# Patent

## Title
Cryptosporidium hominis genes and gene products for chemotherapeutic, immunoprophylactic and diagnostic applications

## Abstract

Cryptosporidium hominis genes and gene products are provided. The genes and gene products are useful for chemotherapeutic, immunotherapeutic, immunoprophylactic and diagnostic applications.

## Background
BACKGROUND OF THE INVENTION
This application is a national stage entry of PCT/US05/31657, International Filing Date: Sep. 7, 2005 and claims priority to Provisional Application 60/607,356, filed Sep. 7, 2004.


STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under Contract Number 5u1a146416 awarded by the National Institutes of Health, NIAID. The Government has certain rights in the invention.


FIELD OF THE INVENTION
The invention generally relates to Cryptosporidium hominis genes and gene products In particular, the invention provides Cryptosporidium hominis genes and gene products for use in chemotherapeutic, immunotherapeutic, immunoprophylactic and diagnostic applications.
BACKGROUND OF THE INVENTION
Cryptosporidium species are the causative agent of cryptosporidiosis, a disease that is characterized by acute gastro-enteritis and diarrhea. The disease is rampant in many developing countries (e.g. in Latin America, Africa, and Asia). However, incidents of cryptosporidiosis occur worldwide and developing countries are not immune to such incidents. For example, an outbreak in Milwaukee, Wis. in the mid 1990's caused over 400,000 human infections. Thus, no site on earth is free from the threat of serious outbreaks of cryptosporidiosis. Further, the National Institutes of Health and the Center for Disease Control classify Cryptosporidium as an important “Category B” agent of potential biological terrorism.
Cryptosporidium species are members of the Apicomplexa. These protozoan pathogens invade host cells using a specialized apical complex, and are usually transmitted by an invertebrate vector or intermediate host. Unlike most Apicomplexans, Cryptosporidium completes its life cycle in a single host and is transmitted by ingestion of oocysts. Two species, C. hominis, and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans whereas C. parvum also infects other mammal species.
Currently, there are no effective treatments for cryptosporidiosis, and control focuses on eliminating oocysts from water supplies. However, the resistance of the organism to common inexpensive water treatments seriously hampers this method of control. Despite decades of sophisticated molecular analysis of this and related organisms (e.g., Plasmodia (malaria parasites), Babesia, Eimeria, Toxoplasma, etc.), no significant advances have been found in treatment or prevention. Thus, traditional approaches of the prior art have thus far failed to identify the proverbial ‘Achilles heel’ of Cryptosporidium, and to provide viable therapies for the prevention or treatment of cryptosporidiosis.
SUMMARY OF THE INVENTION
The present invention is based on the determination of the sequence of the eight chromosome  ̃9.2 Mb genome of C. hominis, and the analysis of that sequence. Using genomics and bioinformatic approaches, a wide array of new targets for therapeutic and diagnostic applications has been identified. Moreover, an accurate view of the overall metabolism of the organism has been obtained, permitting a realistic approach to identification of critical points of attack for therapeutics.
The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Options for both aerobic and anaerobic metabolism are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements and phenotypic differences between these parasites must be due to subtle sequence divergence.
The invention thus provides newly identified genes and gene products of C. hominis that are of use in chemo and immunotherapy, immunoprophylaxis, and diagnostic applications.
It is an object of this invention to provide amino acid sequences as represented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24. It is a further object of this invention to provide nucleotide sequences as represented in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NOS: 25-4000.
The invention further provides a method of detecting and diagnosing C. hominis infection in a patient in need thereof. The method comprises the step of obtaining a biological sample from said patient, and amplifying nucleotide sequences from said sample, and determining infection based on whether there are one or more amplicons are produced.
The invention further provides a method for immunizing a patient against C. hominis infection. The method comprises the step of administering to the patient a non-virulent C. hominis peptide or nucleotide in an amount sufficient to permit the patient to mount an antibody response to the non-virulent C. hominis peptide or nucleotide. By non-virulent, we mean that the peptide or nucleotide does not cause pathology (e.g. symptoms of C. hominis infection).
The invention further provides a method for detecting Cryptosporidium such as C. hominis in a sample, for example, of detecting contamination of a water supply by Cryptosporidium. The method comprises the steps of 1) obtaining a sample; 2) amplifying nucleotide sequences from the sample; and 3) detecting amplicons produced in the step of amplifying. The production of amplicons in the amplifying step indicates the presence of C. hominis in the sample. The method may further include the step of quantifying the amount of amplicons that is produced. This amount will be indicative of the amount of Cryptosporidium (e.g. C. hominis) in the sample. The method may be used, for example, to detect Cryptosporidium contamination in environmental samples such as water. In some embodiments, the sample may include several Cryptosporidium species other than C. hominis. In this case, the determining step permits discrimination between C. hominis and other Cryptosporidium species in the sample.
The invention further provides arrays for assessing the presence or expression of genes in C. hominis, and for detecting the interaction (e.g. binding) of C. hominis macromolecules. The array is a device comprising macromolecules such as nucleic acid probes, peptides, proteins, or antibodies, all of which originate from nucleic acid sequences of C. hominis, i.e. they are identical to or homologous to C. hominis genomic sequences; or are encoded by C. hominis genome sequences; or, in the case of antibodies, they bind to such macromolecules. In particular, the nucleic acid probes include nucleotide sequences homologous to nucleotide sequences of C. hominis. In one embodiment, the nucleic acid probes are at least 70 nucleotides in length.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Schematic representation of putative cellular components and metabolic pathways in C. hominis. The cytoplasmic membrane is indicated by a broad stripe at the top of the figure, and the intracellular compartment is below the stripe. Arrows and lines indicate pathways that are present. Dotted lines indicate multi-step reactions. Numbers indicate the quantity of transporters found for a given class when there is more than one. Important components or pathways that are clearly not present in C. hominis are crossed-out. Steps or components about which the exact nature is unknown or is questionable are shown with question marks. Arrows () and names indicate proposed aerobic parts of the energy metabolism. Abbreviations: ABC, ATP-binding cassette; MRP, multi-drug resistance protein; PEP, phosphoenolpyruvate; THF, tetrahydrofolate; DHF, dihydrofolate; AOX, alternative oxidase; UQ, ubiquinone; NADH DH, NADH dehydrogenase; Hsp70, heat-shock protein 70; Cpn60, chaperone 60; TIM17, translocase of the inner mitochondrial membrane 17; TOM40, translocase of the outer mitochondrial membrane 40; FAS, fatty-acid synthase; PKS, polyketide synthase; GPI, glycosylphosphatidylinositol; IP, inositol phosphate; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; AC, adenilate cyclase; Ado, adenosine; Cyd, cytidine; dThd, deoxythymidine; Urd, uridine; PNO-CPR, pyruvate:NADP+ oxidoreductase fused to cytochrome P450 reductase domain; Narf-like, nuclear prelamin A recognition factor like protein.
FIGS. 2A and B. A, typical amplification curve plots; B, standard curve plot of known amount of C. hominis rRNA.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
The genetic sequence of the eight chromosome  ̃9.2 Mb genome of C. hominis has been determined and analyzed (see Table 1). By using genomics and bioinformatic approaches, a wide array of new targets for therapeutic and diagnostic applications has been identified. In addition, the overall metabolism of the organism has been elucidated, permitting a realistic approach to identification of critical points of attack (i.e. therapeutic targets) for therapeutics directed to the prevention or treatment of cryptosporidiosis. By “therapeutic target” we mean a molecule or pathway, the function of which can be targeted for intervention (e.g. blocking or inhibition) by the administration of agents such as drugs, or by stimulation of a specific immune response (by induction or passive administration (such as an antibody production. Such an intervention results in a decreased ability of the parasite to cause disease or disease symptoms. For example, such intervention may result in total or partial inhibition of the parasite's ability to carry out normal metabolism, to reproduce, to infect cells, to escape from infected cells, to proceed through a normal life cycle, to resist treatment with other disease alleviation agents, to navigate successfully, or to otherwise exert its pathological effects and cause disease.
In addition, C. hominis proteins that have been identified (or non-toxic or inactive fragments thereof) are useful for stimulating an immune system response to antigenic determinants of the proteins in an individual to whom they are administered. The nucleotide sequences and selected amino acid sequences of such proteins are provided in the Sequence Listing, the complete contents of which is hereby incorporated by reference. An immune system response may include increased antibody titers and/or increased cellular immunity, and, in a preferred embodiment, results in immunity to C. hominis. In other words, the proteins, or regions of the proteins encompassing antigenic determinants may be administered as vaccines for immuno prophylaxis. Alternatively, antibodies to antigenic determinants of the C. hominis proteins may also be administered directly to individuals in order to treat or prevent disease symptoms caused by C. hominis, i.e. immuno therapy. In addition, such antibodies may be useful as diagnostic or laboratory tools.
The highly tailored physiology of C. hominis suggests several potential therapeutic targets, examples of which include but are not limited to: 1) transport systems for peptides, amino acids, nucleosides, and sugars; 2) components of glycolysis; 3) the unique prokaryotic FAS1 (fatty acid synthase 1) and PKS1 (polyketide synthase 1); 4) starch and amylopectin biosynthesis or catabolism; 5) nucleic acid or amino acid metabolism; 6) the AOX (alternate oxidase) electron transport system; 7) the TS-DHFR (thymidine synthase-dihydrofolate reductase); and 8) the diverged polyamine synthesis enzymes. Importantly, and unlike other protozoan parasites, no extensive arrays of potentially variant surface proteins were observed, suggesting a possible role for immunoprophylaxis for prevention of cryptosporidiosis.
In addition, the invention provides the DNA sequences that encode numerous ribosomal RNA (rRNA) molecules (SEQ ID NOS: 4003-4014; see Table 14) and transfer RNA (tRNA) molecules (SEQ ID NOS: 4015-4059; see Table 13). These sequences are also provided in the Sequence Listing, the complete contents of which is hereby incorporated by reference. Those of skill in the art will recognize that rRNA and tRNA sequences may have many uses, including but not limited to: detection of Cryptosporidium in clinical samples, diagnosis of cryptosporidiosis in humans and animals, identification of cryptosporidium carriers, detection of Cryptosporidium in water supplies, detection of Cryptosporidium contamination in lakes, rivers or other environmental sites, etc. These targets, in particular the rRNA targets, are very useful for discrimination of Cryptosporidium strains; e.g., C. hominis and C. parvum. Finally, these targets are useful for quantification of Cryptosporidium in any of the above, or other, clinical, water or environmental samples. An example of this is shown in Example 6 below.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
The present invention provides nucleic acid sequences from Cryptosporidium hominis, as provided in the Sequence Listing (and see Table 1). Those of skill in the art will recognize that such sequences may be used for a variety of purposes, including but not limited to translation of the amino acid sequences encoded therein, as probes for diagnostic assay for the detection of C. hominis, for expression in bacterial systems, for expression in fungal or insect cell systems, for generation of recombinant and subunit vaccines or immuno therapeutics, for generation of active enzymes, for purification for structural determination (e.g., standard biochemical and biophysical applications such as NMR, MS, XRay crystallography), for the construction of RNA and protein microarrays, for the construction of nucleic acid libraries, for comparative genome analysis, for phylogeny and taxonomy, for prediction of putative blocking peptide domains, etc. With respect to the nucleic acid sequences disclosed herein, those of skill in the art will recognize that many variants (derivatives) of the sequences may exist or be constructed which would still be suitable for use in the practice of the present invention. For example, with respect to the translation of amino acid sequences from the nucleic acid sequences, due to the redundancy of the genetic code, more than one codon may be used to code for an amino acid. Further, as described below, changes in the amino acid primary sequence may be desired, and this would necessitate changes in the encoding nucleic acid sequences. In addition, those of skill in the art will recognize that many variations of the nucleic acid sequences may be constructed for purposes related to other aspects of the invention, for example: for cloning strategies (e.g. the introduction of restriction enzyme cleavage sites for ease of manipulation of a sequence for insertion into a vector, for rendering the sequence compatible with the cloning system vector or host, for enabling fluorescent or affinity labeling technologies, etc.), for purposes of modifying transcription (e.g. the introduction of specific promoter or enhancer sequences, insertion or deletion of splice signals, for enhancing or negatively regulating transcription levels, for regulating polyadenylation, for controlling termination, and the like), or for modification of active or inactive domains, for elimination or modification of certain activities or domains, for optimizing expression due to codon usage or other compositional biases, for addition of immunologically relevant (enhancing or inhibiting) sequences or for any other suitable purpose. All such variants of the nucleic acid sequences disclosed herein are intended to be encompassed by the present invention, provided the sequences display identity in the range of about 50 to 100%, and preferably about 60 to 100%, or more preferably about 70 to 100%, or even more preferably about 80 to 100%, or most preferably about 90 to 100% or about 95 to 100% to the disclosed sequences. The identity is with reference to the portion of the nucleic acid sequence that corresponds to the original sequence, and is not intended to cover additional elements such as promoters, vector-derived sequences, restriction enzyme cleavage sites, etc. derived from other sources. Those of skill in the art are well-acquainted with methods to determine nucleic acid similarity or identity using simple software alignment tools such as FASTA, the BLAST suite of programs, CLUSTAW, Lineup, Pileup (GCG), or many others.
In addition, the nucleic acids of the present invention are not limited to DNA, but are intended to encompass other nucleic acids as well, such as RNA (e.g. mRNA, tRNA, rRNA, etc.), RNA-DNA hybrids, and various modified forms of DNA and RNA known to those of skill in the art. For example, for use in vivo, nucleic acids may be modified to resist degradation via structural modification (e.g. by the introduction of secondary structures, such as stem loops, or via phosphate backbone modifications, etc.). Alternatively, the nucleic acids may include phosphothioate or phosphodithioate rather than phosphodiesterase linkages within the backbone of the molecule, or methylphosphorothiate terminal linkages. Other variations include but are not limited to: nontraditional bases such as inosine and queosine; acetyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine; stabilized nucleic acid molecules such as nonionic DNA analogs, alkyl- and aryl phosphonates; nucleic acid molecules which contain a diol, such as tetrahyleneglycol or hexaethyleneglycol, at either or both termini; etc. Further, the nucleic acid molecules may be either single or double stranded, or may comprise segments of both single and double strand nucleic acid.
The invention also provides vectors comprising nucleic acid sequences of the invention. Those of skill in the art are well-acquainted with various vectors that may be used e.g. for manipulation of nucleic acid sequences during genetic engineering procedures, for storage of stocks of the nucleic acid, for expression of an amino acid sequence encoded by the nucleic acid, for expression in bacterial, fungal, insect or other host systems, for delivery of DNA vaccines, for amplification of the DNA, for sequence analysis, for molecular interaction studies, etc. Many such vectors are known to those of skill in the art, and include but are not limited to plasmids, adenoviral vectors, various expression vectors (e.g. PTRIEX4, PET41, PET44, and others of the PET series, the pUC vector series, the BlueScript series, derivatives of pBR322 with ColE1 origin of replication, the TOPO vector series, the Gateway vectors, the TET repressor vectors, BAC vectors [pBeloBACs, pCC1BAC, etc.], pcDNA301 and related plasmids with the CMV promoter, pBAC insect vectors, pIEX for insect cells, and many others).
Many of the nucleotide sequences of the present invention represent open reading frames (ORFs) that encode for amino acid sequences, e.g. peptides, polypeptides, and proteins, all of which are also intended to be encompassed by the present invention. In general, for the purposes of the present invention, a peptide comprises about 15 or fewer amino acids, a polypeptide comprises from about 15 to about 100 amino acids, and a protein comprises about 100 or more amino acids, although the terms may be used interchangeably herein. The peptides, polypeptides and proteins of the present invention are generally provided as recombinant molecules, although the amino acid sequences may also be produced synthetically via known peptide synthesis techniques. The peptides, polypeptides and proteins of the present invention are provided in a substantially purified form, i.e. they are generally free of extraneous materials (such as other proteins, nucleic acids, lipids, cellular debris, etc.) and will generally be at least about 75% pure, preferably about 85% pure, and most preferably at least about 90-95% or more pure, as would be understood by one of ordinary skill in the art.
Importantly, the present invention comprehends all amino acid sequences that may be translated from the nucleic acid sequences of the present invention. In general, proteins/polypeptides that are so-translated will be translated from an open reading frame. The invention also encompasses variants (derivatives) of such proteins/polypeptides. For example, variants may exist or be constructed which display: conservative amino acid substitutions; non-conservative amino acid substitutions; truncation by, for example, deletion of amino acids at the amino or carboxy terminus, or internally within the molecule; or by addition of amino acids at the amino or carboxy terminus, or internally within the molecule (e.g. the addition of a histidine tag for purposes of facilitating protein isolation, the substitution of residues to alter solubility properties, the replacement of residues which comprise protease cleavage sites to eliminate cleavage and increase stability, the replacement of residues to form a convenient protease cleavage site, the addition or elimination of glycosylation sites, and the like, for any reason). Such variants may be naturally occurring (e.g. as the result of natural variations between species or between individuals, or as a result of different expression systems used to produce the amino acid sequence, etc.); or they may be purposefully introduced (e.g. in a laboratory setting using genetic engineering techniques). The amino acid sequences may be in a variety of forms, including a neutral (uncharged) forms, or forms which are salts, and may contain modifications such as glycosylation, side chain oxidation or deamidation, phosphorylation and the like. Also included are amino acid sequences modified by additional substituents such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions or the chains, such as oxidation of sulfhydryl groups.
Strategies for improving solubility of cloned proteins are known to those of skill in the art. Such strategies may be used in the practice of this invention, and include: modifying expression conditions (temperature, buffer, nutrients), modification of the promoter or its activity, linking the protein to a different fusion protein that helps it maintain its solubility, expression in the yeast system Pichia pastoris, the insect baculovirus system, or in another eukaryotic organism, etc. Expression of soluble proteins is difficult in bacterial systems, but success is much more common in Pichia, baculovirus or mammalian expression systems.
All such variants of the sequences disclosed herein are intended to be encompassed by the teachings of the present invention, provided the variant protein/polypeptide displays sufficient identity to the original sequences, the original sequence being a sequence as disclosed herein, or an amino acid sequence that can be translated from a nucleic acid sequence disclosed herein, (e.g. from an ORF or portion thereof). Preferably, amino acid identity will be in the range of about 50 to 100%, and preferably about 60 to 100%, or more preferably about 70 to 100%, or even more preferably about 80 to 100%, or most preferably about 90 to 100%, or even 95 to 100%, of the disclosed sequences. The identity is with reference to the portion of the amino acid sequence that corresponds to the original amino acid sequence as translated directly from the nucleic acid sequences disclosed herein, i.e. not including additional elements that might be added, such as sequences added to form chimeric proteins, histidine tags, etc. Those of skill in the art are well acquainted with the methods available for determining the identity between amino acid sequences, for example, FASTA, FASTP, the BLAST suite of comparison software, ClustalW, Lineup, Pileup, or many other alignment software packages.
In addition, such protein/polypeptide variants retain at least about 50 to 100% or more of the activity of the original polypeptide, and preferably about 60 to 100% or more, or more preferably about 70 to 100% or more, or even more preferably about 80 to 100% or more, and most preferably about 90 to 100% or more of the activity of the original sequence. By “activity” we mean the activity or role of the amino acid sequence in C. hominis, which may include but is not limited to: enzymatic activity, activity as a structural component, activity as a transporter protein, activity in signal transduction, role as a membrane component, binding activity, activating activity, transport activity, etc.
In general, the amino acid sequences of the invention are produced in recombinant expression systems. In a preferred embodiment of the present invention, the recombinant system is an E. coli recombinant system, which can be expressed as well in mammalian cells for use, for example, as a DNA vaccine. However, the amino acid sequences may be produced in a variety of other recombinant expression systems. For example, yeast, insect cells (using for example, a baculovirus expression vector), plant cells (e.g. tobacco, potato, corn, etc.), transgenic animals, or mammalian cell culture systems can be used for expression of recombinant proteins. Any appropriate expression system that suitably produces the amino acid sequences of the invention may be used in the practice of the invention. Such systems and their use for the production of recombinant proteins are well known to those of ordinary skill in the art.
In some embodiments, vectors containing nucleic acid sequences (e.g. DNA) that encode the amino acid sequences of the invention will encode a single protein. However, this need not always be the case. Such vectors may contain DNA encoding more than one nucleic acid of the invention, either as separate, discrete sequences, or combined into a single chimeric sequence. For example, in the case of an expression vector, two or more nucleic acids according to the invention may be present in the vector, and the nucleic acids may be expressed separately, resulting in the translation of one amino acid sequence for each nucleic acid. Alternatively, a single polypeptide chain containing more than one amino acid sequence of the invention, or portions of more than one amino acid sequence of the invention, may be combined in tandem. For example, one or more highly antigenic proteins or regions of proteins of the invention may be expressed as a chimera from a single DNA sequence. Alternatively, the amino acid sequences of the invention may be expressed as part of a chimeric protein comprising amino acid sequences from another source, e.g. antigenic sequences known to be useful as adjuvants (e.g. PADRE [and other Pan-DR T helper cell epitope], hepatitis B core antigen, DNA sequences CPG, other chemokines, CTB or cholera toxin B subunit, Ricin B and other plant toxin subunits, LPS or lipopolysaccharide, KLH [key hole limpet hemocyanin], Freund's complete and Freund's incomplete adjuvant, and many other reagents, etc.), sequences that permit targeting of the protein to a specific location within the cell (e.g. nucleus, nucleolus or nuclear membrane, mitochondrion/mitosome/mitochondria-like organelle, membrane, endoplasmic reticulum, golgi, rhoptry, dense granules, calcisomes or acidocalcisomes, and other subcellular organelles compartments, etc.).
The invention also comprehends a cell or cells containing the nucleic acids and/or the amino acid sequences of the invention. For example, the cell may be a host cell that harbors one or more vectors containing nucleic acid sequences of the invention (e.g. DNA or RNA) and/or amino acid sequences of the invention translated from such vectors. Such cells may contain multiple vectors, and the vectors may be the same or different. Further, the cells may be either in vitro or in vivo.
The invention also provides antibodies directed to the amino acid sequences of the present invention. As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins and fragments, and single domain antibodies. Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods that are well-known to those of skill in the art. If desired, the antibodies (whether polyclonal or monoclonal) may also be labeled using conventional techniques.
Such antibodies may be used, for example, for affinity chromatography, immunoassays, and for distinguishing or identifying C. hominis proteins or portions thereof. In a preferred embodiment of the invention, such antibodies may be used therapeutically, e.g. for administration to patients suffering from cryptosporidiosis, or prophylactically in order to prevent cryptosporidiosis in patients at risk for developing the disease.
The invention also comprehends pharmaceutical compositions. The pharmaceutical compositions can comprise polypeptides, antibodies, or nucleic acids of the invention, or combinations of these. The pharmaceutical compositions will comprise a therapeutically effective amount of a polypeptide, antibody, or polynucleotide of the invention. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction of physical symptoms of cryptosporidiosis. The precise effective amount for a subject will depend upon several parameters, including the subject's size, general health, gender, age, etc., and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of those of skill in the art, e.g. a physician. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg of active, therapeutic agent.
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
In addition, pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.
Once formulated, the compositions of the invention can be administered to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated. Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and intranasal, transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
The present invention also encompasses vaccines that provide immunity to disease caused by C. hominis. By “immunity” we mean that administration of one or more proteins, polypeptides or peptides of the present invention to an individual either prevents the development of disease symptoms in that individual when exposed to or infected by C. hominis, or the disease symptoms that develop in the individual are milder than those that would otherwise develop, for example, the disease symptoms that would develop in a matched control individual. Those of skill in the art are well acquainted with the use and meaning of “controls” when comparing results of individuals or populations that have been exposed to different variables (e.g. vaccinated or not).
According to the invention, the vaccine may either be prophylactic (i.e. to prevent or attenuate symptoms of infection) or therapeutic (i.e. to treat disease after infection). Such vaccines comprise one or more of: immunizing antigen(s), immunogen(s), polypeptide(s), protein(s) and nucleic acid(s) from C. hominis (as described herein), usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59TM (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); (3) saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor c[NF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59TM are preferred.
The immunogenic compositions (eg. the immunizing antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for eliciting the production of antibodies, for eliciting a cellular immune response, (or both), and/or for treatment or prevention of disease. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, intranasally, or transdermally/transcutaneously. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648].
In a preferred embodiment of the invention, the proteins that are used in an immunogenic preparation or vaccine of the present invention include at least one of the proteins represented by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 (see Example 2 below).
The present invention also provides tools and methods for the diagnosis of C. hominis infections. Such tools include primers containing nucleotide sequences that specifically hybridize to nucleic acid sequences that are unique to the genome of the C. hominis species. Hybridization of the primers to such a unique sequence permits amplification of the unique sequence (for example, by polymerase chain reaction (PCR)), thus providing a means to specifically identify the presence of C. hominis in biological samples (blood, feces, sputum, urine, bronchoaveloar lavage, etc.). Amplification may be directly from the genome of the organism located in the sample, or from RNA, e.g. from ribosomal RNA (rRNA), which is typically highly expressed and thus more sensitive than DNA as a target. Because the sequences that are amplified are unique to C. hominis, it is possible to distinguish infection by C. hominis from infection with even closely related parasites. By “primer” we mean a nucleotide sequence that hybridizes to another nucleotide sequence of interest, the primer typically being a relatively short nucleotide sequence (e.g. from about 10 to about 100 base pairs) and the nucleotide sequence of interest typically being transcribed from the genome of an organism. PCR amplification techniques are well-known to those of skill in the art
In general, two primers are selected that target sites that flank the sequence of interest for diagnostics or identification. These primers are designed to recognize only the target sequence; i.e., they will hybridize only to the target sequence and to no other sequences. Thus, the sequence is screened against all other known sequences to ensure that there is no other known sequence to which it will hybridize. The primers generally range from 18-30 nucleotides in length (but can be longer or shorter), have Tm's (melting temperatures) that are selected to be compatible with both amplification conditions and with specificity, have little or no internal structure (stem-loop structures caused by internal complementarity), little or not ability to dimerize with themselves, little or no ability to dimerize with the other primer, have few homopolymeric stretches, etc. Many computer programs (e.g., Primer3, Oligo, etc.) are available for these purposes. At times, an internal fluorescent probe is also included for specific use in even more sensitive and automated tests. The internal probe is fluorescently labeled such that it is specifically degraded and therefore fluoresces only if it specifically hybridizes to the target sequence. Alternately, other fluorescent probes can be designed that only fluoresce upon binding specifically to an amplified specific sequence. Thus, several alternative approaches are available for the generation and detection of specific sequences amplified by PCR, and any of these can be applied for diagnostic or identification purposes. (See, for example: Mullis, K., F. Faloona, S. Scharf, R. Saki, G. Horn, and H. Erlich. (1986) Specific enzymatic amplication of DNA in vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biology 51: 263; Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487; Schutzbank T E, Stern H J. (1993) Principles and applications of the polymerase chain reaction. J Int Fed Clin Chem. 1993 July; 5(3):96-105; Erlich H A. (1999) Principles and applications of the polymerase chain reaction. Rev Immunogenet. 1(2): 127-34; Wang, A. M., Doyle, M. V., and D. F. Mark. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA. 1989 December; 86(24): 9717-9721; Kawasaki, E. S., and A. M. Wang. (1989) Detection of gene expression. In: Erlich, H. A., ed., PCR Technology: Principles and Applications of DNA Amplification. Stockton Press, Inc., New York, N.Y., pp. 89-97; Dieter Klein (2002) Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine, 8(6):257-260; Buck G E. (1996) The polymerase chain reaction: a revolutionary new procedure for the laboratory diagnosis of infectious disease. J Ky Med. Assoc. April; 94(4):148-52.)
Because the nucleotide sequences that are being amplified are unique to C. hominis, a positive amplification result is indicative of the presence of C. hominis in the biological sample, and thus of infection by C. hominis. In contrast to current methods of C. hominis detection, the present invention, by elucidating the entire genome of C. hominis, allows the design of a much larger number of such primers. Further, primers provided by the present invention hybridize to sequences that are highly diverse from species closely related to C. hominis. Thus, the accuracy of the diagnostic methods of the present invention are superior to those of the prior art, resulting in very few false positives, and very few missed diagnoses.
The present invention also provides arrays of nucleotide or modified nucleotide, or protein or modified protein sequences, or antibodies to the nucleotides or proteins, for use as probes for assessing the presence or absence of genes, the expression of specific genes, the presence or absence of proteins or immune responses in C. hominis or other Cryptosporidium species. In particular, the arrays of the invention may be used to specifically assess the differential expression of genes any of the various stages of the life cycle of C. hominis, for example, during the sporozoite stage. In order to do so, a biological sample containing C. hominis parasites at a particular stage of development is provided and analyzed using the array. Alternatively, such arrays may be used to monitor C. hominis parasites under other conditions of interest, for example, to monitor gene expression in response to an external stimulus or variable, such as introduction of a drug or other agent into a culture of the parasites; or to monitor the course of a C. hominis infection, either in culture, or in an infected individual. Similarly, the arrays can be used for comparative genome analyses in which the presence or absence of specific genes are probed in different isolates of Cryptosporidium. In order to do so, DNA from the isolate of interest is analyzed using the array. Presence or absence of a particular gene is assessed via hybridization. Using this approach, the array can also be designed to detect specific polymorphisms in genes that have clinical or other relevance. This approach can have diagnostic and clinical importance, or other relevance to infection, treatment or contamination (e.g., water contamination with C. hominis is much more serious than water contamination with other species of Cryptosporidium. The nucleotide probes (usually DNA or a derivative thereof) in an array typically have a length in the range of about 50 to about 90 nucleotides, and preferably about 60 to about 80 nucleotides, and most preferably the probes have a length of about 70 nucleotides. These arrays can be comprehensive; i.e., be comprised of probes for all genes/gene sequences in a genome, or targeted to specific subsets of genes/gene sequences, depending on the application. Peptides, proteins or derivatives thereof (or antibodies thereto) can also be used in similar arrays. In this case, synthetic peptides, proteins (recombinant or otherwise) or derivatives are designed from the gene sequences (Table 1 and others) and used in the array. Again, these peptide/protein arrays can be of various lengths (number of amino acids or derivatives), and they can be comprehensive and include probes for all genes/proteins in the genome, or targeted to specific subsets of genes. These probes, nucleotides, peptides or derivatives thereof, are bound to solid phase surfaces (glass slides, microplates, micro- or nano-beads, etc.) and used to screen processed biological samples for interacting macromolecules. In the case of nucleotide arrays, interactions are usually detecting complementary sequences in a sample by hybridization. In the case of protein/peptide arrays, and in some cases for nucleotide arrays, the array will be used for detection of interacting macromolecules (proteins, antibodies, other nucleic acids, etc.).
EXAMPLES
Example 1
Herein, the  ̃9.2 Mb genome of C. hominis is described. The genome has 32% GC content,  ̃3,994 protein-coding genes, 45 tRNAs, and at least four rRNAs. There is a strikingly high concordance between the genome complement of C. hominis and the requirements imposed by the environmental niches it inhabits. Energy is derived largely from glycolysis and catabolism of starch or amylopectin. Options for both aerobic and anaerobic metabolism are available, the latter requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are quite limited, but an extensive complement of transporters is encoded. Evidence of an apicoplast is absent, but rhoptry, microneme, dense granule, and acidocalcisome related genes are present. A panel of hypothetical signal transduction systems was identified. Comparison of the genomes of the human pathogen C. hominis and the bovine isolate C. parvum shows a high degree of synteny with no evidence of different gene complements. Phenotypic differences between these two parasites therefore seem to be due to very subtle sequence divergences.
The Genome Composition
A  ̃12 fold sequence and  ̃8 fold BAC clone coverage of the genome of C. hominis isolate TU5021 is presented. Alignment2 of these sequences with the HAPPY map3 and chromosomes of the C. parvum genome4 covered 9.1 Mb of the  ̃9.2 Mb. The eight chromosomes range from  ̃0.9− ̃1.4 Mb, and exhibit 31.7% GC content compared to 30.3% and 19.4% for C. parvum and P. falciparum5. The density of repeats of 2-50 bp was approximately one per 2800 bp. The distribution of repeats is biased toward chromosome ends as over 85% are in the telomere proximal thirds of five of the chromosomes. Two octamers, TGGCGCCA and TGCATGCA, over-represented in other apicomplexans3, are  ̃40 and 15 fold over-represented in C. hominis. Interestingly, over 80% of the octamers are localized in non-coding sequences, suggesting a regulatory or other conserved function. 45 tRNAs, 4 or 5 rRNA operons—at least one of each of the two known types, and two clusters of three tandem 5S rRNA genes are present. Like P. falciparum5, two methionine tRNAs are present, suggesting discrete roles in initiation and extension. We estimate 3,994 genes in C. hominis, compared to 3,952 genes in C. parvum and 5,268 in P. falciparum 5 (Table 2). Approximately 60% exhibit similarity to known genes. The distribution of GO annotations for Cryptosporidium, Plasmodium, and Saccharomyces, is remarkably similar, suggesting their phenotypic differences are a reflection of non-conserved or novel gene families of unknown function rather than to functional specialization of conserved gene families. Using ESTs as a guide, we estimate that 5-20% of C. hominis genes have introns.
Energy Metabolism.
C. hominis possesses a highly tailored glycolysis-based metabolism dependent on the host for nutrients and exquisitely adapted for its parasitic life cycle (see FIG. 1). Glycolytic enzymes are present, but the TCA cycle and oxidative phosphorylation (OxPhos) are not. Both an anaerobic pathway using pyruvate: NADP+ oxidoreductase (PNO) and an aerobic pathway using an alternative oxidase (AOX) are available for recycling NAD+ to NADH. In the former, pyruvate is fermented to acetyl-CoA producing NADPH which is then reduced to NADP+, releasing hydrogen, by a Narf like [FE]-hydrogenase, as for Trichomonas 6. Acetyl-CoA is processed by acetate CoAsynthase to produce acetate and ATP, as in Giardia7, yielding four ATP per glucose.
Acetyl-CoA can also be processed to ethanol yielding no additional ATP. Under glucose-limited conditions, conversion of acetyl-CoA to acetate, generating two extra ATP per glucose, might be favored. In excess glucose, pyruvate can be converted to lactate or ethanol to regenerate NAD+ but no additional ATP. C. hominis can also generate ATP by metabolism of glycerol using glycerol-3-phosphate dehydrogenase and triose phosphate isomerase.
C. hominis can convert pyruvate to malate and subsequently to oxaloacetate (OAA), regenerating NAD+. However, malate shuttle enzymes (e.g., aspartate amino transferase) which process OAA to aspartic acid for export from the mitochondrion, are absent. We propose that cytoplasmic malate may be converted to OAA by a mitochondrial membrane bound malate dehydrogenase, like the lactate shuttle of Euglena gracilis8, passing electrons from malate to an electron transport system comprised of elements of Complexes I and III and an alternative oxidase system (AOX) with O2 as electron acceptor and producing no additional ATP.
Enzymes for metabolism of glycogen, starch and amylopectin are present, consistent with suggestions that amylopectin represents an energy reserve for sporozoites9. Lack of glucose-6-phosphate-1-dehydrogenase and other enzymes of the pentose phosphate pathway suggests that, unlike P. falciparum and other apicomplexans10, C. hominis cannot metabolize five-carbon sugars or nucleotides. Components of beta-oxidation (e.g., enoyl-CoA hydratase and acetyl-CoA C-acyltransferase) are also absent, precluding ATP generation from fatty acids. Enzymes for catabolism of proteins are also absent. Major TCA cycle enzymes—isocitrate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase—are absent in C. hominis. Despite the presence of ubiquinol-cytochrome C reductase, NADH dehydrogenase (ubiquinone), H(+)-transporting ATPase, iron-sulfur cluster-like proteins, etc., key components of Complexes II and IV are absent, precluding ATP generation by 0× Phos. Components of 0× Phos that are present (parts of Complexes I and III) probably re-oxidize NADH in a simplified electron-transport chain, as in some plants and protozoa11.
Biosynthesis.
Consistent with previous suggestions (c.f. 12), Cryptosporidium lack enzymes for synthesis of key biochemical building blocks—simple sugars, amino acids and nucleotides. However, starch, amylopectin and fatty acids can be generated from precursors. Interestingly, these C. hominis enzymes exhibit minimal similarity to the known biosynthetic enzymes and therefore represent potential therapeutic targets.
Enzymes of the TCA, urea and nitrogen cycles, and the shikimate pathway are absent, suggesting that Cryptosporidium is an amino acid auxotroph. The shikimate pathway has been proposed as a potential target for glyphosate-based chemotherapy in other parasites including Cryptosporidium13. We found no evidence to support this hypothesis. Enzymes able to interconvert amino acids are encoded in C. hominis. However, unlike P. falciparum5, C. hominis seems to have a full complement of amino acid transporters.
C. hominis lacks enzymes to synthesize bases or nucleosides, but encodes enzymes that convert nucleosides into nucleotides and interconvert nucleotides. As in other parasites, thymidylate synthase and dihydrofolate reductase (DHFR) of C. hominis are encoded as a bifunctional polypeptide, and novel polymorphisms at crucial sites have been proposed to explain Cryptosporidium 's resistance to antifolates (c.f. 14). As previously suggested12, several nucleotide conversion enzymes seem to have prokaryotic origin. Fatty acid biosynthesis in apicomplexans occurs in the apicoplast via a type II system including fatty acid synthase (FAS)15. However, consistent with absence of an apicoplast in Cryptosporidium16, C. hominis encodes large FAS and polyketide synthase (PKS) enzymes, suggesting a type I mechanism (c.f. 17). As previously suggested18, the type I FAS and PKS enzymes of C. hominis have prokaryotic characteristics.
Glycerolipid and phospholipid metabolic pathways for phosphatidylinositol (PI) biosynthesis are available in C. hominis. 1,2-diacylglycerol, an intermediate, is precursor for glycosylphosphatidylinositol (GPI) anchor synthesis. Consistent with previous observations 19, all enzymes required for synthesis of these anchors are encoded in the genome. Polyamines; e.g., putrescine, spermine and spermidine, are critical for cellular viability, and enzymes required for their synthesis are attractive therapeutic targets20. Cryptosporidium can synthesize polyamines using arginine decarboxylase rather than ornithine decarboxylase21 The putative arginine decarboxylase, spermidine synthase and other relevant enzymes encoded by C. hominis are significantly diverged from their homologs and represent potential therapeutic targets.
Signaling and Control Pathways
C. hominis encodes adenylate cyclase, cAMP phosphodiesterase and protein kinase A, suggesting the presence of the cAMP-mediated signalling pathway (Tab.S7). Trimeric G protein, often involved in activation of cAMP mediated signalling, was not found in C. hominis, suggesting that, as for the Kinetoplastida22 and reminiscent of plants, this pathway is independent of this complex in C. hominis. The presence of phosphatidylinositol 3-kinase and phospholipase C suggests that C. hominis utilizes phosphatidylinositol phosphate and Ca2+-mediated regulatory mechanisms. The presence of putative Ca2+ transporters, enzymes associated with acidocalcisomes, and calmodulin imply that Ca2+ transport and sequestering are functional. Protein kinase C receptors suggest that C. hominis has the ability to signal by activation of soluble cytoplasmic receptor-associated kinases.
Organelles
No mitochondrial DNA sequences were found in C. hominis, and both the TCA cycle and OxPhos are absent. However, a double membrane bound organelle generates a proton gradient using cardiolipin and performs some related mitochondrial functions, and mitochondrial marker chaperonin60 was localized to this structure23. Core enzymes of [Fe—S] cluster biosynthesis; i.e. CpFd1, IscU, IscS, mt-HSP70, mtFNR and frataxin, have been reported in Cryptosporidium24, and we were not surprised to observe proteins involved in electron transport. We also used the CDART 25 to identify [Fe—S] domains in HscB (JAC) and ATM1, which are possibly involved in chaperonin activity of Hsp40/DnaJ type and ABC transport. Thus, C. hominis, like another obligate intracellular parasite, the microsporidian Encephalitozoon cuniculi26, contains a minimal set of these proteins. These results imply significant mitochondrial function in C. hominis, and that the previously reported organelle27 is an atypical mitochondrion.
Cryptosporidium apparently lacks an apicoplast16,28, and searches of the C. hominis genome identified no clear apicoplast-encoded genes. Some putative nuclear-encoded apicoplast genes; e.g., T. gondii acetyl-CoA carboxylase 1 precursor29, and P. falciparum adenylyl cyclase30, are present. Others; such as the conserved apicoplast 50S ribosomal protein L33 of Plasmodium, the ribosomal L28 and S9 precursor15 proteins of Toxoplasma, were not found. Together, our data suggest that Cryptosporidium lost an ancestral apicoplast. The presence of D-glucose-6-phosphate ketol-isomerase and 2-phospho-D-glycerate hydrolase that have highest similarity to plant genes and may be derived from ancient algal endosymbionts31 is also suggestive that engulfment of the alga that gave rise to the apicoplast32 preceded divergence of Cryptosporidium from other apicomplexans.
The C. hominis genome encodes multiple proteins specific for components of the apical complex including micronemes and rhoptries. No specific dense granule-associated proteins were observed, probably because these proteins diverge rapidly33. However, proteins implicated in the regulation of transport and enhancement of release of dense granule proteins34 are present. As for Plasmodium, a typical Golgi structure is not apparent in C. hominis 28. However, the presence of secretory organelles implies the existence of a functional endoplasmic reticulum (ER) and Golgi, and C. hominis encodes proteins similar to many related components; including the NSF/SNAP/SNARE/Rab machinery which participates in dense granule release35 and the rhoptry biogenesis mediator AP-136, involved in ER-Golgi-organelle protein traffic. Therefore, the ER-Golgi-organelle machinery of C. hominis is conserved and similar to that of other apicomplexans.
Transporters.
C. hominis exhibits very limited biosynthetic capabilities and is apparently supremely dependent on its ability to import essential nutrients. The genome encodes >80 genes with strong similarity to known transporters and several hundred genes with transporter-like properties. At least twelve sugar or nucleotide-sugar transporters, five putative amino acid transporters, three fatty acid transporters, 23 ABC family transporters including possible multiple drug resistance proteins37, and several putative mitochondrial transporters are present. Other putative transporters for choline uptake, aminophospholipid transport, ATP/ADP transporters, and others with unclear function, were also identified. These transporters represent ideal therapeutic targets.
Comparison of C. hominis and C. parvum Genomes.
Comparison of the genomes of C. hominis and C. parvum showed that the two genomes are very similar; exhibiting only 3-5% sequence divergence with no large insertions, deletions or rearrangements evident. In fact, the gene complements of the two species are essentially identical since the few C. parvum genes not found in C. hominis are proximal to known sequence gaps. Thus, we conclude that the significant phenotypic differences between these parasites are due to functionally significant polymorphisms in relevant protein-coding genes and subtle gene regulatory differences.
Conclusions.
A striking feature of the C. hominis genome is the concordance between its gene complement and metabolic requirements in the environmental niches of its two primary life cycle stages—the quiescent oocyst in the nutrient-poor aerobic environment of contaminated water, and the vegetative parasites in the nutrient-rich anaerobic or microaerophilic environment of the host. Oocysts probably persist by processing stores of complex carbohydrates. Metabolism is likely aerobic via the alternative electron transport system in the unconventional mitochondrion. Consistent with the lack of an energy generating TCA cycle, OxPhos, β-oxidation, and the pentose phosphate pathway, oocysts are relatively inactive, and the two ATP per glucose from glycolysis may provide sufficient energy. In the host, the parasite can import sugars to directly fuel glycolysis, netting two ATP per hexose. In limiting glucose, an additional two ATP per hexose can be generated by converting acetyl-CoA to acetate or via glycerol metabolism. The residual mitochondrion lacks the TCA cycle and OxPhos as expected in an organism that replicates in the anaerobic/microaerophilic environments, and a simplified electron transport system for regenerating reducing power is available. Thus, a glycolysis-based metabolism is sufficient to support Cryptosporidium in all life cycle stages.
Also consistent with the highly tailored Cryptosporidium metabolism are its limited biosynthetic options; i.e., amino acids, nucleotides, and simple sugars cannot be synthesized. The parasite must import these building blocks probably explaining the significant array of transporters present in the genome.
As expected, apicoplast-specific activities are lacking in Cryptosporidium. One hypothesis is that acquisition of the type 1 FAS by a progenitor organism obviated the fatty acid synthesis capabilities of the apicoplast38. Since some apicoplast-related genes remain, our observations suggest that Cryptosporidium diverged from other apicomplexans prior to loss of this organelle.
As previously noted, our analysis shows that Cryptosporidium is a mosaic of sequences from diverse progenitors, including the hypothetical endosymbiont alga which formed the apicoplast, the mitochondrion, and numerous genes acquired from prokaryotes by lateral transfer. Cryptosporidium also exhibits modular gene loss. We assume, based on inference from other apicomplexans and earlier diverging groups like the Euglenozoa, the Heterolobosea, and the jakobids39, that Cryptosporidium progenitors exhibited the TCA cycle, beta-oxidation, OxPhos, amino acid, nucleotide and sugar biosynthesis, fully competent mitochondria, and a functional apicoplast.
Genes associated with these functions are dispersed throughout the genome in Plasmodium and, we assume, in the progenitor. However, these systems seem to have been deleted cleanly in Cryptosporidium, leaving few residual genes or pseudogenes. Thus, its genome is a mosaic resulting from multiple lateral gene transfers and a complex pattern of selective gene deletion.
The highly tailored physiology of C. hominis suggests attractive therapeutic targets. Examples include: 1) transport systems for peptides, amino acids, nucleosides, and sugars, 2) components of glycolysis; 3) the unique prokaryotic FAS1 and PKS1; 4) starch and amylopectin biosynthesis or catabolism; 5) nucleic acid or amino acid metabolism; 6) the AOX electron transport system; 7) the TS-DHFR; and 8) the diverged polyamine synthesis enzymes. Finally, many potential vaccine targets were identified in the C. hominis genome (not shown), and unlike other protozoan parasites, no extensive arrays of potentially variant surface proteins were observed, suggesting a possible role for immunoprophylaxis for cryptosporidiosis.
The availability of the genome sequence of the human pathogen C. hominis represents a critical step forward in our understanding of the biology of this parasite. The gene complement provides very significant insight into its physiology and metabolism, validating previous hypotheses and suggesting others. New obvious targets for chemo- and immunotherapy are already apparent. In short, we anticipate that the availability of the sequence of C. hominis will stimulate very rapid progress in research on this organism, its pathogenicity, and strategies for intervention in the diseases it causes.
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    25. Geer, L. Y., Domrachev, M., Lipman, D. J. & Bryant, S. H. CDART: protein homology by domain architecture. Genome Res. 12, 1619-1623 (2002).
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    29. Zuther, E., Johnson, J. J., Haselkorn, R, McLeod, R. & Gornicki, P. Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. U. S. A 96, 13387-13392 (1999).
    30. Muhia, D. K. et al. Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum. J. Biol. Chem. 278, 22014-22022 (2003).
    31. Dzierszinski, F. et al. The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes. Implications for evolutionary origin of apicomplexans. J. Biol. Chem. 274, 24888-24895 (1999).
    32. Fast, N. M., Kissinger, J. C., Roos, D. S. & Keeling, P. J. Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. Mol. Biol. Evol. 18, 418-426 (2001).
    33. Beyer, T. V., Svezhova, N. V., Radchenko, A. I. & Sidorenko, N. V. Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem). Cell Biol Int. 26, 861-871 (2002).
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    37. Zapata, F., Perkins, M. E., Riojas, Y. A., Wu, T. W. & Le Blancq, S. M. The Cryptosporidium parvum ABC protein family. Mol. Biochem. Parasitol. 120, 157-161 (2002).
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Example 2
Identification of Categories of Genes and Proteins of C. hominis 
Introduction
Genomic sequences from pathogenic microorganisms provide fundamentally new approaches for development of vaccines and chemotherapeutic agents. Thus, reverse vaccinology invokes bioinformatics analyses to identify potential candidates from the genome data by “in silico” analysis. This approach has been successfully applied to identify putative vaccine candidates from bacterial genomes that traditional vaccine development methods have not detected. This approach (see Scarsellie et al, 2005, for review) has recently been applied in several bacterial systems including for example group B streptococci and Neisseria meningitidis. See, for example, the following references: 1) Maione D, Margarit I, Rinaudo C D, Masignani V, Mora M, Scarselli M, Tettelin H, Brettoni C, Iacobini E T, Rosini R, D'Agostino N, Miorin L, Buccato S, Mariani M, Galli G, Nogarotto R, Nardi Dei V, Vegni F, Fraser C, Mancuso G, Teti G, Madoff L C, Paoletti L C, Rappuoli R, Kasper D L, Telford J L, Grandi G. Identification of a universal Group B streptococcus vaccine by multiple genome screen. Science. 2005 Jul. 1; 309(5731):148-50; 2) Scarselli M, Giuliani M M, Adu-Bobie J, Pizza M, Rappuoli R. The impact of genomics on vaccine design. Trends Biotechnol. 2005 February; 23(2):84-91; 3) Serruto D, Adu-Bobie J, Capecchi B, Rappuoli R, Pizza M, Masignani V. Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogens. J. Biotechnol. 2004 Sep. 30; 113(1-3):15-32. Review; 4) Kurz S, Hubner C, Aepinus C, Theiss S, Guckenberger M, Panzner U, Weber J, Frosch M, Dietrich G. Transcriptome-based antigen identification for Neisseria meningitidis. Vaccine. 2003 Jan. 30; 21(7-8):768-75; and, 5) Pizza M, Scarlato V, Masignani V, Giuliani M M, Arico B, Comanducci M, Jennings G T, Baldi L, Bartolini E, Capecchi B, Galeotti C L, Luzzi E, Manetti R, Marchetti E, Mora M, Nuti S, Ratti G, Santini L, Savino S, Scarselli M, Storni E, Zuo P, Broeker M, Hundt E, Knapp B, Blair E, Mason T, Tettelin H, Hood D W, Jeffries A C, Saunders N J, Granoff D M, Venter J C, Moxon E R, Grandi G, Rappuoli R. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science. 2000 Mar. 10; 287(5459):1816-20.
Similarly, systems wide analyses of these pathogens using genomic data leads to identification of potential ‘weak links’ or ‘Achille's Heels’ in the biological processes of the pathogen, which can be exploited for the development of inhibitors of processes essential to the parasite. Gene expression and proteomic technologies complement the “in silico” analysis of the genome data to identify potential vaccine and chemotherapeutic targets. Similar approaches lead to the identification of new biomarkers for the detection of these pathogens and diagnosis of disease caused by them.
C. hominis genes of several types that are of great potential as chemo- or immuno-therapeutics, immuno-prophylactics, and/or detection/diagnostics/quantification agents, have been identified. These genes fall into several general classes: 1) transporters; 2) receptors; 3) surface and secreted proteins; 4) organellar proteins; 5) signal transduction proteins and kinases; 6) critical metabolic enzymes; and 7) specifies specific sequence differences. The application for each of these is outlined briefly below.
Transporters. C. hominis lacks genes and consequently the enzymes and metabolic pathways required for several essential functions: e.g. biosynthesis of simple sugars, amino acids, nucleotides, fatty acids, etc. Thus, these important pathogens are dependent on their environment or host for these essential compounds, and these compounds must be transported across the parasite's membrane by a host of essential transporters. A list of these transporters is given in Table 3.
Knowledge of the sequence of these transporter genes and consequently the protein sequences permits us to predict their functions and to some degree their cellular locations and structures. Specific knowledge of the function of a transporter; e.g., a nucleotide-sugar transporter, provides a means to block the activity of that molecule. Blocking the activity of an essential transporter will prevent the parasite from importing essential compounds and the parasite will die. Thus, these transporters represent ideal targets for chemotherapeutic development.
Similarly, transporters must function at the host-parasite interface; i.e., the parasite membrane exposed to the extracellular domain. This exposed location makes these molecules ideal targets for immunoprophylaxis or immunotherapy. Vaccines developed against these proteins could: 1) protect relevant human populations or animal populations; 2) provide a means to treat infections. Thus, active or passive immunization against pools of these proteins could: 1) prevent infection; 2) provide a therapy for the cryptosporidiosis. The therapeutic effect could result from: 1) killing the parasite via normal immune mechanisms activated by immunization; 2) blocking the activity of the molecule via immune mechanisms and thereby starving the parasite until it is cleared by normal mechanisms. Thus, transporters are ideal targets for vaccines, and could be used for both immuno-prophylaxis and immuno-therapy.
Receptors. Receptors are essential for cell-cell signalling, sensing of the environment, host parasite interactions, uptake of essential nutrients, etc. Receptors by definition are located on the membrane surface in some contact with the extracellular environment. A list of C. hominis receptors is given in Table 4. Blocking of the function of these macromolecules would likely be lethal to the parasite. The extracellular location of these receptors renders them available for attack by the host or passive immune mechanisms, and available for chemotherapeutics unable to cross most membranes. Thus, receptors are excellent targets for immuno-prophylaxis and therapy, or chemotherapy.
Surface and secreted proteins. There are many parasite proteins associated with the surface of the cell, and some that are secreted into the extracellular milieu. A list of over 1000 C. hominis proteins that are associated with the membrane or secreted is given in Table 5. Receptors and transporters will also be members of this group of proteins. Independent of the actual function of these macromolecules, they are amenable to attack by the host immune system or by passive immunization. Thus, these molecules are also idea targets for active or passive immuno-therapy or prophylaxis.
Organellar proteins. C. hominis is a member of the Apicomplexan parasites, and therefore encodes genes that generate apical complex organelles responsible for invasion and pathogenesis of the parasites, and other organelles required for its survival. A list of genes which are involved in organellar biosynthesis and function is given in Table 6. Since these organelles, systems and enzymes are specific to these parasites, they represent ideal targets for therapeutics.
Signal transduction proteins and kinases. These proteins are required for proper, responsive and sensitive gene regulation in the parasite. Lacking the function of these proteins and enzymes, the parasites are unable to respond to their environment properly, or to differentiate correctly. Therefore, these enzymes are required for the viability of this parasite and thereby represent ideal targets for therapeutic (chemo- or immuno-) attack. A list of these proteins and enzymes is given in Table 7.
Critical metabolic enzymes. As outlined above, we have developed a fairly comprehensive understanding of the metabolism of Cryptosporidium simply by analysis of the genes encoded in its genome. Thus, we can see that the parasite is highly dependent on glycolysis for energy, for amino acid interconversion for generation of amino acids, for fatty acid biosynthesis and degradation, for nucleotide synthesis. Blockage or inhibition of these processes would likely be lethal for the parasite. A list of such essential metabolic enzymes and proteins is given in Table 8. These enzymes represent ideal targets for specific chemotherapeutics.
Species specific sequence differences. We now know nearly the entire genome sequence of C. hominis. Thus, we can now easily compare all of its genes to those of related parasites and pathogens. DNA/RNA of these genes provide very specific and easily readable signatures, and these signatures are generally species-specific. Thus, we have identified many sequences that can be useful for identifying C. hominis, and discriminating this parasite from other microbes (Table 9). These signature sequences can also be used for detection, quanitification and diagnostics, as well as for chemotherapeutic targets.
The C. hominis genes disclosed herein and the proteins they encode are newly discovered and have not been used as targets for possible immunotherapeutic purposes, or as targets for chemotherapeutic agents, or as tools for detection/diagnosis of the parasites. As outlined above, there is no immuno- or chemotherapy available for C. hominis. It is essential to develop such therapies. The newly identified genes and the proteins they encode provide a new approach to chemo- and/or immunotherapy. Their advantages include one or more of the following:

    1. they are essential for viability of the parasite
    2. they are localized on the surface of the parasite
    3. they should be accessible to the host immune system or to blocking agents
    4. much is known about these processes (e.g., transport, metabolism, etc.) and analogs that block them can be designed
    5. the proteins/peptides thereof can be synthesized chemically or in recombinant bacteria
    6. the peptides differ significantly from host molecules (both host and parasite homologs have been fully sequenced), and analogs that block the parasite version can be designed so that they do not block any host function.
    7. vaccinogens can be designed so that the host immune response will act only on the parasite protein.


In short, these new genes present novel targets for chemo- and immunotherapy and prophylaxis for Cryptosporidiosis, a disease for which no such agents currently exist. Genome Annotation. The DNA sequence of C. hominis was annotated using a suite of analysis programs to identify important genes. Protein genes and their structural and functional annotations were derived. Approximately 4,000 genes were identified Table 1). Similarly, structural RNA genes (tRNA, rRNA) were identified (not shown).
Vaccinology.
One focus of research in the Cryptosporidium vaccinology project has been application of “in silico” genome analysis to identify potential vaccine candidates from the complete genome sequence of Cryptosporidium hominis. The general steps that are followed are:

    
    
        1. Apply bioinformatics tools to predict possible antigens from the whole genomic sequence.
        2. Focus searches for possible antigens by gene expression analysis, proteomics, and other “systems” approaches.
        3. Clone, express, and purify putative candidates.
        4. Validate the possible protective or biological role of the candidates in animal and “in vitro” models.
        5. Select antigens with positive results for further testing in animals and eventually in humans.

Reverse Vaccinology in Cryptosporidium 

    
    


Identification of potential target antigens using bioinformatics: The initial step was to apply bioinformatic tools to scan the whole genome sequence of C. hominis focusing on proteins that are predicted to be secreted, ER localized, surface-exposed and/or contain antigenic motifs, and that therefore represent potential vaccine targets. Since these proteins are most likely to be exposed to the immune system, they constitute the initial target of the project. This approach has been successfully used for several ne recombinant vaccines (see, for example, Scarsellie et al, 2005, fore review.) Screening of the genome has been carried out using various programs that predict the following characteristics:
presence of a signal peptide
possible membrane protein by presence of transmembrane domains
GPI—anchor domains
antigenicity
similarity to virulence factors characterized in other pathogens
no similarity to human proteins.
role in transport of ions or essential metabolitesions or essential metabolites.
Using various bioinformatics tools, close to 500 Cryptosporidium candidate proteins that are most likely exposed to the immune system were identified (Table 10). Priority is given to those that present a single transmembrane domain, as those are less likely to be toxic to E. coli or other vector hosts used for cloning and expression of the recombinant proteins.
Focus Searches Using Gene Expression and Proteomics
The best antigens for vaccines are likely to be those associated with the membrane during sporozoite stages in the development of the parasite. The sporozoite form of the parasite is responsible for invasion of the intestinal epithelium causing pathology. To identify those proteins, a microarray that targets every gene in the C. hominis genome has been generated. The arrays are used to identify those genes that are expressed in C. hominis sporozoites. These arrays detect mRNAs transcribed from the genes. Therefore, in parallel with the gene expression array work, proteomics analyses are performed. A database containing all C. hominis proteins has been constructed, and C. hominis oocysts and sporozoites are analyzed for those proteins that are expressed in those stages of the parasite. This information permits identification of proteins for vaccinogens.
The array in its current configuration consists of  ̃4,000 70 base synthetic oligonucleotides bound to glass slides. Each of the  ̃4,000 C. hominis genes is represented by a single, specific oligonucleotide in the array. In order to assess expression of these genes in a specific stage, a biological sample containing C. hominis parasites at that stage of development is provided. Total RNA is isolated by standard technology, labeled with fluorescent dyes, and hybridized to the array. A similar RNA sample derived from an alternative stage of the parasite's life cycle is used is labeled with an alternative fluorescent dye and used as a control. Both labeled RNAs are hybridized to the same oligonucleotide array, and the arrays are washed and scanned in a fluorescence scanner. The readout of the scanner provides an estimate of the amount of RNA for each gene that is present in the initial sample and a comparison between the two samples provides an estimate of the relative expression level of each gene in the two samples. Thus, genes that are up or down regulated relative to the control can be identified.
In addition to examining gene expression differences in various life cycle stages, these arrays may be used to monitor C. hominis parasites under other conditions of interest, for example, to monitor gene expression in response to an external stimulus or variable, such as introduction of a drug or other agent into a culture of the parasites; or to monitor the course of a C. hominis infection, either in culture, or in an infected individual.
The 70 base oligonucleotides in the array were designed by ArrayOligoSelectorTM version 3.8.2, (http://arrayoligosel.sourceforge.net/), which selects optimal sequences by:

    
    
        1. Examining every possible 70 mer sequence from every gene;
        2. Using BLASTN (against the whole genome) to check the uniqueness of each 70 mer.
        3. Uniqueness is scored as the theoretical binding energy of a candidate oligo to its most similar genome sequence. The binding energy is calculated using a nearest-neighbour model with the established thermodynamic parameters;
        4. Using the LZW compression algorithm to calculate the sequence complexity score in bytes between the oligo sequence and the its compressed version;
        5. Determining the self-annealing score, calculated as the alignment score of the optimum local alignment between the oligo sequence and its reverse compliment using the Smith-Waterman algorithm;
        6. Calculating the GC content of the oligo;
        7. Choosing, for each gene, the oligo that maximizes uniqueness and sequence complexity, minimizes self-annealing and has GC content closest to specified by the user; also tries to minimize distance to the 3′ end of the gene.
    
    


The oligos were synthesized commercially and validated in the lab, and the process is ongoing. Initial tests were performed as follows:

    
    
        The oligos were reconstituted to 100 pmoles with sterile distilled water;
        Several dilutions (10, 25 and 50 pmoles) were tested for the best printing conditions;
        Two test plates were printed on Corning UltraGAPS slides (amino-silane coated);
        These slides were stained in SYBR Green II and hybridized with Cy-dye labeled RNAs.
    
    


The results of the initial prints indicated that the oligos were intact and the hybrodization was strong. The relatively even staining indicated that that hybridization of each gene was essentially equivalent. Finally, lack of hybridization to the negative control confirms specificity of the hybridization. Thus, use of the array to identify genes that are expressed in C. hominis sporozoites by detecting transcribed mRNAs was validated.
Cloning and Expression of Protein Candidates in Escherichia coli 
In parallel with the gene expression array work, proteomics analyses have also been performed. A database containing all C. hominis proteins has been constructed, and C. hominis oocysts and sporozoites are characterized for those proteins that are expressed in those stages of the parasite. This information permits us to focus on the most likely proteins for vaccinogens.
The cloning and expression of the candidates has been carried out using the pET system from Novagen that allows the cloning of PCR products in a ligase-independent reaction (Ek/LIC cloning). The proteins of interest are expressed as fusion proteins to an epitope tag that permits their detection and purification. Various tags (e.g. His-Tag, GST-tag and Nus-Tag, etc.) are known and available. In addition the Ek/LIC cloning system can be adapted and used in a high throughput pipeline for the cloning and expression of recombinant proteins.
The cloning of PCR products by LIC requires the presence of non-complementary single-stranded overhangs in the vector that anneal to complementary single stranded overhangs on the PCR fragment. In order to create these overhangs the LIC method uses the 3′-5′ exonuclease activity of T4 DNA polymerase, which in the presence of a single deoxyribonucleotide creates very specific 13-14 base single stranded overhangs in both vector and PCR fragment. The primers used in the PCR reaction include 5′ extensions that after the treatment with T4 DNA polymerase generate the overhangs complementary in the fragment to the overhangs created in the vector.







Sense primer:







(SEQ ID NO: 4001)







5′ GAC GAC GAC AAG ATX-insert specific sequence 3′


 


Antisense primer:







(SEQ ID NO: 4002)







5′ GAG GAG AAG CCC GGT-insert specific sequence 3′






After treatment with T4 DNA polymerase in the presence of dATP the PCR fragment is flanked by the overhangs as shown below:








(SEQ ID NO: 4001)









5′ GACGACGACAAGATX-Target protein-xxA



 







(SEQ ID NO: 4002)









AX-Target protein-xxTGGCCCGAAGAGGAG 3′






As a first step, the genes encoding thirteen of these antigens were PCR amplified from genomic DNA. The PCR products were purified and cloned into the vector pTriEX-4, a vector that allows the expression of the proteins as 6X-His and S-tag fusion products in both prokaryotic and eukaryotic hosts. The His-tagged products can be purified through nickel-activated columns. The recombinant plasmids were characterized by restriction enzyme analysis and DNA sequencing to confirm the presence of the Cryptosporidium target gene. Protein overexpression: The recombinant plasmids were used to transform the Escherichia coli strain BL21 (DE3) or other appropriate vector for expression of the fusion proteins. The target genes are cloned into pTriEX-4 under the control of the bacteriophage T7 promoter. Transcription is initiated by the enzyme T7 RNA polymerase which is present in the chromosome of the bacterial host and is under the control of an inducible promoter. In the presence of the inductor, IPTG, T7 RNA polymerase is produced in the bacterial host resulting in the expression of the target protein. This gives this system an excellent way to regulate the production of the target protein under controlled conditions, which is a point to consider in case that the protein has a toxic effect in E. coli. The level of expression is very high and can be 50% of the total protein present in the bacterial host. The expression of the recombinant genes was carried out by culturing bacteria in 3 ml of Overnight Express Instant Tb medium (Novagen) at 37° C. for 12-13 hours. This medium is designed to achieve a high level of expression with IPTG-inducible bacterial systems without the need of monitor cell growth. Expression has been achieved for all but the Zn++ transporter and the Thrombospondin-containing gene TSP1. The Zn transporter seems to be toxic for E. coli as bacterial cultures containing the Zn-transporter clones start to lyse after they reach early stationary phase. Most of the proteins were expressed in E. coli in an insoluble form as inclusion bodies. The exception is the gene encoding the Profilin-like protein.
Therefore, these proteins were expressed using a different vector since the same PCR product can be cloned in different vectors. Two additional epitope tags were assayed, GST and NUS. The proteins expressed from clones in the GST-tag vector were also insoluble. The proteins obtained from the NUS-tag expressed the protein in higher amount compared to the previous two vectors and the fusion proteins can be detected in the soluble fraction by Western blot. Very high levels of expression have been achieved with the Nus-tag clones which results in proteins becoming soluble as detected by gel staining. The results for the experiments are summarized in Table 11.
The nucleotide sequences that encode the proteins, and amino acid sequences of the proteins, are as follows:
1) Zinc transporter (Chro.10338, start position 677, end position 2053) is encoded by the nucleotide sequence:







(SEQ ID NO: 1)







ATGAAAGACTCAGGTCTGGAAAAGCCATTACTTAATGGGAATGGATTTAA


 


AATATTTGCAAGTACAGAGAGTGTTCAGAAGAGGTTAATATATGCAATCT


 


TTTTCTGTCTAGTCTTTACATTGATAGAGGTTGTTGTGGGTATATTATCA


 


AACTCACTGGCACTAATATCAGATGCATCTCACCTCATTTCAGATATATG


 


TAGCTATTTCATTTCTCTGCTTGGTATCCACCTTTCCAAAAGAAAGGCCA


 


CAAACACAATGTCATTTGGTTATAACAGAGCTGAAATATTAGGGGCTTTG


 


TTAAGCATTCTACTAATATGGTTCATGACAATCATGCTTGTTTACGAGGC


 


TATTCAAAGAATGTTATATCCTGTGAATGTTGATGGGTTTTCTATGTTTA


 


TTACCGCTATTTTTGGTACTTTGTCCAACTTATTTATTAGCTTTGTGTTA


 


TCTGTTCACAATCATGGAATAGGTTCAATTGGAGCAGATTGTACCCAACA


 


CAATCATACACATGAACATATGCATGAACACGACTGTAAGCAAGCTCAAA


 


CTCATTTTCAGGATGATTCACTGTATTGCAAAGATCAACAACTAGTAGAA


 


AATCAAGAACAAATTGGAGGAATTAACACTACTTTACTTGAATACCACCA


 


TAGAAGCCAAATGAGAACTAAGGATTTAGATCATGAACTTAATAATTATA


 


CTAATTTAATGAACTCTCCAGTTATAAGAAGAGTCAATTCTGGTTTAAAA


 


GAGTGTTCAGAACGTCAAAATGACTATTCTCATCTCCATAGTAATAATCA


 


CTATCCAAGTAAACATTCCTCTGAACAAGAAAGCTTAGCGCTTAAGTCTG


 


CTTATATCCATGTTTTAGGGGATATTTTACAGAACATCGGAGTAATGATT


 


GCTGGATTACTGATTTTATACAATCCAGCATGGACAATCGCCGATCCTCT


 


ATGTACTATTCTATTCTCCTTCTTTGTCCTCGCAACAACCATCAAAATCC


 


TAAAAGATTCCGCCAATGTTCTAATGGAAGGAGCCCCTATAGGAATTGAT


 


TGTGAATCCATTCAAAACGACTTTCTAAAGCTTTCTTCAGTGCTTGAAGT


 


TCACGATCTACATGTTTGGTCTGTATCTGTTGGAGTTCCTGCATTATCTT


 


GTCATATTGTCGTAGCATCAGAAGATAATGCTAGATTTACATTAAGATAT


 


GCAACGGATCTCTGTCAAAAGAAATATGGAATATTTCACACCACCATTCA


 


AATTGACTATTCTCCAAATAAAGCCACTTGTGAAACAATACATCATCAAA


 


AATGTCTAGTTGGCTCTAATAACCAAAATAAAAGTGAAATTCACCAAATA


 


ATTCATCCCGTTGACTATTCTGCTTAG






and has amino acid sequence:








(SEQ ID NO. 2)







MKDSGLEKPLLNGNGFKIFASTESVQKRLIYAIFFCLVFTLIEVVVGILS


 


NSLALISDASHLISDICSYFISLLGIHLSKRKATNTMSFGYNRAEILGAL


 


LSILLIWFMTIMLVYEAIQRMLYPVNVDGFSMFITAIFGTLSNLFISFVL


 


SVHNHGIGSIGADCTQHNHTHEHMHEHDCKQAQTHFQDDSLYCKDQQLVE


 


NQEQIGGINTTLLEYHHRSQMRTKDLDHELNNYTNLMNSPVIRRVNSGLK


 


ECSERQNDYSHLHSNNHYPSKHSSEQESLALKSAYIHVLGDILQNIGVMI


 


AGLLILYNPAWTIADPLCTILFSFFVLATTIKILKDSANVLMEGAPIGID


 


CESIQNDFLKLSSVLEVHDLHVWSVSVGVPALSCHIVVASEDNARFTLRY


 


ATDLCQKKYGIFHTTIQIDYSPNKATCETIHHQKCLVGSNNQNKSEIHQI


 


IHPVDYSA;






2) Ribosomal protein S19 (CP15) (Chro.60368, start position 13760, end position 14197) is encoded by the nucleotide sequence:








(SEQ ID NO. 3)







ATGGCAGATACTGAACAAAAGAAGAGAACCTTCAGAACTTATAGTTACAG


 


AGGTGTTGACCTCGACAAGCTCCTTACCATGAAATTGGATGAGGTTGTTG


 


AGCTTTTACCAGCACGTAAAAGACGTAAGATAGCCAGAGGTTGTCTTAAC


 


AGAAGAACTGCAGCTTTTATCGCAAAGCTTCGCAAATCTAAGGCTGAATG


 


TCCAATGGGTGAGAAACCTGTTGCTGTTCGTACCCATTTACGTAATATGG


 


TTATCCTCCCAGAAATGGTTGGTTCTGTTGCAGGTGTCTACAATGGTAAG


 


ACTTATGTTACCGTTGAAATTAAGCCAGAAATGATTGGGATGTACCTTGG


 


AGAGTTCTCTATCACCTACAAGCCAGTACGTCATGGTAAGCCAGGTGTTG


 


GTTCAACCAGTTCTTCCAGATTCATTCCTCTAAAGTAA






and has amino acid sequence:








(SEQ ID NO. 4)







MADTEQKKRTFRTYSYRGVDLDKLLTMKLDEVVELLPARKRRKIARGCLN


 


RRTAAFIAKLRKSKAECPMGEKPVAVRTHLRNMVILPEMVGSVAGVYNGK


 


TYVTVEIKPEMIGMYLGEFSITYKPVRHGKPGVGSTSSSRFIPLK;






53) Mucin-like glycoprotein 900 (Chro.70447, start position 855, end position 3963) is encoded by the nucleotide sequence:








(SEQ ID NO. 5)







ATGACAACAACAACAACACCACCATTACCTGATATCGGTGACATTGAAAT


 


TACACCAATCCCAATTGAAAAGATGTTGGATAAGTATACAAGAATGATTT


 


ATGACTATAACAGTGGTTTATTATTAGACTCTAATGATGAACCAATTCCA


 


GGTTCTCAAGCAGGACAAATAGCTGATACAAGCAATTTATTCCCAACTCA


 


AACTCACAAGAGTACTGGTTTACCAATTGATCCAATGGTTGGTCTTCCAT


 


TTGATCCAAAATCAGGTAATTTAGTACATCCATATACCAATCAAACAATG


 


TCTGGTTTATCAGTATCATATCTTGCTGCTAAGAATTTGACAGTTGATAC


 


TGATGAAACCTACGGTTTACCAATTGATACACTCACTGGTTACCCATTAG


 


ATCCAGTTAGTTTGATTCCATTCAATCCAGAAACTGGTGAATTGTTTGAT


 


CCAATCTCAGATGAGATCATGAATGGAACAATTGCAGGTATTGTTTCAGG


 


AATTTCTGCAAGTGAGTCATTATTATCTCAGAAATCAGCTCAAATCGACC


 


CAGCAACAAATATGGTTGTTGGCGAATTTGGTGGATTGTTGAACCCAGCA


 


ACAGGAGTGATGATTCCAGGTTCTTTAGGTCCATCAGAGCAAACTCCATT


 


CTCCCCTGAAATTGAAGATGGTGGTATTATTCCTCCAGAAGTAGCAGCAG


 


CAAATGCTGATAAATTCAAGTTATCTATTCCTCCAAGCGTACCAGAATCA


 


ATTCCAGAAAAGGATCAGAAGATCGATTCTATTTCTGAATTGATGTATGA


 


TATTGAGTCAGGTAGACTTATTGGTCAAGTATCAAAGAGACCAATCCCAG


 


GTTCAATTGCTGGTGATTTGAACCCAATAATGAAGACACCAACACAAACT


 


GACAGTGTAACTGGTAAGCCAATCGATCCAACCACAGGTTTACCTTTCAA


 


TCCACCAACTGGTCATTTGATTAACCCAACAAATAATAATACCATGGATT


 


CTTCATTTGCTGGTGCATACAAATATGCAGTTTCAAATGGTATCAAGACT


 


GATAATGTTTATGGTTTACCAGTTGATGAAATAACAGGTTTGCCAAAGGA


 


TCCAGTCTCAGATATTCCATTTAACTCAACTACAGGTGAATTGGTTGATC


 


CATCAACAGGAAAGCCAATTGACAATTCTACTGCTGGTATTATTAGTGGA


 


AAACCTGGCTTACCACCTATTAAAGATGAAAATGGCAATTTGTTTGATCC


 


ATCAACTAACTTGCCAATAGATGGTAATAACCAATTAATTAACCCAGAAA


 


CCAACAGTACTGTCCCAGGATCAACTTCAGGTTACTACAAAACCAAAGCC


 


AGGAATTCCAGTCAATGGTGGAGGTGTTGTACCTAATGAAGAAGCTAAAG


 


ATCAAGCTGATAAGGGTAAGGATGGATTAATTGTTCCACCAACTAATTCT


 


ATCAATAAGGATCCAGTAACAAATGCTCAATACAGTAATAGTACTGGTAA


 


CATTATTAACCCAGAAACAGGAAAAGTTATTCCAGGCTCACTTCCAGGCT


 


CTCTCAACTATCCATCATTCAATACTCCACAACAAACTGATGAGATTACA


 


GGAAAGCCAGTTGATACTGTTACTGGTTTGCCATATGATCCATCTACAGG


 


TGAAATTATCGATCCAGCAACTAAATTACCAATTCCAGGATCGGTTGCAG


 


GTGATGAAATTCTCACTGAAGTATTGAACATTACAACAGATGAAGTAACA


 


GGTTTGCCAATTGATCCTGAAACCGGTCTTCCAAGAGATCCAGTATCAGG


 


ACTCCCACAACTTCCAAATGGTACTTTGGTTGATCCATCAAATAAAAAAC


 


CAATTCCAGGTTCACATTCTGGATTTATTAATGGTACATCTGGAGAACAA


 


TCACACGAGAAAGATCCAGGTACTGGTAAGCCACTTGATCCAAATACAGG


 


TTTACCATTCGATGAAGATTCTGGTAGTTTAATTAACCCAGAGACTGGAG


 


ATAAACTTCAAGGATCACATTCTGGTACATTTATGCCAGTGCCAGGTAAG


 


CCACAAGGTGAAAATGGAGGTATCATGACACCTGAGCAGATATTGGAAGC


 


ATTAAATAAATTGCCAACAAGTAATGAAGCAAAATATTTCACCAAAACCA


 


AGTTCAGATGCTGTTCCAGACAAACCAACAAATACTTGGTGGAATAAGAT


 


TTCTGGTCAAACCTACCAGGTTGATGGAGAGAAGACTATTCCAGGTTCTG


 


CAGCTTCAGTAATTCACACTGCTCTTGGAACACCAACTCAAACTGATCCA


 


ACAACAGGACTTCCATCTGATCCATCAACAGGTTTACCATTCATTCCAGG


 


ATTTAACGTACTTGTAGATCCTCAGACTGGAGAGCAAATGAAGGGTTCTG


 


TTCCTTATGTTTCATTGTACGTTAAGGAAAAGAACATTGTAACAGAAGCT


 


GCTTATGGTCTACCAGTTGATCCAAAGACTGGTTTCCCAATTGATCCAAT


 


TAGTTACCTCCCATTTGCTAAGAATGGTGAATTAATTGATCCTATCTCTG


 


GTAAATATTTCAGTGGTTCAATTGCTGGATTCATTTCTGGTAAAGCTGGT


 


ACACAATCTAAATCATCTGATGAGTCAGGTAATCCAATTGATCCATCAAC


 


AAATATGCCTTACGATCCAAAAACAGGCAAATTAATTGATCCAGAATCTG


 


GCATTGCTATTGATAATTCTATTTCAGGTGTATTTGCAACTGTACCTGGT


 


ACTGCTGCACCGAAAAAGGGTGGTGTCATTCCAGAGTCAGTTGCAGCTGA


 


GGCAGCAAAGAAATACTTTGCAGCCAATGTTGAAGGAGGAGAAGGAGAAA


 


AAGTTCCACCACCACCAGAATCATCTAGTAACATTGCAATCCAAGCTGCT


 


GGTGGTGCTTCTGCTGCTGTAGGTCTCGTAGCTGCTGGTGTTGGTGCATG


 


GTATGCAAGCAGAAACAGACAAGAAGGAGAAGATGATGATGACTATGCAG


 


ATGGATTTGAAGCAGAATATGAAGAAGAAGAGGAAGAAGAGGGTGATGAA


 


GCAGCAAATGAAACTGTTGTTACAATTGAGCGTGATTCATCATTCTGGAA


 


CGAATCTTAA






and has amino acid sequence:








(SEQ ID NO. 6)







MTTTTTPPLPDIGDIEITPIPIEKMLDKYTRMIYDYNSGLLLDSNDEPIP


 


GSQAGQIADTSNLFPTQTHKSTGLPIDPMVGLPFDPKSGNLVHPYTNQTM


 


SGLSVSYLAAKNLTVDTDETYGLPIDTLTGYPLDPVSLIPFNPETGELFD


 


PISDEIMNGTIAGIVSGISASESLLSQKSAQIDPATNMVVGEFGGLLNPA


 


TGVMIPGSLGPSEQTPFSPEIEDGGIIPPEVAAANADKFKLSIPPSVPES


 


IPEKDQKIDSISELMYDIESGRLIGQVSKRPIPGSIAGDLNPIMKTPTQT


 


DSVTGKPIDPTTGLPFNPPTGHLINPTNNNTMDSSFAGAYKYAVSNGIKT


 


DNVYGLPVDEITGLPKDPVSDIPFNSTTGELVDPSTGKPIDNSTAGIISG


 


KPGLPPIKDENGNLFDPSTNLPIDGNNQLINPETNSTVPGSTSGTTKPKP


 


GIPVNGGGVVPNEEAKDQADKGKDGLIVPPTNSINKDPVTNAQYSNSTGN


 


IINPETGKVIPGSLPGSLNYPSFNTPQQTDEITGKPVDTVTGLPYDPSTG


 


EIIDPATKLPIPGSVAGDEILTEVLNITTDEVTGLPIDPETGLPRDPVSG


 


LPQLPNGTLVDPSNKKPIPGSHSGFINGTSGEQSHEKDPGTGKPLDPNTG


 


LPFDEDSGSLINPETGDKLQGSHSGTFMPVPGKPQGENGGIMTPEQILEA


 


LNKLPTSNEAKYFTKTKFRCCSRQTNKYLVEMKQNISPKPSSDAVPDKPT


 


NTWWNKISGQTYQVDGEKTIPGSAASVIHTALGTPTQTDPTTGLPSDPST


 


GLPFIPGFNVLVDPQTGEQMKGSVPYVSLYVKEKNIVTEAAYGLPVDPKT


 


GFPIDPISYLPFAKNGELIDPISGKYFSGSIAGFISGKAGTQSKSSDESG


 


NPIDPSTNMPYDPKTGKLIDPESGIAIDNSISGVFATVPGTAAPKKGGVI


 


PESVAAEAAKKYFAANVEGGEGEKVPPPPESSSNIAIQAAGGASAAVGLV


 


AAGVGAWYASRNRQEGEDDDDYADGFEAEYEEEEEEEGDEAANETVVTIE


 


RDSSFWNES;






4) CP15/60 (Chro.40225, start position 10793, end position 12259) is encoded by the nucleotide sequence:








(SEQ ID NO. 7)







ATGTTCGAGTTTATTTCAGAAATGTTTCATTCATGTTGCAAATTAAAAAA


 


AAATCAAAAGAATGATGAATACATTTTTATCTTGTGCCCTACCCCAAGTG


 


ATTTAGAAGAGGAATATATTGATCAAGAAGGAAATGTCAAAAAAAAGAAG


 


CTCGAAAAAATTAGAGGAACTGCCAGAAATATTGTCGATAAGGAAATTGT


 


CAGGGAGTGGAGTGGAAGGGAAATTGGAAGCTGTATTTGCTGTCATTTAA


 


TATATGAAGACGAAATGAATGTTTATAGAGCTGATAAATATGGCAGACAT


 


ATTGGTAAGGACCATGAAGAATATGAAGGTAGCCAAACAAGAGAAGAAAA


 


CCGTGTTAACTCAGTTGAATCTCTGAGCTCATATGGCTCAAGAAAACATT


 


TTTCTGAGGAGCCAAATAGCGCAGATTCCAACTCTACCTCAATAAGTTCA


 


GATGAAAATAATAATGCAGTTGAGAATAAGAGTAAAAAAACAAGAGAAAG


 


GAGGAAGTTAAATATCAGTAGATCCCCAAGCGTAATTGAGAAGGAAATAG


 


ATGAAAAAGAAAAGAAGAACAAAAAACTAAAAGAAACAAAAGATGCCAAT


 


AATAAAGAATGCTCCACAATTAGTTCTGATATAAATAATGATATCCATAA


 


CGCGGATGAGAAAACAACTGATAATAGAAATAACAAAAAGCTAGAGAATA


 


CTAATGTAAAGAATGACGAGCAAATTCCATTCTCCGATCAAAAAAAATAT


 


TCTAAATCTTCTCCACTCTCAAAGAATCAATGCCCTCCAAAGTTAGGAAA


 


AAGGCCACCCATGAAGAATGAATTATTGGCTATGAATGGTCAGAAAAACA


 


ATTCACTTAAGTCATCAATTGCAAATAGTAAAAAATGTAGTAAAAAAATA


 


TCAAGTACTCCGAAAAATGAATTTAACAAAATAATTTTGGAAAAAGAAAA


 


GGTAGAAAGTAATTCTCGCGATACTCATAAAGATGACAAAAATCAAACTG


 


GAAATAATAATGACCAGCAAATCAACCACATTACTAGCAGTTCTAATTCT


 


GATAAAGAAATGATTGATAACAGTGGGGAAATTAAATATGAAGAGGAAGA


 


GATGAAGTTTAACAAAGATATTTCTTCGAAAATAATACGTCACAGAGCAT


 


TAATAGGAATTCAAGCCGAAATTATTCTAAAAGATGGATCGACAACGGAC


 


TGTAAAGTTAGCTTCTCAGATGAGGAAGATGATCTTTCATTTATTTGCAA


 


CGATAAAGTTAAAGCTGTTCCTTGGAGTAACATTAAAGAGATTTTTACAA


 


CAAAAAGTGAACTTAGAATGGTGAATACACGAGCACCTATTTTTAAAGAC


 


CCAACATTAATTATTGCACTACATTTAAAAGATACAGGAAATTGTATACC


 


TTTGAAATTTGATTCTAAGAAAAGCAAAGAAGATTTTTTAAATTTCGCCC


 


TCAGAATGATTGGGTAA






and has amino acid sequence:








(SEQ D NO. 8)







MFEFISEMFHSCCKLKKNQKNDEYIFILCPTPSDLEEEYIDQEGNVKKKK


 


LEKIRTARNIVDKEIVREWSGREIGSCICCHLIYEDEMNVYRADKYGRHI


 


GKDHEEYEGSQTREENRVNSVESLSSYGSRKHFSEEPNSADSNSTSISSD


 


ENNNAVENKSKKTRERRKLNISRSPSVIEKEDEKEKKNKKLKETKDANNK


 


ECSTISSDINNDIHNADEKTTDNRNNKKLENTNVKNDEQIPFSDQKKYSK


 


SSPLSKNQCPPKLGKRPPMKNELLAMNGQKNNSLKSSIANSKKCSKKISS


 


TPKNEFNKIILEKEKVESNSRDTHKDDKNQTGNNNDQQINHITSSSNSDK


 


EMIDNSGEIKYEEEEMKFNKDISSKIIRALIGIQAEIILKDGSTTDCKVS


 


FSDEEDDLSFICNDKVKAVPWSNIKEIFTTKSELRMVNTRAPIFKDPTLI


 


IALHLKDTGNCIPLKFDSKKSKLEDFLNFALRMIG






5) Thrombospondin related adhesive protein (TRAP C1) (Chro.10390, start position 4021, end position 6078) is encoded by the nucleotide sequence:








(SEQ ID NO. 9)







ATGAAAAAGTTAATACTTTATTTAGTATTACTACATATATATATTGTTCA


 


GAAATATGTAATATGTTCAAAATTAACTCATTATTCAGTAGGTGGTCATG


 


CATCAACATCAAGAGTGAAGGGAAGAAGTAGTAGTGGTAGTAGTAGTAGT


 


AGTGGCGATTTTAATGTACCAGGATTAAATGGATATTTATGTCCAAGCTA


 


TAATAGAGACCCAAGAGGATTTGGTTGTTTTGGTATGAATACAGCATATA


 


CGGTTAAAAAGAATAGTTGGCAAGAATGTGCAAATCAATGCTATTGGAGT


 


AAATATACAGTATTTGGTAATTGTCAAAGATCTGTATATAATTCAAATAA


 


TAAAGATTGCTATATTAAAAGTGGTGATAACAGATGCGTGAAGTCTCCAG


 


ATGGAATGATTTTAACAAATAGGCAATCATATATGATCGGAGAGTGTGCG


 


ACAACATGTACTGTTTCAACTTGGTCAAGTTGGACTACATGCTCAGGGGT


 


ATGTGGTGAGATGAGATCAAGAACAAGAAGTGTGTTATCATTTCCAAGAT


 


ACGATTATGAATATTGTCCACATCTGATAGAGTATTCAAATTGTGTAGTA


 


CAAAATAAATGCCCAGAAAATTGCCCACAGTATGGGGTTTCAATATTGGG


 


ATGGGGATGTCAGTTTGAATCAACTTTTTCATTTAATAAAAATTTATTTG


 


TTAGTTATGAAGAAGATTGGAGGGGTTGCATGTCAACTTGCAAACAGGAT


 


CCATTTTGTGTAGCTTGGTCGTATAATGCAACTTTATCAGAAGGACCAGA


 


TTCTGTTGGATTTTCAAGAGAATATCGTCCATGTTATACACATAGATTTG


 


CTTCAGGATGTCAAGCTTTAGCACCAGGATGGGTATCAGGTAATAAGAAT


 


ACAATAAATGTTGATTGTGAAACTGGTACTTGTATACATAATGAATGGTC


 


ATCTTGGACAACATGTAAAGATCCTTGTAGTAATACTGAAACAATGAGTA


 


GAAATAGGACAGTAAAGACTGTATCTCAGAATTGGGCAAGTACACCTTGT


 


AGGGATGAGACTCAAATTCAACTTTGTTCAGAAAACCCACAAAGTATTGA


 


AACTTGTAAAACTTGTTTAGTAGGTGGTTGGTCAGAATGGTCAGATTGTT


 


CAACAAGTTGTGGAGAAGGTAATAGAGTTAGAACACGTGAAGTTACTAAA


 


CCTCCATTGAATGGAGATGATTCAACATGCCCAGAATTAATTGAGAAAGA


 


AAGTTGTAATAAAGATGTGGAATGTCCACATGTTCAATGTGATTGGGAGA


 


ATGGTCTTCTTGGTCACCTTGTAGTGTAACTTGTGGATGCGGAACAACTA


 


CAAGAAATAGGGAAGTAAAGGGAGAGAATTGTACAGAATTATCAACAGAA


 


TCAAAGAAGTGTAATTTGGCAAATTGTGACGATAACTCTGCATCATGTAC


 


TGCAGTTATGTCAGTTTGGTCAGAATGGTCAGTTTGTAGTGAGAAATGTG


 


ATCAGGGAGTAGTAAGAAGGTATCGTGATTTTGATTTTACAAAAATTGGG


 


GTTTTTGGTTATAATCCACCCGGTACATCAGAAGAACAAAATAAAGTGAG


 


AGAAATATGCAAGGATACTCCAACATTAGAAGAGGAGCCATGTACTTCAG


 


GAGTTGCATGTACTCCAGGATGTAAATATACTGAATGGAGTACTTGGTCA


 


AGCTGTGATTGTTCTGGAACTCAAACTAGAGATAGAGTTGTTACTTTCCC


 


TGAAGGTGTAATTGATGCAACTTGTCAGAGTTCTAAAGATACAAGATCAT


 


GTAGCAAGCCTGAAGGTTGTACAGAAACTGCTCCAGATTCTGGAGACGCT


 


ACACTTGCCATTGCTATTGGATTACCAGTTGGTATTCTTGGATTATGCAT


 


TATTGCTGGTTCTTTGTTTTTAATTGGTGGGAGATCAGGTGATCAGGAGG


 


AGGATGAGACAAGTTATCAATACTTTGATCAACCTTCTGCTACTTTAGAT


 


CAAGACTCAGAATATGTTCAAGAAATTGGTCCAGAGAGTCAGAACTGGGC


 


TAGTTGA






and has amino acid sequence:








(SEQ ID NO. 10)







MKKLILYLVLLHIYIVQKYVICSKLTHYSVGGHASTSRVKGRSSSGSSSS


 


SGDFNVPGLNGYLCPSYNRDPRGFGCFGMNTAYTVKKNSWQECANQCYWS


 


KYTVFGNCQRSVYNSNNKDCYIKSGDNRCVKSPDGMILTNRQSYMIGECA


 


TTCTVSTWSSWTTCSGVCGEMRSRTRSVLSFPRYDYEYCPHLIEYSNCVV


 


QNKCPENCPQYGVSILGWGCQFESTFSFNKNLFVSYEEDWRGCMSTCKQD


 


PFCVAWSYNATLSEGPDSVGFSREYRPCYTHRFASGCQALAPGWVSGNKN


 


TINVDCETGTCIHNEWSSWTTCKPCSNTETMSRNRTVKTVSQNWASTPCR


 


DETQIQLCSENPQSIETCKTCLVGGWSWSDCSTSCGEGNRVRTREVTKPP


 


LNGDDSTCPELIEKESCNKDVECPHVQCELGEWSSWSPCSVTCGCGTTTR


 


NREVKGENCTELSTESKKCNLANCDDNSASCTAVMSVWSEWSVCSEKCDQ


 


GVVRRYRDFDFTKIGVFGYNPPGTSEEQNKVREICKDT






6) TSP1 domain-containing protein TSP7 precursor (Chrom 60103, start position 12, end position 1982) is encoded by the nucleotide sequence:









(SEQ ID NO. 11)









ATGGATTCAATTAACTTTAGAAGCATTTATATTCCATCAGCAGTGAGGTA



 


TATTATATTACTTTTATTATGGACAATATTTACAAAAAATGTTTATAGTG


 


AAAGTAGTGAAGAAACTTTATTGGGAAGATCAGTATTGGATTTAAACAAG


 


AAAAATACATGTGAATACTATGGAGAGCAGGATGGTATGTTTACTGATTC


 


ATTTCATTCAAGAATATGTATAGTTCCAGAAGATGGATTACATGGAAAAA


 


GGGAATATGAAAATCATCAAAAAAAAACATTTGGAACAATTAGACCAAAT


 


AATAAACAATTATCTGATAATAAATTATATAGGAAAGATGATGATTTAAC


 


TTCTTCAATTGCAGATTTTGATAGTAATTCTGTGAGAATACAGAGAAAAA


 


ACGTGGATTTAGAAGCTATGTTTGGAATAGGAAAAGATAACAACAGAATG


 


AATCTTAATAATGAAGCAATTCAAAGTTTCTATTCAAATAATGAAACAGA


 


AAGCCAAGATAAGAATGCGACAAACGACTATTTTTTATTTAAAGAAGGAC


 


TTTTGAAATTTCAAGAGAAAAAGATATTAAGATATTATTTATATGATGTA


 


GGAAATAAAGTCTATTCAGATACTATAGCTTATCCAGAAAATGTTATATC


 


AGAAAACTGTGCATTTAACTATTTGGGGAATTATGTAGATGTTTATGAAA


 


TTAGTAAAGTATCAGATCCACCAGTAATTTCATGGCCAAATAATCACATA


 


GTTTTTATACACTCTCAGGTAAAATCTGATGGTACATTTAAATTTCAAGT


 


ATATACTAGCTCAGGAGAGATAGGATTTTATTTTGAAGTAACTGATAATA


 


GTTATAAAACAGGTTGTGGTAGTTATTCTAGAGTTGATAAAAGCAAATTT


 


ACTCACTCTGCAAATTCTTTAATTCAAGTTCAATTAGTAAGAAGAAAGTT


 


TGGATTTAATGTATTTGTTGATGGTACTAGAAGAACACAACTAGATATAA


 


TTGATTGTATTGCAAGTGTTCCAACTAAAGTTCAAATAACTAGTGGATCA


 


GGATCACAAATTTATCCAAAAGTAGAAGATTGTCAAATTTCACAGTGGAC


 


AGATTGGTCAACTTGTTCAAAAACTTGCTCAACTGGTTCAAAAGCTAGAT


 


ACCGTTCAGTAATTATGCCAAGTATGAATGGTGGTTTACCATGTCCTAAA


 


TTACTTGATTCAAGTCCTTGTAATGCTGATATTTCATGCTCATCATGCCA


 


ATATTCGGAATGGACAATGTGGGGTGAGTGCTCAGCTACTTGTGGATCAG


 


GATCAACAACCAGAACAAGAAAGTTATTAAGTGCAGCATATTTTATTGAA


 


AGCTGTATTGATACATTCCAATTAAAATCTTGCCATGGTGTTTCTTGCGC


 


TAGTGATTGTATTGTAACAGAATGGTCAGATTGGAGCGAATGTAGTACAA


 


CTTGCGGTGTTGGAAGTCAAATTTCCACAAGATCCATAGTTGTTCCAGAA


 


CAAAATGGTGGAAAATGTGATTATGATCTTAGCAAAATCCAAGAATGCAA


 


TGTTTCTGTTTGCTCCAAGTCTTGTGATCCTTCTCCATGCTTAAATGGCG


 


GTATTTGTAGTGAACTACCAAAGTCAAACTTCGCTTGTACATGCCCGCCA


 


TTTTACGGAGGTGAAACTTGTGATCAATTTGAATTTCCTTGGTGGTTTTA


 


TAGTGTTATAATTGTTTTAGTTGTTTTGGCTATTGGGATATTTTATAAGT


 


CACAAATTTCAAATATAGTTACTCCAAATACAATGGATCCTTCATATGCA


 


GGAGATGGGGATTATGCCTTCAGTCAAGGCCCTGGTCCCCCAGATCCATT


 


GCAAGCAGCAAATGGACAACCTCAATACTACCAAAATACGTATAATTACA


 


ACTATGGGTATTATGACAATTCTAATGAAGGATATTTGGTAAATAATGAT


 


GAAGGAAATTGGATGTACTAG






and has amino acid sequence:









(SEQ ID NO. 12)









MDSINFRSIYIPSAVRYIILLLLWTIFTKNVYSESSEETLLGRSVLDLNK



 


KNTCEYYGEQDGMFTDSFHSRICIVPEDGLHGKREYENHQKKTFGTIRPN


 


NKQLSDNKLYRKDDHLTSSIADFDSNSVRIQRKNVDLEAMFGIGKDNNRM


 


NLNNEAIQSFYSNNETESQDKNATNDYFLFKBGLLKFQEKKILRYYLYDV


 


GNKVYSDTIAYPENVISENSAFNYLGNYVDVYEISKVSDPPVISWPNMHI


 


VFIHSQVKSDGTFKFQVYTSSGEIGFYFEVTDNSYKTGCGSYSRVDKSKF


 


THSANSLIQVQLVRRKFGFNVFVDGTRRTQLDIIDCIASVPTKVQITSGS


 


GSQIYPKVEDCQISQWTDWSTCSKTCSTGSKARYRSVIMPSMNGGLPCPK


 


LLDSSPCNADISCSSCQYSEWTMWGECSATCGSGSTTRTRKLLSAAYFIE


 


SCIDTFQLKSCHGVSCASDCIVTEWSDWSECSTTCGVGSQISTRSIVVPE


 


QNGGKCDYDLSKIQECNVSVCSKSCDPSPCLNGGICSELPKSNFACTCPP


 


FYGGETCDQFEFPWWFYSVIIVLVVLAIGIFYKSQISNIVTPNTMDPSYA


 


GDGDYAFSQGPAPPDPLQAANGQPQYYQNTYNYNYGYYDNSNEGYLVNND


 


EGNWMY






7) Protein similar to riken cDNA 5830420c20 gene (Chrom 60194, start position 15004, end position 16041) is encoded by the nucleotide sequence:









(SEQ ID NO. 13)









ATGAAAGAATCAGGCACAATTAATTATCTAATAACATTTACATTCATTAT



 


TCCTTTCGTACTTTCCCAGTCAACATTATTAAATCTTGGTGCAGGGGGTA


 


TACAGGAAAGGAGGGTTTGCACTGACGAAATGCCATGTAACTTTAGATTG


 


GTTGCTGATTTAGATATGAAGTCAAAGCCAGGGAGTGGGGAAAAGAATTA


 


CAAAAGTTTATTTCAAAAAGGGTCAATAATACAAGACAAAAGGGGCAACT


 


ATCGAGTGTACTGGGGAGAAAGTCTGGAACTTAAAAGCGGATATAATGAA


 


TATGGGAGAGGGATGGAATTAAGTGAGTTGATTTCATATAATGGAATGAT


 


GCTTGCGGGCGACGACCGTACAGGAATAATTTTTGAAATAACTGATGATG


 


GAAAAGGAGTAGCACCAAGATATATATTATCTGAAGGTAATGGAAGAACA


 


GCTAAGGGAATGAAGATTGAGTGGTTTGCTGTAAGAGATGGAATATTGTG


 


GGTTGGCAGTTTTGGAAAAGAGTTCGTATCAAACGGCATAATAGAAAAAA


 


GAGATAATATGTGGGTAGCCACAATTGATAAAAGAGGATATGTTTCACGA


 


TTTAATTGGAGTTTTGTTTATGAAAAAATTAGGAATTCACTGGGGGCGCA


 


ATATCCAGGTTATTGCATTCATGAAGCAGTGATTTGGAGTCATTTAATGA


 


GAAAGTGGATATTTTTACCAAGAAGAGTTAGCTTCGATGAGTATGATGAG


 


GAGAAAGACGAAAAGAGAGGTTCCAATAAAATGATAATTATGACAGATGA


 


TTTTGAAATTCTTGAAATTATTGACGTAGGATTGATAATACCTGAAAGAG


 


GTTTTTCTTCTTTAAAATTTCTTCCTGGGTCGTTTGACCAGATAATAGTT


 


GCAACAAAAAGCGTTGAAGAATCAATTTCAGACACTCAAAAGTCTTTCTT


 


AACTATATTCACAATAAATGGAAAAATTTTAATGGAAGATTTAGAAGTGC


 


CTGGAGACTACAAATACGAGGGGATAGAATTTATATAG






and has amino acid sequence:









(SEQ ED NO. 14)









MKESGTINYLITFTFIIPFVLSQSTLLNLGAGGIQERRVCTDEMPCNFRL



 


VADLDMKSKPGSGEKNYKSLFQKGSIIQDKRGNYRVYWGESLELKSGYNE


 


YGRGMELSELISYNGMMLAGDDRTGIIFEITDDGKGVAPRYILSEGNGRT


 


AKGMKIEWFAVRDGILWVGSFGKEFVSNGIIEKRDNMWVATIDKRGYVSR


 


FNWSFVYEKIRNSLGAQYPGYCIHEAVIWSHLMRKWIFLPRRVSFDEYDE


 


EKDEKRGSNKMIIMTDDFEILEIIDVGLIIPERGFSSLKFLPGSFDQIIV


 


ATKSVEESISDTQKSFLTIFTINGKILMEDLEVPGDYKYEGIEFI






8) SPFH domain/Band 7 family protein (Chrom 50147, start position 5008, end position 5850) is encoded by the nucleotide sequence:









(SEQ ID NO. 15)









ATGTCGAGAATTGAAAAAGGTTTTAACATCTTAGCTAACTTGGGGATAAT



 


GCTGGTTGCAGGTGGAAGCATTTTGGCGTCTAATAGTATGTATAACGTGG


 


ATGCTGGACACAGAGCAATAAAGTTCTCAAGAATACATGGAGTTCAAAAA


 


AGGATTTATGGAGAAGGAACTGATTTTATGCTACCTTGGATTGAAAGACC


 


AGTGATATTTGACATCAGAGCGAGACCTCGAGTTGTTGTATCTCTAACGG


 


GAAGTAAAGATCTTCAAATGGTGAATATTACGTGCAGAGTTTTATCAAGG


 


CCTGATAAAGATAAGCTGGTGGAAATATATAGAAATATAGGGCTGGATCA


 


CGATGAGAAGATTCTTCCATCAATAATTAATGAAGTGCTAAAATCAGTAG


 


TGGCTCAGTATAATGCTTCTCAACTACTAACTATGAGAGAAGACGTGAGC


 


AAAACAATCCGAGATTTACTGGTAAAAAGAGCCCAAGAGTTTAATATTAT


 


TCTGGATGATGTCTCTTTAACCCATTTAAGCTTTTCTCAGGATTATGAAA


 


AGGCTGTAGAGTCCAAGCAAGTTGCTCAACAACAAGCAGAAAGAGCGAAA


 


TATCTTGTTCTCAAGGCAAACGAAGAGAAAAAAAGTACTATTATTAAGGC


 


TGAAGGCGAAGCAAAAGCTGCAAAACTAATTGGAGATGCGATAAATGAGA


 


ATCCTGCCTTTATTGCTGTTAAACAGGTGGAGACTTATAGAGAAATCTCT


 


AATATTTTAGCAAAATCAACTTCTAAATCGCTTATAAATCTTTCATCATT


 


TTTGCCAAACCTCCCAAATAGTAATTTACAATCATCTTGTTAG






and has amino acid sequence:









(SEQ ID NO. 16)









MSRIEKGFNILANLGIMLVAGGSILASNSMYNVDAGHRAIKFSRIHGVQK



 


RIYGEGTHFMLPWIERPVIFDIRARPRVVVSLTGSKDLQMVNITCRVLSR


 


PDKDKLVEIYRNIGLDHDEKILPSIINEVLKSVVAQYNASQLLTMREDVS


 


KTIRDLLVKRAQEFNIILDDVSLTHLSFSQDYEKAVESKQVAQQQAERAK


 


YLVLKANEEKKSTIIKAEGEAKAAKLIGDAINENPAFIALKQVETYREIS


 


NILAKSTSKSLINLSSFLPNLPNSNLQSSC






9) UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T4 protein (Chrom 70457, start position 44914, end position 46158) is encoded by the nucleotide sequence:









(SEQ ID NO. 17)









ATGAGGAACGCATTCCCTCTCGGGCTGTCAATATGTTATTTGATGTTGAA



 


AGTTGCATTGACTACTTTTGTTTTTGGGTCAAAAGAAGAGTTTACAACAC


 


TCCCTAGAGAATTAATAAACTCTTGGTTGGAAGAAAATGAATATGCTGGA


 


TTATATGGAAAATCAGATATTTTTTCAATTGTAATAATACCAGACTGTGA


 


AGATAATGAATTGATTGATGTTACAATAAATAGTATACTTTTGACTGCAA


 


ATCGAAATTTACTTCATGAAATAATAATTATTTCAAATGATTGTCAAGAC


 


TCTGGAAAAGATATTAAAAGTTATTTGGGTGAGAAATTCTTGGATAAGCC


 


TTTGATTAAAATAATTGAGACTGAATTACAAGAATTGGGAGAATTACAGA


 


ATCTTGGAGCAAATAATTGAACTGGGGAAATCATTTTATTTGTTCCGTCT


 


GCAACTCTTTTTCCAAAAAATTGGATGTCACCAATAATGAGGAGTTTAAG


 


TGATAATTATAAATCAATAATAGTTCCAAGATTTAAAAAATTGAATAAAA


 


ACAAATGGGCATTTTCGAACAATGATCCTGTATATTCACCAAAAATGATG


 


TTCACAAAAGAATTTGAATTAACAAATATCCATACATTAGATAATAAAGT


 


TCCAATGTTCTATTCAAAAATCTTTGCAATAACAAAATCATGGTGGTTAA


 


ATATATCAAAGCTTTCAGATCCAACAATTAACCTGATATTCAAAACGAGT


 


ATTAACTTTGATATTTCTCTAAGATCATGGAATTGTGGTGGGCGAGTAGC


 


TCAGATAGCAGAACTGTCATTTGGTGTAACTAAGGTAAAAATCTCACAAC


 


CTTCGTTAGAAATAAGACAAGTTCTATTGGAATCTTGGATAGATGAGCCA


 


ACCAAGCAGATGATTATGAATAATAGTGAGAAGCTGGCTAACTATATGAA


 


ACTATCATCAGGATTATTTGAGGTATTGATAAATAAACGTAAGGAACTCA


 


TTAAAGAGTATGAATGTGACCAAAAGTCAATATTTACTTCAAAATTTTAT


 


AATGAGTTGAGCGAATTTGGACTAATAGAATATCCAAAAAGTCAAATAGT


 


TTTTAGTGGTAATGGCAAATGCTTCACATTAATTGGGAACGAGAAAAAAA


 


GTGGAGAAAAGAATTTCGAGTTAAAGTTATCAGAATGTAAACCAAATGAA


 


AATGCACAAATATTCTACATTGACAATGAGAGTAAGTTGATCTAG






and has amino acid sequence:









(SEQ ID NO. 18)









MRNAFPLGLSICYLMLKVALTTFVFGSKEEFTTLPRELINSWLEENEYAG



 


LYGKSDIFSIVIIPDCEDNELIDVTINSILLTANRNLLHEIIIISNDCQD


 


SGKDIKSYLGEKFLDKPLIKIIETELQELGELQNLGANNSTGEIIILFVP


 


SATLFPKNWMSPIMRSLSDNYKSIIVPRFKKLNKNKWAFSNNDPVYSPKM


 


MFTKEFELTNIHTLDNKVPMFYSKIFAITKSWWLNISKLSDPTINLIFKT


 


SINFDISLRSWNCGGRVAQIAELSFGVTKVKISQPSLEIRQVLLESWIDE


 


PTKQMIMNNSEKLANYMKLSSGLFEVLINKRKELIKEYECDQKSIFTSKF


 


YNELSEFGLIEYPKSQIVFSGNGKCFTLIGNEKKSGEKNFELKLSECKPN


 


ENAQIFYIDNESKLI






10) ABC transporter ATP-binding protein (Chro.70309, start position 5325, end position 7046) is encoded by the nucleotide sequence:









(SEQ ID NO. 19)









ATGCTTATTATAAACGGAGCAATTAATTGTGCTAGAACTGATATGTCGAT



 


TCGAATTCAAATTGATTTAAGGATGTGGCTAACTAATTTGATTTTGAAAC


 


AATATTATTCTGATTTAACTTACTATCAGTTCTCAATAAATAAAACGATT


 


GATAATCCTGACCAAAGAATTGGAGAGGACATTTCACTGTTTTCATCACA


 


TTTATTATTGTTAATATGTCGTTGTATAGACAACTTATTTGACTTTTTTG


 


TTTATTCAATCTTGCTATACAATGTTAATTTCAAATTGTTTATTTCAGCA


 


ATTATTTATTCTTGTTTTGGCACATTTTTAACTGCTAAATTAGGCATGAA


 


TATAATATTATTAAAAGTTCAAGAAAAAAAGCTTGAAAGTGATTTTAGAT


 


ATTCAATTATGAGAGTTGGTGAAAATGCAGAAAATGTTGCAATGTATGGA


 


GGAGCTCAGTGCGAGATGGAAAGACATGAACAAATTTTAAATTCATTGCT


 


TCTCAACTTAACTACGAAAAGATCATTTGAATCTAAAATGGGACTCTTTG


 


GAAGTATTTTTAGAAACCTAATACGTGTTCTGCCTATTGCGGTTATTTCA


 


GGAGACTACTTTTCAGGAAATATCCAACTTGGAAGAATTAATCAGTGTAG


 


CCTTGCATTTAACAGTATAGTTGAAGATATTTCAATTTTGGTCAATACTT


 


TTAGAGAAATAAGTAATCTACTTTCTTCAATAGATAGAGTAGGACATTTT


 


ATTGCATTGATGGCAGATAACTATATTGAATCTCAATCTATAAATATTGG


 


AGAGAAATTGATTAGTAGTTTTGAGAGTGATTCTAAAACTAGCAAAAAAA


 


TTGATTTCTTACATCTTGAAAGCGAGTTTTCAAGACAAATGAAGGAAAAA


 


TCTTTAGAATTTAAACTTAATTTCTCGACAGGAATTGCTTCTAAGTCATT


 


AAAAAATTGTGTTAAATTAGAATTTACAAACACTCAGGGGAAATCTTCGG


 


TTAATATACGTGGAAAAATTAGATCAGTAATATGGCCAGAACCAAAAATT


 


AAGTTTGAAAGTGTATCGATTAATACTCCGGAAGAATATCCCAGGAAACT


 


TCTTTTTAATATAAACTTTATGATTGAACAAAGCGATAAAGTCTTAATAA


 


CAGGGGACTCCGGTGTTGGGAAATCATCACTCTTAAAGGTAATTTGTGGG


 


ATTTGGAATAATGGGTCAGGAAATATTTATAGGCCACCTTCTAGTGAATT


 


ATTATTTATACCGCAAAAACCCTACTGTACCCAAGCAACGCTAAGGGAAC


 


AACTGTTTTATCCGCAAATACCCTCAATTAAAACTAATGGTTATGAATAT


 


AAAAATAAGGAAGAACTAGATTCATATCTATTGAAAATTCTTGAGGAAGT


 


TGGGCTGAAATATCTATGTGATCGACTTTCTGAAAGTGAAACAGTTAATT


 


GCTTAGACACCATTAAAGACTGGTCAACAATACTTTCGCTTGGAGAACAG


 


CAAAGACTTGCATTTGCAAGAATATTTATTTTCAAACCATCTATTTGTTT


 


CCTTGATGAAGCTACAAGTGCGTTAGACATGGAGACTGAAACAAAATTAT


 


ATTCAATGCTAAATAAAAAAAACTTTACATACGTTAGTGTAGGTCACAGG


 


CCCTCAATATCAATATTTCACAACAAAAAAGTCCTTATAAAAAATGGTAA


 


TATAATTTTTGAATGTATATGA






and has amino acid sequence:









(SEQ ID NO. 20)









MLIINGAINCARTDMSIRIQIDLRMWLTNLILKQYYSDLTYYQFSINKTI



 


DNPDQRIGEDISLFSSHLLLLICRCIDNLFDFFVYSILLYNVNFKLFISA


 


IIYSCFGTFLTAKLGMNIILLKVQEKKLESDFRYSIMRVGENAENVAMYG


 


GAQCEMRRHEQILNSLLLNLTTKRSFESKMGLFGSIFRNLIRVLPIAVIS


 


GDYFSGNIQLGRINQCSLAFNSIVEDISILVNTFREISNLLSSIDRVGLF


 


IALMADNYIESQSINIGEKLISSFESDSKTSKKIDFLHLESEFSRQMKEK


 


SLEFKLNFSTGIASKSLKNCVKLEFTNTQGKSSVNIRGKIRSVIWPEPKI


 


KFESVSINTPEEYPRKLLFNINFMIEQSDKVLITGDSGVGKSSLLKVICG


 


IWNNGSGNIYRPPSSELLFIPQKPYCTQATLREQLFYPQIFSIKTNGYEY


 


KNKEELDSYLLKILEEVGLKYLCDRLSESETVNCLDTIKDWSTILSLGEQ


 


QRLAFARIFIFKPSICFLDEATSALDMETETKLYSMLNKKNFTYVSVGHR


 


PSISIFHNKKVLIKNGNIIFECI






11) Sporozoite cysteine-rich protein (Chro.60102, start position 11337, end position 12380) is encoded by the nucleotide sequence:









(SEQ ID NO. 21)









ATGTTAGAATTTAGACAATATGATTTAAAGTTTATGAAAATGAAAAGAAT



 


TTTGTATTTTATATTAATACATATTCTTATATTTAATATTTTAGAAATAA


 


ATTCTTTACCACCAAGTTTTAGTTGGACAAAAGCATGGAAAGATATTACC


 


AGTGAAGGGTTAGTATATACATTTAGTTCAAATAAGCTACCTTGGTATTC


 


TGGAGTATCTTTTAGGATTGTGGGTAAATTTAACGCAGAAAATGATAAAG


 


AAACTTTGGTAACAATTCAGAATGGTGATTTATACCACTGTAAGTTGATT


 


ATAAATTTTGCAGCACAAACAGTAGATGTGGAATCTACAGGATATACTGC


 


AGAAGAGAGATGGGCTAGATCTTATGCTTACTTTCCATTTCCATATAAGC


 


CAAAATTGATGGATCTTGACTTGGTAGTTGAGAAATTAAGATGGCCAGGA


 


GGGTTTTACTTTTATATTTCAGGAAGTGGACCATATTATCCTTGTCATAG


 


CATAGTGTATTCAAATGTGAATAAATTAACTTTTGGTAATGGACAAAATA


 


ACTTTAGTAAATATAAAATTACAAGAAATGTTCCTTTGGCAGATCCTTAT


 


AGAAGGACTTATTTCTGGGACGAATTTCAACAAAGATACTATTTTGATGA


 


TAAAAATTTGTATTATGTAAATAGCACCGGAATTGATGAGAAAATCTGGA


 


GTACCAAATGGTGATAGAATTCCAAAACATTATAAATCTTGGCCAGAAGA


 


ACTAGAAATACATGTACATTCAGCAAGTATGTACCCAGTTAATGATAAAA


 


GATACGGATGGGGAGGTACGGTAGCAGTATTTACAAGCGATCAGAGTCAG


 


TTTTATTATAGAATGAATGGATTTTTTGCAACTTTGTCAAGTAATTCATA


 


TTGTTTAAGTTCGAGTGTATTATTAAGTGGGACAAGTTATACAGTTAGTG


 


GAGATTATCCTTTTGATTTTGATAATCCAGGTCAACCTTTCAATGTAAGT


 


TTACTTATGATTATTAAGATAATAAGCCTATTTATTATGAGGTAG






and has amino acid sequence:









(SEQ ID NO. 22)









MLEFRQYDLKFMKMKRILYFILIHILIFNILEINSLPPSFSWTKAWKDIT



 


SEGLVYTFSSNKLPWYSGVSFRIVGKFNAENDKETLVTIQNGDLYHCKLI


 


INFAAQTVDVESTGYTAEERWARSYAYFPFPYKPKLMDLDLVVEKLRWPG


 


GFYFYISGSGPYYPCHSIVYSNVNKLTFGNGQNNFSKYKITRNVPLADPY


 


RRTYFWDEFQQRYYFDDKNLYYVNSTGIDEKSGVPNGDRIPKHYKSWPEE


 


LEIHVHSASMYPVNDKRYGWGGTVAVFTSDQSQFYYRMNGFFATLSSNSY


 


CLSSSVLLSGTSYTVSGDYPFDFDNPGQPFNVSLLMIIKIISLFIMR






12) 19K sporozoite antigen “profilin” (Chro.30189 start position 2297, end position 2785) is encoded by the nucleotide sequence:









(SEQ ID NO. 23)









ATGTCTGAATGGGATGATATGGTCAAAGAATGGTTAATTGACACCGGTAG



 


TGTATGTGCTGGTGGTCTTTGTTCAATAGATGGTGCATTCTATGCTGCTT


 


CTGCTGATCAAGGTGATGCCTGGAAGACTCTTGTTAGAGAAGATCATGAA


 


GAAAATGTTATTCAATCCGACGGAGTTTCAGAGGCTGCTGAATTAATTAA


 


TGATCAAACTACACTATGCCAAGCTATCTCTGAGGGTAAGGCACCAAACG


 


GCGTTTGGGTCGGAGGAAACAAATATAAGATTATCCGCGTAGAGAAGGAC


 


TTCCAACAAAACGATGCTATTGTTAATGTTACATTCTGTAACAAACCTCA


 


AGGTGGATGTTTTTTAGTTGATACTCAAAACGGTACTGTTGTCGTTGCGG


 


TTTACGACGAATCCAAAGATCAATCATCAGGTAATTGCAAGAAGGTTGCT


 


TTGCAACTGGCCGAGTACCTCGTATCTCAGGGATACTAA






and has amino acid sequence:









(SEQ ID NO. 24)









MSEWDDMVKEWLIDTGSVCAGGLCSIDGAFYAASADQGDAWKTLVREDHE



 


ENVIQSDGVSEAAELINDQTTLCQAISEGKAPNGVWVGGNKYKIIRVEKD


 


FQQNDAIVNVTFCNKPQGGCFLVDTQNGTVVVAVYDESKDQSSGNCKKVA


 


LQLAEYLVSQGY






Example 3
Immunotherapy
C. hominis proteins have been overexpressed in E. coli and isolated, and are tested in model systems to examine their abilities to induce a protective immune response. Animal models of Cryptosporidium infection have demonstrated the role of both cell and humoral immune responses (1, 2). Thus, mice with targeted disruptions of genes encoding immunological important molecules have established the role of these proteins in the immune response against Cryptosporidium (1-7). Other studies of the immune response against Cryptosporidium use neonatal mice and mice lacking functional T or B cell responses (1, 8, 9). These studies have demonstrated how an immune response is induced in mice that are not immuno-competent. Adult mice are resistant to the infection with Cryptosporidium but the injection of an antibody against Interferon Gamma (IFN γ) prior to infection render them susceptible to Cryptosporidium (10, 13). IFN γ plays an important role in the induction of an immune response against many microorganisms, and many studies have shown that it is one of the key components of the immune response against Cryptosporidium infections (11-17). Thus the temporary neutralization of IFN γ by the administration of the antibody in adult mice is an excellent tool to study and characterize the immune response in the context of an immunocompetent animal (10).
The IFN knockdown mouse model follows a pattern of infection very similar to that seen in severe clinical infections (10). During the infection the animals lost weight and then regained it during recovery. This is also similar to what is observed in metabolically stressed children during and after diarrheal diseases. The predominant site of infection was the small intestine as it has been observed in animal and human severe infections. No parasites can be detected by day 30, thus the level of infection achieved is enough to generate an immune response, which also it is demonstrated by the detection of cellular responses against Cryptosporidium antigen extracts and proteins with lymphocytes obtained from the treated animals. Thus this model offers the possibility to study the potential role of Cryptosporidium as the animal can mount a parasite specific immune response (10).
We use this model (and alternative models such as a malnourished mouse model, unpublished) to study the potential role of Cryptosporidium recombinant proteins and plasmid DNA containing Cryptosporidium genes to induce an immune response. Our protocol to carry out this study is outlined in brief below. Subject mice are divided in groups: Group I mice are immunized with a pool of recombinant antigens (or plasmids in the case of DNA vaccine) in adjuvant (Freund's or other as appropriate); Group II (control animals) are immunized with Freund's adjuvant alone (or plasmid with no insert in the case of a DNA vaccine). The immunological responses in each group are followed and the response to challenge is measured. A sample immunization schedule is outlined below:

    
    
        1) Day 0: Immunization with a pool of recombinant antigens (2-3 μg each), DNA (20-100 μg) or controls. Immunization is performed intraperitoneally or intranasally.
        2) Day 14: Boost Immunization with the same antigens (protein, DNA, control). Boost is administered intranasally.
        3) Day 28: Anti-IFN-γ Ab inoculation, intraperitoneally, 2 h prior to Cryptosporidium infection.
        4) Day 28 (2 hr after inoculation of anti-IFN-γ Ab): Infection with Cryptosporidium. 

The immune response and protection to infection is monitored. A sample protocol is outlined below:

        1) Serum samples are collected at day 0, 7, 14 and 28 to monitor the immune response. Antibody levels against fusion proteins and Cryptosporidium extract are measured by immunoblotting and indirect immunofluorescence.
        2) Fecal IgA responses are measured before and after vaccination and infection.
        3) Fecal shedding of Cryptosporidium is monitored by fluorescent microscopy and real time PCR three times per week after infection
        4) Cytokine and lymphoproliferation responses are examined by isolation of mesenteric lymphocyte node and spleen cells to detect cytokine responses and lymphoproliferation against antigens (those that can be purified) and against a Cryptosporidium preparation.
    
    


These results indicate proteins which induce an antibody (mucosal or humoral) or cellular immune response. More important, these results indicate which immune response is protective and reduces infection (and shedding) by the exposed animal.
REFERENCES FOR EXAMPLE 3


    1. Riggs M W. Recent advances in cryptosporidiosis: the immune response. Microbes Infect. 2002. (10): 1067-80.
    2. Lean I S, McDonald V, Pollok R C. The role of cytolines in the pathogenesis of Cryptosporidium infection. Curr Opin Infect Dis. 2002. 15(3): 229-34.
    3. Ehigiator H N, Romagnoli P, Borgelt K, Fernandez M, McNair N, Secor W E, Mead J R. Mucosal cytokine and antigen-specific responses to Cryptosporidium parvum in IL-12p40 KO mice. Parasite Immunol. 2005. 27(1-2): 17-28.
    4. Chen W, Harp J A, Harmsen A G. Cryptosporidium parvum infection in gene-targeted B cell-deficient mice. J. Parasitol. 2003. 89(2): 391-3.
    5. Davami M H, Bancroft G J, McDonald V. Cryptosporidium infection in major histocompatibility complex congeneic strains of mice: variation in susceptibility and the role of T-cell cytokine responses. Parasitol Res. 1997. 83(3): 257-63.
    6: Lacroix S, Mancassola R, Naciri M, Laurent F. Cryptosporidium parvum-specific mucosal immune response in C57BL/6 neonatal and gamma interferon-deficient mice: role of tumor necrosis factor alpha in protection. Infect Immun. 2001. 69(3): 1635-42.
    7. McDonald S A, O'Grady J E, Bajaj-Elliott M, Notley C A, Alexander J, Brombacher F, McDonald V. Protection against the early acute phase of Cryptosporidium parvum infection conferred by interleukin-4-induced expression of T helper 1 cytokines. J Infect Dis. 2004. 190(5): 1019-25.
    8. McDonald V, Deer R, Uni S, Iseki M, Bancroft G J. Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice. Infect Immun. 1992. 60(8): 3325-31.
    9. Kuhls T L, Greenfield R A, Mosier D A, Crawford D L, Joyce W A. Cryptosporidiosis in adult and neonatal mice with severe combined immunodeficiency. J. Comp Pathol. 1992. 106(4): 399-410.
    10. Theodos C M, Sullivan K L, Griffiths J K, Tzipori S. Profiles of healing and nonhealing Cryptosporidium parvum infection in C57BL/6 mice with functional B and T lymphocytes: the extent of gamma interferon modulation determines the outcome of infection. Infect Immun. 1997 65(11): 4761-9.
    11. You X, Mead J R. Characterization of experimental Cryptosporidium parvum infection in IFN-gamma knockout mice. Parasitology. 1998. 117: 525-31.
    12. Mead J R, You X. Susceptibility differences to Cryptosporidium parvum infection in two strains of gamma interferon knockout mice. J. Parasitol. 1998. 84(5): 1045-8
    13. Griffiths J K, Theodos C, Paris M, Tzipori S. The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum. J Clin Microbiol. 1998. 36(9): 2503-8.
    14. Pollok R C, Farthing M J, Bajaj-Elliott M, Sanderson I R, McDonald V. Interferon gamma induces enterocyte resistance against infection by the intracellular pathogen Cryptosporidium parvum. Gastroenterology. 2001. 20: 99-107.
    15. Aguirre S A, Perryman L E, Davis W C, McGuire T C. IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of CD4+alpha beta+IFN− gamma+ and CD4+alpha beta+IL-4+lymphocytes in gut-associated lymphoid tissue during resolution of infection. J. Immunol. 1998. 161: 1891-900.
    16: Lehmann J, Enssle K H, Lehmann I, Emmendorfer A, Lohmann-Matthes M L. The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice. J Interferon Cytokine Res. 2000. 20: 63-77.
    17. Lacroix S, Mancassola R, Naciri M, Laurent F. Cryptosporidium parvum-specific mucosal immune response in C57BL/6 neonatal and gamma interferon-deficient mice: role of tumor necrosis factor alpha in protection. Infect Immun. 2001. 69: 1635-42.


Example 4
Development of Chemotherapeutic Agents
The approach to the development of chemotherapeutic intervention strategies parallels the selection of candidate vaccinogens using genome sequence data and informatics approaches. The rationale is that weak links in the parasite's biology can be identified by examining its genome. For example, several hundred likely essential transporter proteins that are responsible for importing essential nutrients into the parasite have been identified (see Table 3). Without these transporters, the parasite is unable to obtain required building blocks and therefore is unable to survive. Inhibition of one or more transporters (for example, by small molecule inhibitors or by antibodies) is a useful strategy for prevention or treatment of the C. hominis related disease.
The process for development of these agents includes the following steps:

    
    
        1) Informatic pathway annotation and analysis to identify the processes known to be active in the parasite. This work has been completed, although updates are included with technology advances.
        2) Network and topological analysis are applied to identify nodes that are considered ‘critical elements’ or ‘essential steps’. This analysis involves a series of mathematical and computational steps to identify those proteins that, when inhibited, are most likely to result in death or growth inhibition of the parasite. Examples of such ‘critical elements’ include transporters for essential nutrients (ions, amino acids, nucleotides, sugars, etc.) for which no substitute is available, essential steps in energy metabolism (e.g., glycolysis, regeneration of NADP, etc.), intermediary metabolism (e.g., carbohydrate anabolism and catabolism), biosynthesis (nucleotide interconversion, amino acid interconversion, synthesis of amylopectin, fatty acid biosynthesis), and other essential pathways.
        3) Genes that encode proteins that are deemed likely to be ‘critical elements’ according to step 2, are cloned and expressed in bacterial systems as outlined above. The gene products (proteins) are then tested for activity and examined for inhibition by libraries of potential inhibitory compounds. This is performed in collaboration with pharmaceutical companies that maintain batteries of such compounds.
        4) In parallel with #3, the proteins are crystallized and subjected to X-Ray crystallography to identify their 3D structures.
        5) The 3D structures are used in ‘rational drug design’ to identify categories of potentially inhibitory compounds.
    
    


For example, we have identified the zinc transporter (see above) as a focus of these investigations. Cryptosporidium, like all living organisms, requires divalent cations like zinc. This compound must be actively imported into the cell. In the absence of zinc, or if the organism is unable to import it from its surroundings, Cryptosporidium will die. Thus, we have cloned and expressed the Cryptosporidium zinc transporter and will now search for compounds that inhibit its activity. In parallel, the structure of the transporter will be determined to further guide selection of potential inhibitory compounds.
Example 5
Development of DNA Vaccine
Plasmid DNA was also isolated using the EndoFree plasmid Giga Kit (Qiagen) and is used as an antigen to be injected in mice to test their protective role against Cryptosporidium. Animal models of Cryptosporidium infection have demonstrated the role of both cell and humoral immune responses (1, 2). Thus, mice with targeted disruptions of genes encoding immunological important molecules have established the role of these proteins in the immune response against Cryptosporidium (1-7). Other studies of the immune response against Cryptosporidium use neonatal mice and mice lacking functional T or B cell responses (1, 8, 9). These studies have demonstrated how an immune response is induced in mice that are not immuno-competent. Adult mice are resistant to the infection with Cryptosporidium but the injection of an antibody against Interferon Gamma (IFN γ) prior to infection render them susceptible to Cryptosporidium (10, 13). IFN γ plays an important role in the induction of an immune response against many microorganisms, and many studies have shown that it is one of the key components of the immune response against Cryptosporidium infections (11-17). Thus the temporary neutralization of IFN γ by the administration of the antibody in adult mice is an excellent tool to study and characterize the immune response in the context of an immunocompetent animal (c.f., 10).
The IFN knockdown mouse model follows a pattern of infection very similar to that seen in severe clinical infections (10). During the infection the animals lost weight and then regained it during recovery. This is also similar to what is observed in metabolically stressed children during and after diarrheal diseases. The predominant site of infection was the small intestine as it has been observed in animal and human severe infections. No parasites can be detected by day 30, thus the level of infection achieved is enough to generate an immune response, which also it is demonstrated by the detection of cellular responses against Cryptosporidium antigen extracts and proteins with lymphocytes obtained from the treated animals. Thus this model offers the possibility to study the potential role of Cryptosporidium as the animal can mount a parasite specific immune response (10).
We use this model (and alternative models such as a malnourished mouse model, unpublished) to study the potential role of Cryptosporidium recombinant proteins and plasmid DNA containing Cryptosporidium genes to induce an immune response. Our protocol to carry out this study is outlined in brief below. Subject mice are divided in groups: Group I mice are immunized with a pool of recombinant antigens (or plasmids in the case of DNA vaccine) in adjuvant (Freund's or other as appropriate); Group II (control animals) are immunized with Freund's adjuvant alone (or plasmid with no insert in the case of a DNA vaccine). The immunological responses in each group are followed and the response to challenge is measured. A sample immunization schedule is outlined below:

    
    
        1) Day 0: Immunization with a pool of recombinant antigens (2-3 μg each), DNA (20-100 μg) or controls. Immunization is performed intraperitoneally or intranasally.
        2) Day 14: Boost Immunization with the same antigens (protein, DNA, control). Boost is administered intranasally.
        3) Day 28: Anti-IFN-γ Ab inoculation, intraperitoneally, 2 h prior to Cryptosporidium infection.
        4) Day 28 (2 hr after inoculation of anti-IFN-γ Ab): Infection with Cryptosporidium. 

The immune response and protection to infection is subsequently monitored. A sample protocol is outlined below:

        5) Serum samples are collected at day 0, 7, 14 and 28 to monitor the immune response. Antibody levels against fusion proteins and Cryptosporidium extract are measured by immunoblotting and indirect immunofluorescence.
        6) Fecal IgA responses are measured before and after vaccination and infection.
        7) Fecal shedding of Cryptosporidium is monitored by fluorescent microscopy and real time PCR three times per week after infection
        8) Cytokine and lymphoproliferation responses are examined by isolation of mesenteric lymphocyte node and spleen cells to detect cytokine responses and lymphoproliferation against antigens (those that can be purified) and against a Cryptosporidium preparation.
    
    


These results indicate proteins which induce an antibody (mucosal or humoral) or cellular immune response. More important, these results indicate which immune response is protective and reduces infection (and shedding) by the exposed animal.
REFERENCES FOR EXAMPLE 5


    1. Aguilar-Be I, da Silva Zardo R, Paraguai de Souza E, Borja-Cabrera G P, Rosado-Vallado M, Mut-Martin M, Garcia-Miss Mdel R, Palatnik de Sousa C B, Dumonteil E. Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis. Infect Immun. 2005 February; 73(2):812-9.
    2. Aguiar J C, LaBaer J, Blair P L, Shamailova V Y, Koundinya M, Russell J A, Huang F, Mar W, Anthony R M, Witney A, Caruana S R, Brizuela L, Sacci J B Jr, Hoffman S L, Carucci D J. High-throughput generation of P. falciparum functional molecules by recombinational cloning. Genome Res. 2004 October; 14(10B):2076-82.
    3. Wu S Q, Wang M, Liu Q, Zhu Y J, Suo X, Jiang J S. Construction of DNA vaccines and their induced protective immunity against experimental Eimeria tenella infection. Parasitol Res. 2004 November; 94(5):332-6.
    4. Tborra S, Soto M, Carrion J, Alonso C, Requena J M. Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis. Vaccine. 2004 Sep. 28; 22(29-30):3865-76.
    5. Sagodira S, Iochmann S, Mevelec M N, Dimier-Poisson I, Bout D. Nasal immunization of mice with Cryptosporidium parvum DNA induces systemic and intestinal immune responses. Parasite Immunol. 1999 October; 21(10):507-16.
    6. Sagodira S, Buzoni-Gatel D, lochmann S, Naciri M, Bout D. Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA. Vaccine. 1999 May 14; 17(19):2346-55.
    7. Jenkins M, Kerr D, Fayer R, Wall R. Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen. Vaccine. 1995 December; 13(17):1658-64.
    8. Huygen K. Plasmid DNA vaccination. Microbes Infect. 2005 May; 7(5-6):932-8.
    9. Barouch D H, Letvin N L, Seder R A. The role of cytokine DNAs as vaccine adjuvants for optimizing cellular immune responses. Immunol Rev. 2004 December; 202:266-74.
    10. Sukumaran B, Madhubala R. Leishmaniasis: current status of vaccine development. Curr Mol. Med. 2004 September; 4(6):667-79.
    11. Barry M A, Howell D P, Andersson H A, Chen J L, Singh R A. Expression library immunization to discover and improve vaccine antigens. Immunol Rev. 2004 June; 199:68-83.
    12. Leifert J A, Rodriguez-Carreno M P, Rodriguez F, Whitton J L. Targeting plasmid-encoded proteins to the antigen presentation pathways. Immunol Rev. 2004 June; 199:40-53.
    13. Howarth M, Elliott T. The processing of antigens delivered as DNA vaccines. Immunol Rev. 2004 June; 199:27-39.
    14. Xu F, Ulmer J B. Attenuated salmonella and Shigella as carriers for DNA vaccines. J Drug Target. 2003; 11(8-10):481-8.
    15. Aguirre S A, Perryman L E, Davis W C, McGuire T C. IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of CD4+alpha beta+IFN−gamma+ and CD4+alpha beta+IL-4+lymphocytes in gut-associated lymphoid tissue during resolution of infection. J Immunol. 1998. 161: 1891-900.
    16. Lehmann J, Enssle K H, Lehmann I, Emmendorfer A, Lohmann-Matthes M L. The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice. J Interferon Cytokine Res. 2000. 20(1): 63-77.


Example 6
Detection and Diagnostics
Detection and diagnosis of C. hominis infection are very difficult and are typically based on microscopic examination (of water supplied or stool, etc.). A small number of previously characterized genes are now used for detection and diagnosis. These genes differ from their homologs in closely related parasites and therefore form the basis of a genetic signature. However, current protocols are limited in scope and could result in errors in identification. We have compared all the genes of C. hominis to many other related parasites, have identified important signature nucleotide sequences, which serve as the basis for the development of improved diagnostic tools. The signature nucleotide sequences are unique to C. hominis (see Table 9). Detection of the unique, signature sequences using nucleotide probes specific for them, provides a highly reliable method of detecting the presence of C. hominis in abiological sample, and thus for the diagnosis of a C. hominis infection.
As important as detection and diagnosis, it is important to quantify Cryptosporidium in an infected sample to assess the seriousness of the disease or contamination. Thus, Cryptosporidium are common in the environment and small quantities of the organism in water samples or in fecal samples are not uncommon. Using the signature nucleotide sequences described in Table 9, it is possible to both specifically identify and to accurately quantify levels of Cryptosporidium in essentially any biological or environmental sample.
An example of such use is described below.
Real Time PCR(RT-PCR) Analysis of Detection, Quantification and Diagnosis
The example below used TaqMan® assay system technology (Applied Biosystems, Foster City, Calif.), but any fluorescent real time PCR technology (of many that are commercially available) can be similarly applied (1, 2, 3). In brief, oligonucleotide primers and probes specific for sequences of Cryptosporidium small subunit ribosomal RNA gene and the pyruvate kinase genes were designed (4, 5) using Primer Express® version 2.0 software. These sequences were selected among the many available in Table 9 because of historical precedent. However, nearly all of the sequences described in Table 9 could be used by one trained in the art as specific targets in these analyses. For each target, two primers (forward and reverse) flanking one internal probe are synthesized. The primers are synthesized without modified bases or labels. The probes are synthesized with 5′ end linked FAM (6-carboxyfluoresceine) and 3′ end fluorescent TAMRA (6-carboxytetramethylrhodamine) dyes. These fluorescent dyes are commonly used for TaqMan® assay system technology, but many other labeling systems are equally applicable.
Primers and Probes for the Small Subunit rRNA Gene and the Pyruvate Kinase Gene.











Small Subunit rRNA





Name (forward): 18S#2-295F



Sequence:



CAGCTTTAGACGGTAGGGTATTGG
(SEQ ID NO: 4060)



 



Name (reverse): 18S#2-368R



Sequence:



TCTCCGGAATCGAACCCTAAT
(SEQ ID NO: 4061)



 



Name (probe): 18S#2-324T



Sequence:



CCCGTTACCCGTCATTGCCACG
(SEQ ID NO: 4062)



 



Pyruvate kinase










Name (forward): pyruvate kinase-1016F












Sequence:





GGCCAACAAGGGCAGAAA
(SEQ ID NO: 4063)



 










Name (reverse): pyruvate kinase-1091R












Sequence:





TCTCCAGATAGCATAACACAATCTGA
(SEQ ID NO: 4064)



 










Name (probe): pyruvate kinase-1040T












Sequence:





ATGTTGCAAACGCTGTTTTAGATG
(SEQ ID NO: 4065)






Synthesis of all primers and probes were performed at the VCU Nucleic Acids Research Facilities.

We tested the effectiveness of the primers above to detect parasite sequences in total RNA of the human cell line HCT-8 infected with Cryptosporidium. However, with insignificant modifications, this procedure is equally effective for detection and quantification of Cryptosporidium in other samples (e.g., water samples, fecal samples, sputum or bronchalveolar lavage, serum, etc.). In addition, this procedure is equally effective if the target is DNA instead of RNA. Thus, this example is provided as a general validation of this approach to Cryptosporidium detection and quantification.
Live (LV) and heat inactivated (HI) parasites were used to infect monolayers of cultured HCT-8 human cell line. RNA was isolated from these infected cell lines using standard procedures for purification of total RNA. Assays to quantify the Cryptosporidium RNA in these cells using the primers described above were performed in VCU's Nucleic Acids Research Facilities in an ABI Prism® 7900 Sequence Detection System (SDS) (Applied Biosystems, Foster City, Calif.) using the TaqMan® One Step PCR Master Mix Reagents Kit (P/N: 4309169). Again, any other real time PCR system would be equally applicable to this technological approach. All samples were processed in triplicate following standard procedures established in the Nucleic Acids Research Facilities.
The FIG. 2 shows typical amplification curve plots (A) generated by these samples and a standard curve plot of known amounts of Cryptosporidium rRNA, for measuring the absolute quantities of Cryptosporidium small sufigurebunit rRNA in the samples (B). The table below shows the numerical results of the parasite RNA quantification. These results showed that the Cryptosporidium RNA is present only in the samples in which infection was mediated with live parasites. As expected, heat inactivated parasites do not infect, and no parasite RNA is observed. Moreover, the results indicate that the amount of Cryptosporidium RNA present varies from approximately 5-30 picograms, permitting a direct calculation of the number of parasites present in the sample.
C. hominis is the primary agent of human cryptosporidiosis. C. parvum is a common cause of disease in animals, and an occasional problem in humans. It is important to differentiate between these two (and other) strains of Cryptosporidium. Our technology enables the ready discrimination of these isolates. Thus, we have demonstrated that we can use the sequences we have described in Table 9 for differentiating C. parvum from C. hominis. In brief, the same technology outlined above; e.g., RT PCR using primer and probe sets specific for C. hominis or C. parvum are used to amplify RNA (or DNA) purified from a sample (water, fecal material, other sample). In these amplifications, we can not only detect and discriminate different Cryptosporidium strains (e.g., C. hominis from C. parvum), but each can be concurrently quantified.
REFERENCES FOR EXAMPLE 6


    1. Wang, A. M., Doyle, M. V., and D. F. Mark. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA. 1989 December; 86(24): 9717-9721.
    2. Kawasaki, E. S., and A. M. Wang. (1989) Detection of gene expression. In: Erlich, H. A., ed., PCR Technology: Principles and Applications of DNA Amplification. Stockton Press, Inc., New York, N.Y., pp. 89-97.
    3. Dieter Klein (2002) Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine, 8(6):257-260.
    4. Xu P, Widmer G, Wang Y, Ozaki L S, Alves J M, Serrano M G, Puiu D, Manque P, Akiyoshi D, Mackey A J, Pearson W R, Dear P H, Bankier A T, Peterson D L, Abrahamsen M S, Kapur V, Tzipori S, Buck G A. (2004) The genome of Cryptosporidium hominis. Nature 431:1107-12.
    5. Abrahamsen M S, Templeton T J, Enomoto S, Abrahante J E, Zhu G, Lancto C A, Deng M, Liu C, Widmer G, Tzipori S, Buck G A, Xu P, Bankier A T, Dear P H, Konfortov B A, Spriggs B F, Iyer L, Anantharaman V, Aravind L, Kapur V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304(5669):441-5.


Example 7
Nucleotide Arrays for Detection of Cryptosporidium Genes
The array consists of  ̃4,000 70 base synthetic oligonucleotides bound to glass slides. Each of the  ̃4,000 C. hominis genes is represented by a single, specific oligonucleotide in the array. In order to assess presence or absence of these genes in a biological sample containing putative Cryptosporidium parasites, DNA is isolated by standard technology, labeled with fluorescent dyes, and hybridized to the array. A similar DNA sample derived from a known Cryptosporidium sample is labeled with an alternative fluorescent dye and used as a control. Both labeled DNAs are hybridized to the same oligonucleotide array, and the arrays are washed and scanned in a fluorescence scanner. The readout of the scanner provides an estimate of the amount of DNA for each gene that is present in the initial sample and a comparison between the two samples. Thus, Cryptosporidium genes that are present or absent are determined relative to the control.
The 70 base oligonucleotides in the array were designed by ArrayOligoSelectorTM version 3.8.2, (http://arrayoligosel.sourceforge.net/), which selects optimal sequences by:

    
    
        1. Examining every possible 70 mer sequence from every gene;
        2. Using BLASTN (against the whole genome) to check the uniqueness of each 70 mer.
        3. Uniqueness is scored as the theoretical binding energy of a candidate oligo to its most similar genome sequence. The binding energy is calculated using a nearest-neighbour model with the established thermodynamic parameters;
        4. Using the LZW compression algorithm to calculate the sequence complexity score in bytes between the oligo sequence and the its compressed version;
        5. Determining the self-annealing score, calculated as the alignment score of the optimum local alignment between the oligo sequence and its reverse compliment using the Smith-Waterman algorithm;
        6. Calculating the GC content of the oligo;
        7. Choosing, for each gene, the oligo that maximizes uniqueness and sequence complexity, minimizes self-annealing and has GC content closest to specified by the user; also tries to minimize distance to the 3′ end of the gene.
    
    


The approximately 4000 oligos and controls designed in this fashion were synthesized commercially and validated in the lab. In the FIG. 3, a hybridization of the array with DNA from C. hominis and C. parvum was performed to identify C. hominis genes that are present or absent in C. parvum. Therefore, DNA was purified from C. hominis and C. parvum, labeled with the fluorescent dyes Cy3 and Cy5, respectively, by indirectly incorporating amino-allyl (aa)-dUTP (Ambion) followed by coupling with fluorescent dyes. Briefly, four micrograms of genomic DNA from each of the two Cryptosporidium species was digested with restriction enzyme Hae III, translated with Klenow Exonuclease-free polymerase using random hexamers (pdN6 from Pharmacia) in the presence of aminoallyl-dUTP, and purified to remove the unincorporated nucleotides. The amino-allyl-dUTP labeled DNA samples were dried, dissolved in 0.1 M NaHCO3, pH 9.0, and fluorescently labeled by coupling of the amino allyl-dUTP to Cy3 or Cy5 dyes essentially as described by the manufacturer (Amersham Pharmacia Biotech). The labeled DNAs were hybridized to the oligonucleotide array, which contains approximately 4000 probes representing each of the known C. hominis genes. These results demonstrate that the gene complements of C. hominis and C. parvum show expected similarities, but permit identification of specific genetic differences.
While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein. 







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LENGTHY TABLES




The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08114976B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

## Claims

1. An isolated nucleic acid molecule encoding the polypeptide set forth as SEQ. ID. NO:8.

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule is set forth as SEQ. ID. NO. 7.