCRII vector individually and sequenced.
  • M129/B9 represents the reference strain and Sl, L2, RJLl and JL are clinical isolates from patients in San Antonio and Dallas.
  • RJLl 1112 ⁇ T ⁇ G) and 1174 (T ⁇ C) .
  • Bolded gs shown were introduced by site directed mutagenesis in order to express CARDS protein in E. coli.
  • JL SEQ ID NO:3
  • JL had only one change at aa position 37i Ilc - *Scr .
  • RJLl had one more additional change (comparing to JL) at aa position 392 Trp ⁇ Arg
  • L2 had one more additional change (comparing to JL) at aa position 245 Asp ⁇ Gly .
  • DNA fragments were generated by digesting plasmid pCR-cards with Nde ⁇ and BamHI and ligated into pET19b to generate p ⁇ T-cards.
  • the plasmid was transformed into competent E. coli BL21 (DE3) cells grown to a density of 2 X 10 9 cells/ml at 37 ° C in standard LB broth containing 100 ⁇ g/ml ampicillin (Sigma- Aldrich). Induction of recombinant protein synthesis was accomplished by addition of 100 ⁇ M of isopropyl thio ⁇ -galactopyranoside (Sigma-Aldrich), and bacteria were incubated for 3 h at 37 C under aeration at 220 rpm.
  • mice were immunized subcutaneously with 50-100 ⁇ g of recombinant total CARDS protein suspended in complete Freund's adjuvant (no peptides or truncated domains). Individual mice were boosted three times with the same amount of recombinant antigen in incomplete Freund's adjuvant at 14-day intervals. Serum samples were collected and used for immunological characterization. Monoclonal antibodies were produced using recombinant CARDS toxin and hybridoma supernatants were screened for immunoreactivity with CARDS protein and truncated peptides.
  • Domain 1 (SEQ ID NO:69): Primer 1 and 372D1R
  • PWLPTPG 201 IATPVHLSIP OAASVADVSE GTSASLSFAC PDWSPPSSNG ENPLDKCME 251 KIDNYNLQSL PQYASSVKEL EDTPVYLRGI KTQKTFMLQA
  • the D 1 PCR fragment (SEQ ID NO:74) encoding the cards first 249 amino acids (SEQ ID NO:69) was cloned into the E. coli His lo -tagged expression vector, pET19b (Novagen), using N del and Ban ⁇ HI restriction sites incorporated into the oligonucleotide primers used to amplify this nucleic acid 5' tttttacatatgccaaatcctgttag 3' (SEQ ID NO:72) and 5' ggatcctctacgcaatgcatttgtctag 3' (SEQ ID NO:65).
  • a recombinant plasmid was used to transform E. coli strain BL21 ( ⁇ DE3). Transformants were grown to mid-log phase before inducing Dl expression by addition of IPTG to a final concentration of 1 mM. After four hours, cells were harvested by centrifugation at 800Og for 15 minutes at 4 0 C and the pellet was resuspended in 50 mM phosphate buffer ph 8.0, containing 300 mM NaCl, 10 mM imidazole and complete, EDTA-free protease inhibitor (Sigma).
  • Fractions containing purified protein were desalted using PlO columns (Amersham Biosciences) with TG buffer (20 mM Tris-Cl, pH 7.4, 5% glycerol) and concentrated using YM-10 (Amicon) membranes. Protein concentrations were estimated using a BCA protein assay kit (Pierce) and the protein was aliquoted and stored at -8O 0 C.
  • Confluent HEp-2 cells were incubated with medium alone or in the presence of 40 ⁇ g/ml CARDS protein for 16 hours at 37 0 C. Cells were washed and incubated with fresh medium at 37 0 C for four hours. Cell free extracts (CFE) were prepared and assayed for ADP-ribosylation (CFE were incubated with 40 ⁇ g/ml CARDS protein for one hour with 0.1 ⁇ M [ 32 P]NAD in 10OmM Tris pH 7.5, 2OmM DTT). The reaction mixture was precipitated with 10% TCA and proteins were resolved in a 4-15% gradient gel by SDS-PAGE and transferred to nitrocellulose membrane for autoradiography. As shown in the autoradiogram in Figure 2, the CARDS toxin exhibits ADP ribosylating activity. Cytopathology in Chinese Hamster Ovary (CHO) cells
  • CPE Cytopathogenic effects
  • Baboons Individual animals were instilled with active toxin or non-toxin diluent into the right lower lobe by direct endoscopic placement. Baseline lavages were obtained from the contralateral lobes of each animal and follow-up bilateral lavages were obtained 24 and 48 hours after the initial inoculation to investigate systemic effects. Subsequently, animals were sacrificed and airways evaluated in both groups by histochemistry and by assessment of inflammatory responses in bronchoalveolar lavage (BAL).
  • BAL bronchoalveolar lavage
  • inflammatory cytokines such as interleukin 6 (IL-6)
  • IL-6 interleukin 6
  • Chemokines RANTES and IL-8 showed increases of 10 fold and 300 to 500 fold, respectively. No substantial stages in cytokine/chemokine profiles were observed in the negative control animal.
  • mice Individual animals were intranasally inoculated with CARDS toxin and monitored for changes in lung histopathology and BAL-associated inflammatory responses. Mice showed very striking peribronchiolar infiltration of lymphocytes, mononuclear cells and neutrophils. As in the case of baboons, marked lung injury was observed in the lungs and other airway tissues. The proliferation marker, MIB-I, was markedly reduced in CARDS toxin-treated mice, indicating a very powerful shut down of cell proliferation in contrast to non-toxin treated control animals. For example, IL-6 and IL-12 increased within the first six days of intranasal introduction of CARDS toxin by 8 fold and 20 fold, respectively. CARDS toxin can mimic the course of active mycoplasma infection both in terms of cytopathology and cytokine/chemokine responses.
  • the CARDS TX is considered as a singular entity (i.e., an individual chemical molecule) it is no longer a virulence factor but an immuno-modulator.
  • CARDS TX stimulates the immune system to induce a predictable immune response of cytokines and chemokines, etc., that is not being utilized for the survival of the pathogen. This has profound implications because many pathogens subvert the immune system by down-modulating, inhibiting, or misdirecting the cytokine, chemokine, and immune signaling responses designed to protect the host. Therefore, an immuno-modulator (i.e., CARDS TX) that is capable of inducing a specified immune response that a different pathogen needs to actively subvert in order to cause disease, could prevent disease caused by that pathogen.
  • CARDS TX an immuno-modulator
  • Plague pneumonia is a rapidly progressive pneumonia caused by Yersinia pestis that causes death in mice or humans in 2-4 days, depending on the susceptible species and infectious dose. Untreated, 100% of infected animals will die from the disease. In a mouse model, all mice are dead in 2-3 days from an infection with 500-800 bacteria given intranasally (IN).
  • the yersinia bacterium possesses numerous virulence factors that actively modulate the host innate response, thereby allowing yersinia to survive and cause disease.
  • One common feature is the early inhibition of inflammatory responses.
  • CARDS TX administered intranasally induces a pro-inflammatory cytokine response in the bronchial alveolar lavage fluid (BALF) within days 1 and 2 of application to mice and baboons.
  • BALF bronchial alveolar lavage fluid
  • Y. pestis initially induces an anti-inflammatory response at early time points (0-36 hours) and then a proinflammatory response > 36 hrs post infection.
  • Differential expression of cytokines continued in CARDS TX treated mice out to at least day 6.
  • mice When mice were treated with various doses of CARDS TX intranasally (IN) and then challenged IN 4 days later with a lethal high or a lethal low dose of Yersinia pestis, improved mouse survival was observed, indicating that CARDS TX stimulated an immune response that reduced the degree of Y. pestis virulence capabilities.
  • the dose of CARDS TX was optimized and 1 ⁇ g of toxin IN four days prior to infection gives the best improved survival in these studies.
  • the present invention provides a chimeric protein or polypeptide comprising, consisting essentially of and/or consisting of a CARDS toxin or biologically active fragment or domain thereof and a ligand for contacting the CARDS toxin or biologically active fragment or domain thereof with a target cell.
  • a nucleic acid molecule that encodes a chimeric protein of this invention, as well as a vector and/or cell comprising the nucleic acid molecule.
  • a biologically active fragment of the CARDS toxin can be a fragment as described herein that retains one or more biological activities of the CARDS toxin, such as toxin activity.
  • a biologically active fragment of this invention can also be a domain of the CARDS toxin, as described herein.
  • the chimeric protein can comprise a "toxin domain," which is a protein or functional fragment thereof that has toxic activity (e.g., ADP-ribosylating activity) and/or cytopathology inducing activity) on a cell as described herein, hi some embodiments, in addition to a toxin domain of a CARDS toxin, the chimeric protein of this invention can comprise a toxin domain of another toxin, which can be, but is not limited to a toxin domain of diphtheria toxin, ricin, Pseudomonas exotoxin, colicin, anthrax toxin, tetanus toxin, botulinum toxin, saporin, abrin, bryodin, pokeweed anti- viral protein, viscumin and gelonin.
  • a toxin domain of another toxin, which can be, but is not limited to a toxin domain of diphtheria toxin, ricin, P
  • a chimeric protein of this invention can comprise more than one (e.g., e, 3, 4, 5, or more) toxin domains or functional fragments thereof, which can be present in any order and/or in any combination in the chimeric protein.
  • toxin domains When multiple toxin domains are present, they can be immediately adjacent to one another, separated by one or more targeting moieties (antibody/ligand) and/or translocation domains, and/or separated by linkers.
  • the moieties of the chimeric protein of this invention can be present in any order, multiplicity and/or combination relative to one another.
  • the chimeric proteins of this invention can also be modified by use in vivo by the addition of a blocking agent at the amino and/or carboxy-terminal end, to facilitate survival of the chimeric protein in vivo.
  • blocking agents of this invention include, but are not limited to, additional related and/or unrelated peptide sequences that can be attached to either end of the chimeric protein. Blocking can be carried out either chemically during synthesis of the chimeric protein of by recombinant DNA technology according to protocols well known in the art.
  • the ligand moiety of the chimeric protein of this invention can be an antibody that specifically reacts with an antigen on a cell surface, such that the antibody will bind to the surface of a cell possessing the antigen (a target cell), thereby bringing the CARDS toxin moiety of the chimeric protein in contact with the cell.
  • the CARDS toxin moiety can be internalized by the target cell and the CARDS toxin or biologically active fragment thereof is active in the target cell, resulting in damage to and/or death of the target cell.
  • the ligand moiety of the chimeric protein can be an antibody to a cancer antigen, which can be an antigen that is present only on the surface of a cancer cell and/or it can be a cancer-associated antigen that is present on the surface of a cancer cell in an amount greater than the amount of antigen that would be present on the surface of a non-cancerous (e.g., normal) cell.
  • a cancer antigen can be an antigen that is present only on the surface of a cancer cell and/or it can be a cancer-associated antigen that is present on the surface of a cancer cell in an amount greater than the amount of antigen that would be present on the surface of a non-cancerous (e.g., normal) cell.
  • a cancer antigen of this invention can include, but is not limited to HER2/neu and BRCAl antigens for breast cancer, MART-1/MelanA, gplOO, tyrosinase, TRP-I, TRP-2, NY-ESO-I, CDK-4, ⁇ -catenin, MUM-I, Caspase-8, KIAA0205, HPVE7, SART-I, PRAME, and pl5 antigens, members of the MAGE family, the BAGE family (such as BAGE-I), the DAGE/PRAME family (such as DAGE-I), the GAGE family, the RAGE family (such as RAGE-I), the SMAGE family, NAG, TAG-72, CA125, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPVl 6 E7), the SSX family, HOM-MEL-55, NY- COL
  • MAGE family include, but are not limited to, MAGE-I, MAGE-2, MAGE-3, MAGE-4 and MAGE-11.
  • GAGE family include, but are not limited to, GAGE- 1, GAGE-6. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9: 684-693, Sahin et al. (1997) in Curr. Opin. Immunol 9: 709-716, and Shawler et al. (1997), the entire contents of which are incorporated by reference herein for their teachings of cancer antigens.
  • the cancer antigen can also be, but is not limited to, human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC- 18, the Ha-ras oncogene product, carcino-embryonic antigen (CEA), the raf oncogene product, CA- 125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), prostate-specific membrane antigen (PSMA),prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, ⁇ -HCG, gp43, HSP-70 , pl7 mel, HSP-70, gp43, HMW, HOJ-I,
  • the cancer antigen of this invention can also be an antibody produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma; hairy cell leukemia), a fragment of such an antibody, which contains an epitope of the idiotype of the antibody, a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or complementarity determining region (CDR) of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR.
  • the cancer antigen of this invention can be a single chain antibody (scFv), comprising linked VH, and V L domains, which retains the conformation and specific binding activity of
  • the present invention is in no way limited to the cancer antigens listed herein.
  • Other cancer antigens be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506, the entire contents of which are incorporated by reference herein.
  • the present invention further provides a method for treating cancer, a method for killing a tumor cell and/or a method for reducing the size of a tumor in a subject, comprising administering to the subject and/or contacting cancer cells (e.g., tumor cells) of the subject with a chimeric protein of this invention and/or a nucleic acid encoding a chimeric polypeptide of this invention.
  • cancer cells e.g., tumor cells
  • the cancer to be treated by administration to a subject of a chimeric polypeptide and/or nucleic acid encoding a chimeric polypeptide of this invention can be, but is not limited to, B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasia, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, adrenal cancer, anal cancer, colorectal cancer, endometrial cancer, esophygeal cancer, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, kidney cancer, laryngeal cancer, niedulloblastoma, mesothelioma, neuroblastoma, oropharyngeal cancer, osteosarcoma, parathyroid cancer, thyroid cancer, penile cancer, pituitary cancer,
  • the ligand of this invention can be a ligand that specifically binds a receptor on a muscle cell, which can include, but is not limited to, an antibody reactive to N-CAM (neuronal cell adhesion molecule) (Sigma Chemical Company, St. Louis, Mo.), an antibody reactive with the muscle-specific antigen, Leu- 19, an antibody reactive with dystrophin (Sigma) and an antibody reactive with a nicotinic acetylcholine receptor (nAchR).
  • N-CAM neurovascular cell adhesion molecule
  • nAchR nicotinic acetylcholine receptor
  • the nAch receptor and antibodies generated thereto are readily available from publicly accessible depositories. (See, e.g., U.S. Pat. No. 5,192,684, ATCC Nos.: HB 8987 (mAb 64), HB 189 (mAb 270), and TIB 175 (niAb 35), all of which are incorporated herein by reference.)
  • the present invention also provides methods of treating a muscle spasm, (e.g., a focal muscle spasm; a facial wrinkle), comprising contacting a muscle cell of the muscle in spasm with a chimeric protein of this invention.
  • a muscle spasm e.g., a focal muscle spasm; a facial wrinkle
  • Such methods can be used, for example, to treat blepharospasm, cervical dystonia, hand dystonia, limb dystonia, hemifacial spasm, bruxism, strabismus, VI nerve palsy, spasmodic dysphonia and/or oromandibular dystonia, as well as any other disease or disorder associated with muscle spasm that is now known or later identified.
  • a method of treating a muscle spasm according to this invention can include, for example, administering (e.g., by intramuscular injection) an effective amount of the chimeric polypeptide and/or nucleic acid encoding the chimeric polypeptide of this invention to the muscle of a subject.
  • the chimeric polypeptide and/or nucleic acid of this invention can also be administered with an effective amount of botulinum toxin, either alone and/or as part of a chimeric polypeptide and/or encoded by a nucleic acid molecule.
  • a "muscle spasm” includes a brief, unsustained contraction and/or a paroxysmal, spontaneous, prolonged contraction of one or more muscles, which are responsive to treatment involving selective destruction of one or more muscles at the site of the muscle spasm.
  • the production of any type of antibody and/or antibody fragment can be carried out according to well developed in the art.
  • a humanized or “chimeric” antibody is an immunoglobulin molecule comprising a human moiety and a non-human moiety, in any combination.
  • variable region of a humanized immunoglobulin molecule can be from a non-human (e.g., murine) source and the constant region can be from a human source.
  • the humanized antibody can have the antigen-binding specificity of the non- human source and the effector function of the human source (see, e.g. U.S. Patent No. 5,482,856, the entire contents of which are incorporated by reference herein). Protocols for the production of human antibodies are well known (see, e.g., U.S. Patent No. 5, 001, 065, the entire contents of which are incorporated by reference herein).
  • the chimeric protein of this invention can comprise a translocation or internalization domain of a toxin protein, e.g., to facilitate delivery of the toxin moiety into the target cell-
  • the translocation domain can be from the CARDS toxin described herein and/or the domain can be from any other toxin that has a translocation domain.
  • toxins include but are not limited to diphtheria toxin, colicin, delta-endotoxin, anthrax toxin, tetanus toxin, botulinum toxin and Pseudomonas exotoxin.
  • the chimeric proteins of this invention are produced by methods well known in the art.
  • an antibody or ligand that allows for specific targeting of the CARDS toxin to a specific cell type or population can be selected according to art- known procedures for the specific therapeutic effect desired.
  • the antibody or ligand can be joined via a covalent or non-covalent bond to a CARDS toxin and/or biologically active fragment thereof as described herein.
  • the moieties can be attached, joined or connected by any of a number of means well known to those of skill in the art.
  • the chimeric protein of the present invention can be recombinantly expressed as a single-chain fusion protein comprising both antibody and toxin according to methods well known in the art.
  • the toxin moiety can be joined, linked or conjugated directly, or through a linker (spacer), to the ligand.
  • a "linker” as used herein, is a molecule that is used to join two molecules. The linker is capable of forming covalent bonds or high-affinity non-covalent bonds to both molecules. Suitable linkers are well known to those of ordinary skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers and/or peptide linkers. The linkers can be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine).
  • the procedure for attaching a toxin to an antibody or other polypeptide targeting molecule can vary according to the chemical structure of the toxin.
  • Immunoglobulin molecules contain a variety of functional groups; e.g., sulfhydryl (--S), carboxylic acid (COOH) and free amine (NH 2 ) groups, which are available for reaction with a suitable functional group on a toxin.
  • the antibody/ligand and/or toxin can be derivatized to expose or attach additional reactive functional groups.
  • the derivatization can involve attachment of any of a number of linker molecules (including commercially available linker molecules, e.g., from Pierce Chemical Company, Rockford lll), Ih some embodiments, a bifunctional linker having one functional group reactive with a group on the toxin and another functional group reactive with a group on an antibody/ligand, can be used. Derivatization can also involve chemical treatment of the toxin or antibody/ligand (e.g., by glycol cleavage of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups).
  • the free aldehyde groups on an antibody can be reacted with free amine or hydrazine groups on a toxin to form the chimeric protein (see, e.g., U.S. Pat. No. 4,671,958. the entire contents of which are incorporated by reference herein).
  • Procedures for generation of free sulfhydryl groups on an antibody or antibody fragment are also known (see, e.g., U.S. Pat. No. 4,659,839).
  • Many procedures and linker molecules for attachment of various compounds including toxins are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos.
  • the linker molecule is m-Malimidobenzoyl-N-hydroxysuccinimideester (MBS) which can be used to prepare chimeric proteins as described, for example, in Youle and Nevelle. Proc. Natl. Acad. ScL 77(9):5483-5486 (1980).
  • MFS m-Malimidobenzoyl-N-hydroxysuccinimideester
  • the present invention further provides a chimeric protein comprising a linkage that is cleavable in the vicinity of or within the target site and that can be used when the toxin is to be released at the target site. Cleavage of the linkage to release the toxin from the antibody/ligand can be facilitated by enzymatic activity and/or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • cleavable linkers are known in the art (see, e.g., U.S. Pat. Nos.
  • SPDP is a reversible NHS-ester, pyridyl disulfide cross- linker used to conjugate amine-containing molecules to sulfhydryls.
  • Another chemical modification reagent is 2-iminothiolane, which reacts with amines and yields a sulfhydryl.
  • Water soluble SPDP analogs such as Sulfo-LC-SPDP (Pierce, Rockford, 111.) can also be used.
  • SMPT is a reversible NHS-ester, pyridyl disulfide cross-linker developed to avoid cleavage in vivo prior to reaching the target cell or site.
  • the chimeric protein of this invention can also be produced according to standard protocols for recombinant DNA technology.
  • the chimeric proteins can be produced in any number of well-defined expression systems and purified according to act-known standards for in vivo administration.
  • a nucleic acid encoding the chimeric protein can be administered to a subject and/or to a cell of a subject of this invention and the chimeric protein can be produced in the cell or in the subject.
  • the chimeric proteins and nucleic acids encoding them can be administered in vivo or ex vivo.
  • the chimeric protein of this invention can be present in a composition, which can be a pharmaceutical composition comprising the chimeric protein and/or nucleic acid molecule encoding a chimeric protein in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable includes a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the chimeric polypeptide and/or nucleic acid without causing substantial deleterious biological effects or interacting in a deleterious manner in the subject and/or with any of the other components of the composition in which it is contained.
  • the dose of the chimeric protein and/or nucleic acid encoding the chimeric protein will depend upon the properties of the particular chimeric polypeptide employed, e.g., its activity and biological half-life, the concentration of chimeric polypeptide in the formulation, the site and rate of administration, the clinical tolerance and characteristics (e.g., sex, gender, species, age, size, weight, overall condition) of the patient involved, the nature and severity of the disease or disorder to be treated or altered, etc., as are well know considerations in the art.
  • chimeric protein and/or nucleic acid encoding the chimeric protein of this invention can be administered to a subject in a variety of ways (e.g., orally, intravenously, subcutaneously, intramuscularly, intratumorally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratrachaeally and/or intrapulmonarily.
  • ways e.g., orally, intravenously, subcutaneously, intramuscularly, intratumorally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratrachaeally and/or intrapulmonarily.
  • the chimeric polypeptide of this invention can be administered to a subject by, e.g., injection, into a muscle or into a tumor, in an amount ranging from about 1 ng to about 500 mg.
  • An effective amount can be determined by one of skill in the art, using art-known teachings such as those provided in MacDonald and Glover ("Effective tumor targeting: Strategies for delivery of armed antibodies” Current Opinion in Drug Discovery and Development 8:177-183 (2005) and Michl and Gress ("Bacteria and bacterial toxins as therapeutic agents for solid tumors" Current Cancer Drug Targets 4:689-702 (2004).
  • the chimeric protein can be administered at the site of the neuromuscular junctions of the muscle to be treated.
  • the chimeric proteins of the present invention can be administered alone, in combination, and/or in conjunction with a conjugated and/or an unconjugated form of a different toxin (e.g., botulinum toxin).
  • a conjugated and/or an unconjugated form of a different toxin e.g., botulinum toxin.
  • the chimeric protein and/or nucleic acid can be administered once or it can be administered periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • the dose should be sufficient to treat or ameliorate known symptoms or signs (e.g., release of muscle spasm; decrease in tumor size or reduction in tumor cell count) without producing unacceptable toxicity to the subject.
  • An effective amount is an amount that provides either subjective relief of at least one symptom and/or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • the chimeric protein and/or nucleic acid can also be administered via microspheres, liposomes or other microparticulate delivery systems placed in certain tissues, including blood.
  • testing of any of the chimeric proteins and nucleic acids encoding chimeric proteins of this invention for cytotoxicity both in vitro and in vivo can be carried out according to protocols well described in the art.
  • methods of determining cytotoxicity of a chimeric protein of this invention used to treat muscle spasms are described in U.S. Patent No. 6,780,413, the entire contents of which are incorporated by reference herein.
  • Methods of determining the cytotoxicity of a chimeric protein of this invention used to treat cancer and/or to kill tumor cells are described in U.S. Patent No. 6,846,484, the entire contents of which are incorporated by reference herein.
  • Mycoplasma penetrans toxin The present invention provides a protein of M penetrans that is shown herein to have activities similar to the CARDS toxin. To identify this protein, sequence of M. penetrans proteins were examined for a motif found inADP-ribosylating bacterial toxins, which is the presence of two essential amino acid residues, which are arginine at position 28 and glutamic acid at position 156. The protein MYPE9110 was identified as having this motif. In order to express a full length protein in E. coli BL21 (DE3) LpxM, all 7 UGAs encoding tryptophan in MYPE9110 were changed to UGG. The recombinant protein was then purified using nickel column chromatography.
  • CHO cell lysates were incubated with recombinant M YPE9110 in 1 mM ATP, 1 mM GTP, 1OmM thymidine, 1OmM DTT, 5OmM Tris (pH 7.4) with 5% glycerol and lO ⁇ M 32 P labeled NAD (lOCi/mmol).
  • the reaction volume was incubated at 37 0 C for 30 minutes, followed by the addition of 20% TCA to precipitate proteins. This mixture was then spun at 13,00Og for 10 minutes.
  • MYPE9110 is another member of the ADP-ribosylating, vacuolating toxins unique to mycoplasmas. Role of mycoplasma toxins in airway disorders and asthma
  • Certain aspects of this invention are based on the identification of the CARDS TX as an important mediator of asthma, thus allowing for the development of effective strategies to diagnose, treat and prevent asthma and related airway diseases in a substantial population of both children and adults.
  • Asthma is a complex disease that afflicts over 15 million Americans. Despite the apparent increase in prevalence of disease within our population, asthma is still a poorly understood disease. This is in part due to the complex mixture of genetic factors, environmental stimuli, and immune system status that impacts disease development and progression.
  • Asthma is a complex disease since it involves genetic predisposition, environmental factors, and an interaction with the immune status in the development and progression of the disease. Over 15 million Americans are afflicted with this disease and despite the use of potent medications, between 16- 17% of patients experience continuous daily and frequent nocturnal symptoms.
  • One underappreciated and controversial factor in the etiology of asthma is the role that atypical bacterial infections, such as those caused by Mycoplasma pneumoniae, play in initiating, exacerbating and prolonging airway-related symptoms and pathologies.
  • Multiple lines of evidence directly link M. pneumoniae to the pathogenesis of asthma beyond its role as a precipitating factor in acute exacerbation of asthma. In children, M.
  • pneumoniae infections have been shown to induce chronic lung damage for prolonged periods after the resolution of respiratory tract symptoms. Studies have demonstrated abnormal pulmonary function tests in up to 50% of children and abnormalities of the lung in 37% of children months to years after an episode of M. pneumoniae respiratory infection. Mycoplasma pneumoniae is also known to induce a number of inflammatory mediators implicated in the pathogenesis of asthma. IgE, IL- 4, and IL-5 have been shown to be significantly elevated in children with M. pneumoniae infections, suggesting that M. pneumoniae can induce a Th2 like cytokine response. In adults, M. pneumoniae has been detected in a large percentage of patients with stable moderately severe chronic asthma.
  • the CARDS TX of this invention which is the first ADP-ribosylating (and vacuolating) toxin associated with any pathogenic human or animal mycoplasma, represents the first bonaflde virulence factor identified that provides an association between M. pneumoniae infection and acute and chronic airway disease and extra-pulmonary manifestations.
  • Project 1 Mycoplasma pneumoniae rCARDS TX as mediator of airway dysfunction in mice
  • the present invention provides evidence for a role of the CARDS TX in the pathogenesis associated with airway inflammation, airway obstruction and airway hyperreactivity associated with respiratory M. pneumoniae infection. Additional studies are being conducted to identify the role played by the CARDS toxin in the ability of M pneumoniae to induce acute asthma exacerbations and in the deleterious long-term effects of mycoplasma respiratory tract infection. Therapeutic interventions directed against CARDS TX can then be developed to treat and/or prevent M. pneumoniae-associated reactive airway disease and asthma. The specific aims of these studies are to 1) understand the specific contribution of active CARDS TX to the airway obstruction, hyperreactivity, and inflammation observed in M.
  • Active CARDS TX induces histologic pulmonary inflammation in mice (and baboons) consistent with M. pneumoniae infection, while heat-inactivated toxin does not.
  • BALB/c mice were inoculated intranasally once with either active recombinant CARDS TX, heat-inactivated rCARDS TX, or the toxin carrier solution (controls).
  • Mouse lungs were examined at 2, 4, and 6 days post-inoculation. Lungs were removed, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and H&E stained. The lungs from mice given CARDS TX demonstrated acute histologic inflammation similar to that seen with M. pneumoniae infection in BALB/c mice.
  • Lungs from mice inoculated with heat-inactivated toxin demonstrated minimal inflammation, and lungs from control mice inoculated with the toxin carrier solution had no histologic abnormalities.
  • the histologic scoring system developed by Cimolai et al. (1992) for experimental M. pneumoniae infection the greater the score the greater the inflammation
  • the CARDS TX treated lungs had significantly greater inflammation than the control mice.
  • Active CARDS TX induces the production of cytokines/chemokines in the lower respiratory tract of mice and baboons.
  • BAL bronchoalveolar lavage fluid
  • Active rCARDS TX induces airway obstruction and airway hyperreactivity.
  • mice were inoculated with rCARDS TX as described herein and airway obstruction and airway hyperreactivity (AHR) were measured before and after aerosolized methacholine, respectively, by plethysmography (enhanced pause) in nonsedated, unrestrained mice.
  • Enhanced Pause is a dimensionless value that represents a function of the ratio of peak expiratory flow to peak inspiratory flow and a function of the timing of expiration. Penh does not directly measure airway resistance. Adler and colleagues
  • mice infected with M. pneumoniae develop a specific antibody response to CARDS TX.
  • mice inoculated with M pneumoniae to produce respiratory tract infection have been shown to develop specific anti-CARDS TX serum IgG antibody. Studies will be carried out to determine if antibodies to the CARDS TX are protective, thus leading to the development of vaccines or to immune-based therapies specific for acute and/or chronic M pneumoniae respiratory infection associated with reactive airway disease or asthma. Patients who fully recover from naturally acquired or experimentally induced M pneumoniae respiratory disease are generally resistant to reinfection or rechallenge. Previous immunization strategies against M. pneumoniae have only offered partial protection and are thought to have been hampered by a lack of knowledge of the basic virulence mechanism responsible for disease (Bearliest, 1984). The CARDS TX of the present invention presents new opportunities for immunization and treatment strategies against M. pneumoniae.
  • Azithromycin therapy had no significant effect on BAL concentrations of IL-6 and INF- ⁇ in the applicants' M. pneumoniae model, while significant reductions in these cytokines were demonstrated with clarithromycin (a macrolide), cethromycin (a ketolide), and NVP- PDF713 (a peptide deformylase inhibitor) therapy (Hardy et al, 2003; Rios et al, 2004; Rios et al, 2005; Fonseca-Aten et al, 2005b).
  • azithromycin may interact with mycoplasma virulence properties (such as CARDS TX protein production) in a different manner than these other inhibitors of protein synthesis.
  • mycoplasma virulence properties such as CARDS TX protein production
  • Antimicrobial structural differences are known to produce differential inhibitory effects on protein synthesis and 5OS ribosomal subunit assembly (Mabe et al, 2004).
  • azithromycin therapy did not improve airway obstruction in the applicants' model, from an inimunopathogenic perspective; this may have been a functional consequence of the lack of significant reduction in BAL concentrations of IL-6 and/or INF- ⁇ with azithromycin therapy.
  • the BALB/c-ovalbumin model of allergic asthma will be used to test the following specific aims: 1) determine the contribution of the CARDS TX to the pathogenesis of M. pneumoniae associated allergic asthma using established murine models; 2) investigate the role of CARDS TX in the pathogenesis of asthma associated with M. pneumoniae infection. Mice will be infected with wild type M. pneumoniae or M. pneumoniae with a null mutation in the CARDS TX gene. Pathogenesis will be evaluated in the BALB/c mouse model with and without ovalbumin-induced airway hyper-responsiveness, to elucidate the role of CARDS TX in the context of the infectious model; and 3) investigate the activity of CARDS TX in vivo.
  • CARDS TX-induced gene expression, localization/co-localization, and biochemical activity in vivo will also be carried out, using the BALB/c mouse model with and without ovalbumin-induced airway hyper-responsiveness.
  • CARDS TX elicits cytopathology in mammalian cells.
  • PCR was used to amplify the cards tx open reading frame from M. pneumoniae clinical isolate S 1.
  • the amplified gene was cloned into a bacterial expression vector and recombinant protein expressed and purified.
  • CHO cells cultured in the presence of exogenous rCARDS TX displayed characteristic rounding and distinct vacuolization with disruption of monolayer integrity. Both low concentrations of rCARDS TX (10-50 ng/ml) and high concentrations of rCARDS TX (10-50 ⁇ g/ml) elicited overt cytopathological effect (CPE) but with different time requirements. Heat inactivation of rCARDS TX preparations (30 minutes at 6O 0 C) abolished CPE, which reinforced the cytotoxic properties of 'heat labile' CARDS toxin.
  • CPE cytopathological effect
  • Nasal washes were collected from 40 of the patients and serum from 42. Three to eight weeks later "convalescent" NW and serum samples were also obtained. Additionally, sera from 23 chronic asthmatics ages 6-40 years were included in the analysis. NW and sera were analyzed for antibodies to the CARDS TX or the Pl adhesin protein and by PCR for the CARDS TX or Pl gene. Pl is the major adhesin of M. pneumoniae and is considered the 'gold standard' for molecular diagnosis of M. pneumoniae. CARDS TX antibodies can be detected in the serum of 55% of acute asthmatics, 48% of the convalescent asthmatics, and 70% of the chronic asthmatics (Table 1).
  • TX Pl 11/42 (26.19%) 1/23 (4.35%) 6/23 (26.09%) l
  • Individual serum samples were analyzed by ELISA against equimolar concentrations of recombinant CARDS TX or recombinant Pl protein. Results represent the fraction of positives per total analyzed. Positive and negative results were determined based on the analysis of statistical significance of ELISA results from known positive and negative serum samples. Note that acute and convalescent categories represent the same group of patients, who presented with acute asthma and were re-studied 6-8 weeks later. The chronic patients represent a separate group who presented with clinically stable chronic asthma.
  • cytokines Concentrations of cytokines were evaluated in the bronchial alveolar lavage fluid (BALF) of BALB/c mice that were sedated and intranasally (IN) inoculated with 50 ⁇ g of rCARDS TX, or carrier solution as a negative control. After two days, BALF was collected and concentrations of cytokines and chemokines determined using the Luminex system or ELISA. We examined levels of IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-IO, IL-12, IL-13, KC, TNF- ⁇ , IFN- ⁇ , GM-CSF 3 IPlO, TGF- ⁇ , MIP-Ia, MIG and MCP-I.
  • This immunologic profile represents inflammatory, THl, TH2, growth factor, and chemotatic cytokines for which associations have been made with M. pneumoniae respiratory disease (7, 8, 18, 25, 38, 41, 45, 53).
  • Statistically significant differences (p ⁇ 0.05) in cytokine concentrations ranging from 4 fold to 800 fold were observed for IL-12 (6 fold), TNF- ⁇ .(600 fold, IL-6 (800 fold), GM-CSF (4 fold), IL-l ⁇ . (5 fold), KC (4 fold), and Mipl ⁇ .(8 fold).
  • No significant differences between carrier solution and CARDS TX in BALF were observed for IL-2, IL-4, IL-5, IL-10, IFN- ⁇ , MCP-I, MIG, and IP-10.
  • This cytokine profile which is consistent with the profile observed in the mouse model of M. pneumoniae infection, is indicative of a ThI type inflammatory response and clearly implicates CARDS TX as a mediator of proinflammatory responses in the mouse airway. Based on what has been observed with M. pneumoniae infections, it was important to examine the histopathology that follows CARDS TX intoxication. Lungs of mice used for cytokine analysis were prepared for histopathology and scored using the system of Cimolai et al. (9). To determine the impact on lung pathology, lungs were removed and fixed in 10% neutral buffered formalin (NBF) prior to being embedded in paraffin, sectioned, and stained with H&E. No histologic abnormalities were present in control lungs, and heat inactivated toxin treated lungs showed minimal numbers of lymphocytes in peribronchiolar and perivascular sites).
  • NNF neutral buffered formalin
  • mice were infected IN with 10 7 CFU of wild-type (WT) M. pneumoniae or cards tx null mutant. After 4 days and 18 days, lungs were harvested for immunohistochemistry and histopathology. Fresh frozen tissues (lungs) were prepared as described previously (16, 17) and 7 ⁇ M sections were cut and stained for the presence of M. pneumoniae and CARDS TX.
  • CARDS TX is produced in infected cells in vivo, and significant amounts of CARDS TX appear to accumulate in lungs during infection. Toxin can also be detected in areas of the lung where there is no apparent M. pneumoniae staining, suggesting that it is diffusible within infected tissues. Intranasal application of CARDS TX in mice leads to pronounced cellular infiltration to BALF.
  • mice treated with CARDS TX showed a robust infiltration of cells into BALF, whereas animals treated with heat-inactivated CARDS TX remained at baseline levels. Only slight differences were observed when either carrier solution or heat-inactivated TX was used as a control.
  • Cellular levels of infiltration were determined by directly counting cells in BALF using a hemacytometer. FACS analysis of cells in BALF of intoxicated animals revealed a 3-5 fold increase in numbers of GRl+ granulocytes and a 3-10 fold increase in CDl lb+, BM8+ macrophages over the course of the experiment. Numbers of CD4+ and CD8+ T-cells, as well as CD 19+ B-cells, did not change.
  • CARDS TX induces changes in airway resistance and hyperresponsiveness to methacholine treatment.
  • the presence and titer of antibodies reactive against the CARDS toxin and the frequency of cards tx PCR positivity may be considered to be key indicators of disease status.
  • This project will evaluate the prevalence of antibodies to CARDS toxin and detect cards tx DNA by PCR in nasal lavage, sputum and serum in various groups of patients with acute and chronic asthma. Chronic stable asthmatics, patients with acute exacerbation of asthma and a group of asthmatics with refractory asthma will be evaluated.
  • the sensitivity of the CARDS toxin to the "gold' standard Pl assay for M. pneumoniae and the cellular and cytokine responses will be evaluated in these groups of asthmatic patients.
  • Two adult baboons underwent bronchoscopy using an Olympus bronchoscope after sedation and oral intubation. Both animals were lavaged with 20 ml of saline with a return of 10 ml of BALF. Following acquisition of baseline BALF specimens from the left lower lobe, one animal received 50 ⁇ g of rCARDS TX in carrier fluid in the right lower lobe (RLL) bronchus, and the control baboon received the same amount of heat inactivated CARDS TX in the RLL. Thereafter, BALF specimens were obtained on days one and two from both lower lobes.
  • M. pneumoniae strains and other Mycoplasma species were grown in SP4 broth, harvested at mid-to-late exponential growth phase and stored at -80°C. Sputum samples were collected from healthy individuals, and known amounts (in triplicate) of SP4-broth grown M. pneumoniae cells were added. These 'spiked' test preparations were subjected to DNA extraction utilizing a commercial kit (QiaAmp DNA Blood Mini Kit, Qiagen). Based on the cards tx sequence, unique regions were selected for generation of oligonucleotide primers. Distinct bands of expected size were observed with a detection limit of 10 X 10 "15 g, which is equivalent to ⁇ 11 genome copies (816 kbp).
  • PCR was also employed to amplify cards tx sequences from NWs of pediatric patients with chronic wheezing, and in 3 of 8 specimens examined, unequivocal evidence was obtained for the presence of cards tx DNA.
  • the current PCR assay for M. pneumoniae which amplifies unique Pl adhesin gene sequences, was weakly positive in only one patient.
  • Anti-CARDS TX antibodies and PCR products are present in acute, convalescent, and chronic pediatric asthma patients.
  • CARDS TX or Pl gene sequences as determined by PCR indicates the presence of M. pneumoniae (or at least M. pneumoniae DNA) in specific tissues or body fluids.
  • a CARDS TX PCR assay was used to detect the presence of CARDS TX gene sequences in this patient population, which was compared to the existing 'gold standard' published Pl PCR assay.
  • the CARDS TX gene was amplified in serum of 17% of acute asthmatics, 13% of convalescent asthmatics and 4% of chronic asthmatics.
  • Pl was present in serum of 5% of acute asthmatics and in 0% of convalescent or chronic asthmatics (Table 2).
  • CARDS TX gene When NW material was examined by PCR, CARDS TX gene was present in 30% of acute asthmatics, and 13% of convalescent asthmatics. Pl was detectable in 5% of acute asthmatics and undetectable in convalescent asthmatics. NW samples from chronic asthmatics were unavailable.
  • Serum and nasal wash samples from each category were subjected to PCR for cards tx or pi genes as described herein for studies with simulated clinical sputum samples and chronic asthma patient-derived nasopharyngeal washes (NWs).
  • Acute and convalescent categories represent the same group of patients, who presented with acute asthma and were re-studied 6-8 weeks later.
  • the chronic patients represent a separate group who presented with clinically stable chronic asthma. N/A, not available.
  • M. pneumoniae produces CARDS TX in lungs.
  • mice were infected intranasally (IN) with 10 7 CFU of the Sl clinical strain of M. pneumoniae or cards tx null mutant. After 4 days and 18 days, lungs were harvested for immunohistochemistry (using anti-CARDS TX polyclonal antibodies raised against recombinant protein) and histopathology. Fresh frozen lung tissues were prepared and stained for the presence of M. pneumoniae and CARDS TX.
  • CARDS TX and card tx gene and their potentially superior role as a diagnostic target compared to existing reagents for M. pneumoniae should help improve the clinical management M. pneumoniae-associated asthma. More significantly, CARDS TX may be the etiologic factor in M. pneumoni ⁇ e-associated asthma and other related airway diseases.
  • the cards tx gene and anti-CARDS TX antibodies are present in serum and respiratory secretions of asthmatics, as well as people with M. pneumoniae associated pneumonia. Using a primate animal model, applicants have shown that CARDS TX elicits cytokine and chemokine profiles and histopathological changes observed with M. pneumoniae infection. Similar results were observed in murine models.
  • CARDS TX-mediated ADP- ribosyl transferase (ART) activity through detecting CARDS TX minimal domain(s) and amino acids essential for enzymatic activity; and identifying the mammalian proteins that are ADP-ribosylated by CARDS TX.
  • Transcriptional and proteomic analysis will be carried out of CARDS TX in wild type strains and in M. pneumoniae strains having their CARDS TX promoter fused to GFP and luciferase under different environmental conditions.
  • CARDS TX epitopes that serve as antigenic and diagnostic determinants in humans and mice will be mapped and those epitopes capable of inducing neutralizing antibodies will be identified. The goal is to develop effective strategies to diagnose, treat and prevent asthma and related airway diseases in a substantial population of both children and adults.
  • the amino-terminal region of the CARDS TX exhibits amino acid identity (27% of amino acids 1 to 226) with Bordetella pertussis toxin Sl subunit. However, the remaining amino acid residues of MPN372 (amino acids 227 to 591) shared no homologies with other subunits of pertussis toxin, any known toxins, or reported protein sequences.
  • PCR was used to amplify the mpn372 coding region from M. pneumoniae clinical isolate Sl as described herein.
  • the sequence of mpn372 has eight UGA codons at amino acid positions 148, 195, 233, 364, 392, 450, 462 and 508. Therefore, single strand overlap extension polymerase chain reaction (SOE-PCR) was used for site-directed mutagenesis to change each UGA codon into UGG in order to express full-length rCARDS TX protein in E. coli.
  • SOE-PCR single strand overlap extension polymerase chain reaction
  • pET- MPN372 was used to transform E. coli strain BL21 ( ⁇ DE3). Transformants (5000 ml culture) were grown to mid-log phase before inducing CARDS TX expression by addition of IPTG to a final concentration of 1 mM.
  • rCARDS TX was purified in a single step elution with 25OmM imidazole, desalted and concentrated. Protein concentrations were estimated using a BCA protein assay kit (Pierce), aliquoted and stored at -80 0 C.
  • nmLPS non-myristylated LPS
  • nmLPS non-myristylated LPS
  • coif 2 and Salmonella 5 results in strains which grow normally but produce a non-myristylated LPS (nmLPS); the latter exhibits markedly diminished ability to induce cytokine production in vitro and in vivo 53 .
  • nmLPS non-myristylated LPS
  • a ipxM mutant E. coli strain BL21 ⁇ DE3 was constructed 54 . All mycoplasma recombinant proteins in applicants' laboratory, such as CARDS TX and elongation factor-Tu (EF-Tu), are currently expressed and purified from /pxM-inactivated E. coli BL21 (DE3) 54 (provided by Dr. Jean-Francois Gauchat).
  • recombinant proteins are routinely passed through sequential polymixin columns to reduce remaining 'endotoxin' contamination before use on cell lines and in animal experiments described herein.
  • Limulus assays are performed to determine endotoxin concentrations in each recombinant preparation. Endotoxin is measured with the Limulus amebocyte lysate system (Associates of Cape Cod, East Falmouth, MA) according to the manufacturer's directions. All preparations of rCARDS TX and other recombinant mycoplasma proteins/peptides are analyzed for endotoxin levels.
  • recombinant preparations which axe measured at concentrations of 50 ⁇ g recombinant protein/50 ⁇ l contain endotoxin levels at or below the minimal detection levels of the assay (0.1 EU/ml).
  • the assay is linear over a range of 0.1-1.0 EU/ml.
  • IP complete Freund's adjuvant intraperitoneally
  • CARDS TX Primary amino acid sequence alignment shows homologies between PTX Sl subunit (>27% identity over >239 residues) and CARDS TX. Although bacterial ADP- ribosylating enzymes do not share extended amino acid conservation, they exhibit a conserved active site scaffold (Table 3). For example, alignment of the primary amino acid sequence of CARDS TX with PTX Sl and others indicates that CARDS TX contains three conserved motifs common to bacterial ARTs, which are necessary for catalysis 55"57 . These three motifs are: i) a potential catalytic glutamate observed at position 132.
  • Mono-ADP- ribosylation is a post-translational modification of proteins, shared by eukaryotes and prokaryotes, which modulates protein function.
  • Mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose group of ⁇ -nicotinamide adenine dinucleotide (NAD + ) to a specific amino acid in target protein acceptors with the simultaneous release of nicotinamide 58 .
  • rCARDS TX elicits cytopathology (CPE) in Chinese Hamster Ovary (CHO) cells.
  • CHO cells were used as the indicator system since PTX is known to elicit a rounded and clustered appearance in these cells 59 .
  • Endotoxin-def ⁇ cient E. coli 54 were used to generate high yields of rCARDS TX with little to no detectable endotoxin.
  • CHO-Kl cell (ATCC) monolayers were grown to 60-75% confluence in 25-ml flasks using F12-K medium supplemented with 5% fetal bovine serum.
  • This medium was replaced by fresh F12-K medium w/o serum but containing filter-sterilized (0.22 ⁇ m) endotoxin free rCARDS TX, or heat- inactivated rCARDS TX (30 minutes at 100 0 C) or rEF-Tu Mp as negative controls for 2 to 4 hours in a CO 2 incubator at 37°C. Then, serum was added to each culture and incubation continued for 16-48 hours. CHO cells were observed every 6-12 hours for morphological changes. CHO cells cultured in the presence of exogenous rCARDS TX displayed characteristic rounding and distinct vacuolization with disruption of monolayer integrity.
  • Cytopathology was slow to develop at low concentrations of rCARDS TX (10- 50 ng/ml), requiring approximately 16-28 hours, while higher concentrations of rCARDS TX (10-50 ⁇ g/ml) elicited overt CPE in 4-12 hours.
  • Heat inactivation of rCARDS TX preparations abolished CPE, which reinforced the cytotoxic properties of 'heat labile' CARDS TX (i.e., endotoxin is heat stable under these conditions).
  • rEF-Tu preparations (additional negative control) were without cytopathologic effect.
  • rCARDS TX possesses ADP-ribosyltransferase activity.
  • rC ARDS TX ART activity of rC ARDS TX was assayed by determining incorporation Of 32 P ADP-ribose moiety (from ⁇ [ P] NAD) into indicator cell proteins as described for PTX 60 . Briefly, CHO-Kl cell extracts were incubated with (5 to 40 ⁇ g) or without rCARDS TX for 10-30 min in 100 ⁇ l of reaction mixture containing 0.1M Tris-HCl (pH 7.5), O.lmM ATP, O.lmM GTP 5 1OmM thymidine, 2OmM dithiothreitol (DTT) and lO ⁇ M ⁇ 32 P-NAD (10 Ci/mmol).
  • Radiolabeled proteins were TCA precipitated and dissolved in SDS-PAGE sample buffer and heated for 3 min at 100°C. Aliquots were subjected to SDS-PAGE through 4% to 12% NuPAGE gels (Invitrogen) using MES or MOPS as running buffers and standard protocols. After electrophoresis, gels were transferred to nitrocellulose membranes, dried and exposed to Kodak X-OMAT film for 24 h.
  • CHO cell extracts from non-CARDS TX treated cells possessed very weakly radiolabeled bands with apparent molecular masses ranging from 20 to > 90 kDa; i.e., mono-ADP ribosylation occurs in CHO cells via 'inherent' mono ARTs.
  • CHO cell extracts treated with CARDS TX possessed intensely radiolabeled, ADP-ribosylated proteins with apparent molecular masses at 45, 28, 26 and 21 kDa. Studies were undertaken to determine whether sulphydryl agents might influence CARDS TX activity since many bacterial ADP-ribosylating toxins undergo enzymatic activation following reduction of a disulfide bridge 61 and the primary structure of CARDS TX contains six cysteine residues.
  • ADP-ribosylation was markedly increased by the presence of DTT, which suggests that CARDS TX ADP- ribosyltransferase activity is DTT-dependent, similar to cholera 62 and pertussis toxins 63 .
  • the absence of GTP and ATP revealed less noticeable effects on ADP-ribosylation of target proteins.
  • intact CHO and HEp-2 cells were grown in 25 ml tissue culture flasks, incubated with or without CARDS TX (5 to 40 ⁇ g) at 37°C for 16h, and cell extracts were prepared. To these cell extracts, additional rCARDS TX was added along with reaction mixture containing 32 PNAD.
  • Cell extracts prepared from non-CARDS TX treated intact cells contained three prominent radiolabeled proteins with apparent molecular masses of 45, 42 and 26 kDa that were specifically ADP-ribosylated by rCARDS TX.
  • Analysis of host proteins from the intoxicated, intact cell preparation i.e., HEp-2 cells pre-incubated with 5 to 40 ⁇ g rCARDS TX for 16 h
  • CARDS TX gene mpn372 (only one copy per genome) was amplified from four clinical M. pneumoniae isolates (designated Sl, L2, Jl and RJLl) plus reference laboratory strain (B9/M129) by means of primers, which included 10 bp upstream of the start codon and 10 bp downstream of the stop codon. 10 ng of chromosomal DNA from each strain underwent 12 PCR cycles in the presence of high fidelity Taq DNA polymerase as follows: 94°C for 30 s (denaturation), 56°C for 1 min (annealing), 68°C for 2 min (extension).
  • Polymerase chain reaction products were analyzed on 0.8 % agarose gel and compared with 1 kb-plus DNA ladder molecular weight markers (Invitrogen). Amplified fragments were eluted using a Qiagen elution kit, cloned into a TOPO TA cloning vector and transformed into TOP 10 cells. Plasmids with inserts were sequenced by automated DNA sequencing performed on an Applied Biosystems DNA Sequencer model 373A by means of plasmid specific primers (M13 forward and reverse). Sequences were analyzed and translated to amino acids and compared with reference strain M129 using the 'blast two sequences program'.
  • Nucleotide and translated amino acid sequences of CARDS TX from reference strain B9/M129 showed 100% perfect match to published sequences of M. pneumoniae (M129) genome.
  • the translation of nucleotide sequences of clinical isolates revealed changes in amino acids at positions 38, 245, 308, 371, 391 and 392. All clinical isolates exhibited changes at amino acid position 371 (Ile ⁇ Ser). JL had only one change at amino acid position 371 (Ile- ⁇ Ser).
  • RJLl had one additional change (compared to JL) at aa position 392 (Trp ⁇ Arg).
  • L2 had one additional change (compared to JL) at aa position 245 (Asp ⁇ Gly).
  • Sl had three additional changes (compared to JL) at aa positions 38 (Leu ⁇ Pro), 308 (Ser ⁇ Pro) and 391 (Phe ⁇ Ser). These differences are interesting and can serve to link sequence polymorphisms with epidemiological and pathogenic observations in the future.
  • CARDS TX gene locus and promoter mapping Up- and downstream, the CARDS TX locus is flanked by genes encoding hypothetical proteins of unknown function. In contrast to mpn372, adjacent proteins are encoded by the complementary strand, and the presence of all corresponding transcripts has been demonstrated by RT-PCR. This organization of genes suggested a monocistronic transcript for mpn372, and promoter mapping was performed. A transcriptional start of mpn372 message was detected by primer extension (PE). Based on M.
  • PE primer extension
  • pneumoniae genomic DNA sequences 8 forward primer 5'- ACCACAGCAGCGAACCGAGAATGG-3' (SEQ ID NO:113) and reverse primer, MPN372PE, 5'-GCTGTGGGTGTTTCTGAAGTGGAA-S' (SEQ ID NO: 114) were designed.
  • the corresponding upstream region was PCR amplified and cloned, and purified plasmid DNA was used to generate the sequence ladder.
  • the same P-labeled reverse primer (MPN372PE) was used in PE to identify the transcriptional start (5'- TTTTGAAGTTTTTAATTTGTAAAATTTCATTTTTTAAAAATG-S') (SEQ ID NO:115). AU products were electrophoresed on 6% sequencing gels.
  • a short 5' untranslated region (portion of transcript between transcriptional start and ATG-codon) was located 5 nucleotides downstream of the presumed -10 element. The identical single transcriptional start was detected when RNA was isolated at different mycoplasma growth phases and used in PE.
  • M. pneumoniae CARDS TX was expressed in E. coli and rCARDS TX was purified under endotoxin-free conditions. Endotoxin-free rCARDS TX was shown to elicit cytopathology in CHO cells and possesses ADP ribosyltransferase (ART) activity.
  • the CARDS TX gene, mpn372 or cards tx is not organized in an operon and has its own transcriptional start at 10 bp upstream of the start ATG codon.
  • the mpn372 gene was sequenced in different clinical isolates, which indicated the occurrence of limited polymorphisms.
  • CARDS TX is localized in the cytosolic and membrane fractions and some of the CARDS TX is surface exposed but no toxin is detected in the supernatant of in vitro grown mycoplasmas in SP4 broth.
  • Contact of M. pneumoniae with mammalian cells or the in vivo microenvironment provides a considerably different set of signals that up-regulate CARDS TX transcription and translation.
  • M. pneumoniae genes of special relevance to this invention were measured: five genes (mpn372 [cards txj, tuf, gap, pdhA and pdhB) encoding proteins involved in M. pneumoniae binding to host targets, i.e., SP-A or fibronectin, and two housekeeping genes (enolase [eno] and NADH oxidase [nox] which serve as normalizers. Transcriptional levels of these genes were compared by DNA array at different mycoplasma growth phases. Gene-specific primers were designed based on the published M. pneumoniae genome.
  • transposons are used to generate insertional mutations for mapping and/or functional studies, transposons or their insertional elements may continue to transpose around the genome, resulting in multiple insertions and preventing clear interpretation of experimental data 70 . This is particularly relevant when transposons are used in heterologous genetic systems (such as mycoplasmas) where control of the transposase may be compromised.
  • minitransposons have been constructed with an antibiotic resistance marker flanked by inner and outer inverted repeat transposon sequences. In this case, the transposase gene is located outside the inverted repeats to trap the transposon at the initial site of integration 70 .
  • transposon pMT85-mini7>24007, was used (provided by Dr.
  • ⁇ pMT85-m.miTn4001 has a colEl plasmid origin of replication and a selectable gentamicin/kanamycin marker within Tn4001 to allow for direct rescue of chromosomal sequences adjacent to the insertion site.
  • Specific properties of pMT85- ffi ⁇ Tn4001 were described in a poster presentation by the Herrmann lab (International Organization for Mycoplasmology Meeting, July 2005, Athens, Georgia).
  • pMT85- v ⁇ x ⁇ Tn4001 was transformed into competent mycoplasmas by electroporation as described 69 . After growth in selective medium (2 passages), the complete mixture of amplified transposon mutants was collected and M.
  • pneumoniae cell suspensions were passed through 25-gauge needles several times and filtered through membrane filter units with 0.45- ⁇ m-pore size (Millipore) to remove mycoplasma aggregates. Then, mycoplasma cells were diluted in SP4 medium and plated on SP4 agar containing 80 ⁇ g/ml gentamicin. Chromosomal DNAs from individual Tn-integrated colonies were Hindffl digested, self-ligated and transformed in E. coli ToplO. Plasmid DNAs were isolated and subjected to DNA sequencing in order to identify the place of integration of the transposon in the chromosomal DNA of M. pneumoniae. Recently, among hundreds of colonies screened, a M.
  • pneumoniae transposon mutant was isolated that had integration in the cards tx gene.
  • mini-Th- 4001 integration was confirmed, and SDS-PAGE/immunoblot analysis confirmed that CARDS TX synthesis was absent in the mutant strain.
  • CARDS TX is believed to be up-regulated when M. pneumoniae contacts host cells or in vivo microenvironments; based upon observations from the studies described herein.
  • mini-Tn-4001 a CARDS TX null mutant of M. pneumoniae was isolated. Collection of M. pneumoniae infected patient sera. Acute and convalescent-phase sera obtained during an outbreak in 1993
  • test sera were diluted in 1% BSA in PBS.
  • Individual wells of Immulon 4 HBX Immunoplates (Dynox) were coated at 4°C overnight with 50 ⁇ l of rCARDS TX (or ⁇ PDH-BM P or ⁇ EF-TUM P for comparative purposes), and diluted (50 ng to 3 ⁇ g/well) in carbonate-bicarbonate buffer (32 mM Na 2 C ⁇ 3 , 64 mM NaHCO 3 ). Plates were washed, 100 ⁇ l of 1 % (wt/vol) BSA in PBS added to each well, and plates further incubated for 2 h at room temperature. After washing, 50 ⁇ l of diluted human sera were added to each well.
  • Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol 97:286-92. Barile, M. F. 1984. Immunization against Mycoplasma pneumoniae disease: areview. Isr J Med Sci 20:912-5.
  • Cimolai N., G. P. Taylor, D. Mali, and B. J. Morrison. 1992. Definition and application of a histopathological scoring scheme for an animal model of acute Mycoplasma pneumoniae pulmonary infection. Microbiol Immunol 36:465-78. Fonseca Aten M, Rios AM, Mejias A, Chavez-Bueno S, Katz K, Gomez AM,
  • Mycoplasma pneumoniae induces chronic respiratory infection, airway hyperreactivity, and pulmonary inflammation: a murine model of infection-associated chronic reactive airway disease. Infect Immun. 70: 649-654.
  • Mycoplasma pneumoniae pneumonia model a microbiologic, histologic, immunologic, and respiratory plethysmographic profile. Infect Immun. 69: 3869-
  • Chlamydia pneumoniae in asthma effect of clarithromycin. Chest. 121 : 1782-1788.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Pulmonology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des toxines mycoplasma, des domaines/fragments biologiquement actifs de ces toxines, des anticorps pour ces toxines, des protéines de fusion thérapeutiques comprenant ces toxines et/ou des domaines/fragments biologiquement actifs de ces toxines et des acides nucléiques codant pour ces toxines et ces protéines de fusion. Cette invention concerne aussi des techniques de traitement et/ou de prévention de maladies et de pathologies au moyen de ces compositions.
PCT/US2006/012266 2005-04-07 2006-03-31 Procedes et compositions pour toxines mycoplasma WO2006110367A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/887,995 US20090104185A1 (en) 2005-04-07 2006-03-31 Methods and Compositions for Mycoplasma Toxins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US2005/011897 WO2006110132A1 (fr) 2005-04-07 2005-04-07 Methodes et compositions pour les exotoxines mycoplasma pneumoniae
USPCT/US2005/011897 2005-04-07

Publications (2)

Publication Number Publication Date
WO2006110367A2 true WO2006110367A2 (fr) 2006-10-19
WO2006110367A3 WO2006110367A3 (fr) 2006-12-21

Family

ID=37087319

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2005/011897 WO2006110132A1 (fr) 2005-04-07 2005-04-07 Methodes et compositions pour les exotoxines mycoplasma pneumoniae
PCT/US2006/012266 WO2006110367A2 (fr) 2005-04-07 2006-03-31 Procedes et compositions pour toxines mycoplasma

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2005/011897 WO2006110132A1 (fr) 2005-04-07 2005-04-07 Methodes et compositions pour les exotoxines mycoplasma pneumoniae

Country Status (1)

Country Link
WO (2) WO2006110132A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838691A (zh) * 2022-10-08 2023-03-24 苏州大学附属儿童医院 抗人肺炎支原体cards tx单克隆抗体mm03h

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040191260A1 (en) * 2003-03-26 2004-09-30 Technion Research & Development Foundation Ltd. Compositions capable of specifically binding particular human antigen presenting molecule/pathogen-derived antigen complexes and uses thereof
ZA200305980B (en) * 2001-02-12 2007-01-31 Res Dev Foundation Modified proteins, designer toxins, and methods of making thereof
GB0108024D0 (en) * 2001-03-30 2001-05-23 Chiron Spa Bacterial toxins

Also Published As

Publication number Publication date
WO2006110367A3 (fr) 2006-12-21
WO2006110132A1 (fr) 2006-10-19

Similar Documents

Publication Publication Date Title
US8088894B2 (en) Methods and compositions for Mycoplasma pneumoniae exotoxins
EP1666058B1 (fr) Méthode pour la détection de l'infection par Pseudomonas aeruginosa
EP1219711B1 (fr) Lawsonia intracellularis vaccin
US9176133B2 (en) Immunogenic proteins of Leptospira
KR20010104693A (ko) 신규한 스트렙토코커스 항원
KR20010052767A (ko) 백신
Valentine et al. Identification of three highly attenuated Salmonella typhimurium mutants that are more immunogenic and protective in mice than a prototypical aroA mutant
US8324354B2 (en) Environmentally regulated genes of Streptococcus suis
US20090104185A1 (en) Methods and Compositions for Mycoplasma Toxins
US20020102242A1 (en) Compositions and methods for administering pneumococcal DNA
Gianfaldoni et al. Sortase A confers protection against Streptococcus pneumoniae in mice
WO2006110367A2 (fr) Procedes et compositions pour toxines mycoplasma
US8586046B2 (en) Methods and compositions for mycoplasma pneumoniae exotoxins
AU2004278035B2 (en) Methods and compositions for Mycoplasma pneumoniae exotoxins
Kai was supported in part by a Health Science Research Grant for Re-search on Emerging and Re-emerging Infectious Diseases, Ministry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06749143

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 11887995

Country of ref document: US