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WO1990002564A1 - Vaccin de diagnostic utilisant des proteines homologues a des proteines de thermochoc de trypanosoma cruzi - Google Patents

Vaccin de diagnostic utilisant des proteines homologues a des proteines de thermochoc de trypanosoma cruzi Download PDF

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Publication number
WO1990002564A1
WO1990002564A1 PCT/US1989/003955 US8903955W WO9002564A1 WO 1990002564 A1 WO1990002564 A1 WO 1990002564A1 US 8903955 W US8903955 W US 8903955W WO 9002564 A1 WO9002564 A1 WO 9002564A1
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Prior art keywords
protein
organism
derived
native protein
dna
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PCT/US1989/003955
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English (en)
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Elizabeth Dragon
Daryl Faulds
Stacey Sias
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Codon
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Publication of WO1990002564A1 publication Critical patent/WO1990002564A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to vaccines and diagnostics and more particularly to vaccines and diagnostics which employ proteins and/or fragments and/or derivatives thereof having homology to heat shock proteins of Trypanosoma cruzi.
  • Heat shock proteins sometimes referred to as stress proteins, have been found in a wide variety of cells, and have been generally described in an
  • Figure 1 provides the gene and derived, amino acid sequence for the Hsp70 antigen of T. cruzi.
  • Figure 2 provides an alignment of heat shock proteins from a variety of organisms: 1. M. hyopneumoniae, 2. Bacillus megaterium, 3. Escherichia coli, 4. T. cruzi, 5. T. cruzi, 6. Rat, 7. Xenopus laevis 8. human, 9. chicken, 10. Zea mays, 11. Serratia marcescens.
  • Figure 3 provides a restriction map of pMYCO16 containing the full length gene for the Hsp70 antigen of M. hyopneumoniae.
  • Figure 4 provides an intermediate plasmid for the expression of the Hsp70 antigen of M.
  • Figure 5 provides the gene and derived amino acid sequence for the Hsp70 antigen of M.
  • Figure 6 provides restriction map of pMYCO29 which is a low level expression plasmid containing the full length gene for the. Hsp70 antigen of M.
  • Figure 7 provides a restriction map of pMYCO31 which is a high level expression plasmid containing the full length gene for the Hsp70 antigen of M. hyopneumoniae.
  • Figure 8 provides a restriction map of pCAM101 containing the trpT176 gene.
  • Figure 9 provides a restriction map of pMYCO32 which is an expression plasmid containing the full length gene for the Hsp70 antigen of M.
  • Figure 10 provides a restriction map of pMGA4 which is an expression plasmid containing the full length gene for the Hsp70 antigen of M. gallisepticum.
  • Figure 11 provides the gene and derived amino acid sequence for the Hsp70 antigen of M.
  • Figure 12 provides a restriction map of pMGA10 which is an expression plasmid containing the full length gene for the Hsp70 antigen of M.
  • Vaccines are disclosed for the protection against organisms which comprise a physiologically acceptable carrier with a protein which is capable of eliciting an antibody which recognizes at least one epitope of a native protein present in the organism, the native protein having at least 50% homology with a heat shock protein of T. cruzi.
  • a protein capable of eliciting an antibody which recognizes at least one epitope of a native protein present in the organism the native protein having at least 50% homology with a T. cruzi heat shock protein.
  • processes for determining an organism in a host which comprise contacting a sample derived from a host containing an organism or suspected of containing an organism with an antibody or antibody fragment which recognizes at least one epitope of a native protein present in the
  • the native protein having at least 50% homology with a heat shock protein of T. cruzi; and determining protein present in the organism bound to the antibody.
  • the native protein referred to above may be derived from a species of Mycoplasma, Mycobacteria or Trypanosoma, provided that the native protein is not derived from Trypanosoma cruzi.
  • the native protein of Mycoplasma derivation is one selected from the group consisting of M. mycoides, M. bovis, M. bovigenitalium, M. bovoculi, M. bovirhinis, M. dispar, M. hyorhinis, M. hyosynoviae, M. hyopneumoniae, M. gallisepticum, M. pneumoniae, and M. synoviae, most preferably from M. hyopneumoniae and M. gallisepticum.
  • the native protein of Mycobacteria derivation is preferably one selected from the group consisting of M. bovis, M. leprae, and M. tuberculosis.
  • the recombinant sequence of nucleic acid encoding the heat shock proteins of M. hyopneumoniae and M. gallisepticum is also disclosed.
  • heat shock proteins and/or fragments and/or derivatives thereof may be employed in a vaccine to protect against an organism containing such heat shock protein.
  • RNA sequences encoding for a heat shock protein of an organism may be employed as a diagnostic for
  • a vaccine for protecting against an organism which includes a heat shock protein
  • the vaccine includes a protein capable of eliciting an antibody which recognizes at least one epitope of a heat shock protein of the organism which heat shock protein of the organism has at least 50% homology with a heat shock protein of Trypanosoma cruzi (T. cruzi).
  • a process for protecting against a disease caused by an organism which includes a heat shock protein by administering to a host at least one protein capable of eliciting an antibody which recognizes at least one epitope of a heat shock protein of the organism which heat shock protein of the organism has at least 50% homology with a heat shock protein of Trypanosoma cruzi (T.
  • an antigen or protein has at least 50% homology with a heat shock protein of T.
  • cruzi as used herein, means that on a position by position basis, at least 50% of the amino acids of the heat shock protein of T. cruzi are also present in the antigen or protein.
  • the heat shock protein or polypeptide of T. cruzi with which an antigen or protein is to have at least 50% homology is at least one of the T. cruzi heat shock proteins having a molecular weight of about 70 kD, or about 85 kD or about 65 kD, preferably the heat shock protein having a molecular weight of about 70 kD.
  • the T. cruzi heat shock protein having a molecular weight of about 70 kD may be prepared as described in Example 1.
  • T. cruzi heat shock protein having a molecular weight of about 85 kD is described by
  • the protein which is present in the organism and which is at least 50% homologous to a T. cruzi heat shock protein will sometimes be referred to herein as the "homologous protein” or the “homologous heat shock protein”.
  • the protein employed in formulating the vaccine for protection against an organism may be identical to a homologous protein present in the organism to be protected against, or may be a fragment or derivative of such homologous protein, provided that the protein which is used in the vaccine is capable of eliciting an antibody which recognizes at least one epitope of the homologous protein.
  • the protein employed in the vaccine may be only a portion of the homologous protein present in the organism or may have one or more amino acids which differ from the amino acids of the homologous protein in the organism or may be the homologous protein (or fragment or derivative thereof) fused to another protein.
  • protein which is capable of eliciting an antibody which recognizes at least one epitope of a native protein present in the organism, said native protein having at least 50% homology with a heat shock protein of T. cruzi (such protein present in the organism is what is sometimes referred to as the homologous protein) encompasses the homologous protein present in the organism or a fragment of such
  • homologous protein or a derivative of such homologous protein or a fusion product of such homologous protein (or fragment or derivative thereof) with another protein.
  • proteins included in the vaccine may include more or less amino acids or amino acids different from the amino acids of the homologous protein present in the organism.
  • the protein or proteins employed in the vaccine may be identified and produced by recombinant techniques. More particularly, the DNA (or RNA) encoding for a T. cruzi heat shock protein is
  • RNA DNA encoding for a T. cruzi heat shock protein
  • homologous DNA organism having the requisite homology is sometimes referred to herein as the "homologous DNA”.
  • the homologous DNA of the organism identified by such probe is employed to produce homologous protein of the organism by recombinant techniques.
  • the DNA encoding for the protein of Figure 1 may be suitably labeled, for example with 32 P, by procedures known in the art to thereby provide a probe for identifying DNA in the organism having at least 50% homology with the DNA sequence encoding for the protein of Figure 1.
  • Figure 2 presents an alignment of the amino acid sequences of Hsp70 proteins from a number of species.
  • the amino acids are depicted by their single letter abbreviations. Stretches of sequence identical in all examined species were identified (denoted by upper case text in the consensus sequence depicted below the individual sequences).
  • the DNA sequences which could encode these conserved sequences were determined.
  • the 17-mer nucleotide sequences having low coding degeneracy serve as universal oligonucleotide probes for Hsp70 genes.
  • the probing conditions selected are such that hybrids are identified in which there is at least 50% homology between the selected DNA probe which encodes for a T. cruzi heat shock protein and the DNA being probed for in the organism. Such probing is done at relatively low stringency. Low stringency is achieved by known methods such as reduced temperature and increased salt concentrations (e.g., hybridizing at 37oC and 5-6 X standard salt-citrate buffer or 5-6X standard salt-EDTA-Tris buffer).
  • the selected homologous DNA of the organism may be included in any of a wide variety of vectors or plasmids for producing a protein to be employed in formulating a vaccine against the organism.
  • vectors include chromosomal, nonchromosomal and
  • the appropriate DNA sequences may be inserted into the vector by a variety of procedures.
  • DNA sequences are inserted into an
  • promoter operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter as representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL promoter and other promoters.
  • promoters known to control expression of genes in prokaryotic and eukaryotic cells or their viruses.
  • the expression vector also contains a
  • the vector may also
  • the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as
  • dihydrofolate reductase or neomycin resistance for eukaryotic cell culture or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequences as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate hosts there may be
  • bacterial cells such as E. coli.
  • Salmonella typhimurium fungal cells, such as yeast; animal cells such as CHO or Bowes melanoma; plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the expression vehicle including the appropriate DNA sequences for the protein to be described
  • the t-RNA inserted at the selected site may include a DNA or gene sequence which is not part of the gene coding for the protein.
  • the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in expression or improves purification or permits increases in the immunonogenicity.
  • renaturation of proteins can be found in the scientific literature and include; 1) denaturation (unfolding) of improperly folded proteins using agents such as alkali, chaotropic agent, organic solvents, and ionic
  • the vaccine which includes a protein of the type hereinabove described may be employed in a vaccine for protecting against diseases caused by a wide variety of organisms.
  • Table 1 provides representative examples of such organisms.
  • species of Trypanosoma, Mycoplasma and Mycobacteria are described herein. Heat shock proteins for Mycobacteria are known. Young et al., P.N.A.S. (USA), 85:4267-4270 (1988).
  • a host may be protected against a disease caused by a certain organism by incorporating into the vaccine a protein which is capable of eliciting
  • the protein which is capable of eliciting such antibodies may correspond to the homologous protein of the organism or may be a fragment or derivative thereof.
  • the active protein may correspond to the homologous protein of the organism or may be a fragment or derivative thereof.
  • the protein which is included in the vaccine would be one or more heat shock proteins of T. cruzi or a fragment or derivative thereof capable of eliciting antibodies which recognize an epitope of T. cruzi heat shock protein.
  • the host which is protected is dependent upon the organism against which protection is sought. In general, the host is an animal (either a human or nonhuman animal) which is subject to a disease caused by the organism. Thus, for example if the organism against which protection is sought is one which is known to cause disease in man, then the vaccine
  • the vaccine including the active protein or proteins would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to cause a disease in a nonhuman animal, then the vaccine including the active protein would be administered to a human host. If the organism is known to
  • the active protein is employed in the vaccine in an amount effective to provide protection against the disease caused by the organism against which protection is sought.
  • each dose of the vaccine contains at least 5 micrograms and preferably at least 100 micrograms of the active protein. In most cases, the vaccine does not include the active protein in an amount greater than 20 milligrams.
  • protection or “protecting” when used with respect to a vaccine means that the vaccine prevents the disease or reduces the severity of
  • the active protein is employed in conjunction with a physiologically acceptable vehicle to provide protection against the organism.
  • a physiologically acceptable vehicle for vaccines in carriers, there may be mentioned: mineral oil, alum, synthetic polymers, etc.
  • Vehicles for vaccines are well known in the art and the selection of a suitable vehicle is deemed to be within the scope of those skilled in the art from the teachings herein. The selection of a suitable vehicle is also dependent upon the manner in which the vaccine is to be administered.
  • the vaccine may be in the form of an injectable dose and may be administered
  • intra-muscularly, intravenously, or by sub-cutaneous administration It is also possible to administer the vaccine orally by mixing the active components with feed or water; providing a tablet form, etc.
  • a vaccine may also be formulated by use of an antibody elicited in response to a homologous protein of the organism.
  • the protein and/or antibody used in the vaccine is essentially free of the organism; i.e., cellular matter.
  • a diagnostic kit and/or assay for determining an organism which employs in the assay and/or kit an antigen which is recognized by an antibody elicited by a protein of the organism which has at least 50% homology with a T. cruzi heat shock protein, as hereinabove described, i.e., a
  • the antigen employed as a diagnostic may be obtained or produced as hereinabove described with reference to the active protein included in the
  • a diagnostic assay and/or reagent for determining an organism which includes and/or employs an antibody (or fragment thereof) which recognizes an antigen of the organism to be determined, which antigen of the organism has at least 50% homology with a heat shock protein of T.
  • the antibody employed in the assay and/or assay kit may be either a polyclonal or monoclonal antibody elicited in response to a homologous protein.
  • the antibody employed in the diagnostic assay and/or kit is elicited in response to a protein and/or fragment and/or derivative thereof having at least 50% homology with a heat shock protein of T.
  • a diagnostic kit and/or assay for determining an organism which includes a homologous protein may be formulated to determine such organism by a variety of procedure.
  • the organism may be determined by a so-called sandwich assay kit or assay for
  • determining the organism by determining in a sample (derived from a host containing or suspected of containing the organism) antibody elicited in response to a homologous protein of the organism.
  • antigen of the type hereinabove described is contacted with the sample under conditions at which any of such antibody present in the sample is immunobound to the antigen, which antigen is
  • Antibody bound to such antigen may then be determined by use of an appropriate tracer comprised of a ligand bound or recognized by such antibody labeled with a detectable marker or label .
  • the ligand of the tracer may be, for example, an antibody bound by or recognized by the bound antibody.
  • the marker may be any one of a wide variety of labels (for example a radioactive label, an enzyme label, a chromogen label, etc.).
  • ELISA sandwich assay format in which a plastic microtiter plate is coated with an antigen of the type described (one which is recognized by antibody elicited in response to homologous protein of the organism) and sample derived from a host suspected of containing the organism is incubated with the coated antigen. After appropriate washing, labeled
  • immunoglobulin antiglobulin to the host species which is suspected of containing the organism
  • detectable enzyme for example
  • horseradish peroxidase or alkaline phosphatase is incubated with the antibody bound by the coated
  • an agglutination assay may be employed in which case particles, such as polystyrene beads, coated with the appropriate antigen is mixed with appropriate sample, and presence of antibody is detected by agglutination.
  • an antibody of the type hereinabove described may be employed to directly determine a homologous heat shock antigen or protein of the organism to be
  • a sample derived from a hostcontaining or suspected of containing the organism
  • a sandwich assay by contacting the sample with an antibody (or fragment thereof) which recognizes the homologous heat shock antigen of the organism, which antibody is preferably supported on a solid support.
  • an antibody or fragment thereof
  • Such contacting is effected under conditions which will immunobind the homologous heat shock antigen (if present) to the antibody.
  • bound antigen may be determined by use of a tracer comprised of a ligand (which is bound by or recognizes the homologous antigen) labeled with a detectable marker or label.
  • the tracer may be
  • the antibodies capable of recognizing a homologous protein of the organism may be a monoclonal and/or polyclonal antibody.
  • the package may include other materials useful in the assay, for example, tracer, buffers, standards, etc., in appropriate reagent containers.
  • an assay and/or reagent kit for determining the presence of an organism which includes or employs a DNA probe which encodes for a protein of the organism having at least 50% homology with a heat shock protein of T. cruzi as hereinabove described.
  • the DNA probe which is used may be all or a portion of the DNA which encodes for a homologous protein. If a portion of the DNA which encodes for a homologous protein is employed, such DNA portion should include a portion of the DNA which encodes for a variable region of the homologous protein.
  • the DNA probe is employed under conditions whereby hybridization is accomplished over at least a portion of the DNA which encodes for a variable region (preferably a hypervariable region) of the homologous protein.
  • the hydridization may be performed with a suitably labeled form of the DNA (for example 32 P, although other detectable labels, including non- radioactive labels may be used) in a procedure
  • Log phase cells were harvested by centrifugation and washed twice with cold PSG (20 mM sodium phosphate, pH 7.4, 0.9% NaCl, 1.0% glucose).
  • Parasites were harvested from culture by centrifugation and washed several times with PSG (20 mM sodium phosphate, pH 7.4, 0.9% NaCl, 1.0% glucose). Epimastigotes were resuspended at a concentration of 10 9 /ml in PEG/EGTA buffer (20 mM Tris-HCl, pH 7.6, 25 mM EGTA, 50 mM MgCl, 25mM CaCl, 1.0% Triton-X100, and 4mM dithiothreitol), plus 250 u/ml of RNAS in (Promega Biotec, Madison, WI), incubated on ice for 20 min., centrifuged at 8000 x g for 15 minutes at 4oC.
  • the supernatant containing the RNA was phenol extracted 3 times, then extracted once with chloroformisoamyl alcohol (24:1) and ethanol precipitated.
  • the pellet (nuclei and kinetoplasts) was resuspended at a
  • RNAse digestion the sample was extracted once with phenol, once with chloroformisoamyl alcohol, and then precipated with ethanol. The size of the DNA was determined to be greater than 20 kilobase pairs (kb) on agarose gels. Trypomastigote DNA and RNA was prepared in an identical manner except that the parasites were resuspended at a concentration of 5 x 10 9 /ml.
  • Oligo(dT)-cellulose chromatography (Aviv and Leder, J. Immunol., 127:855-859, 1972). Total RNA was loaded onto an oligo (dT)-cellulose column (Type 3,
  • Tris-HCl pH 7.5, 1 mM EDTA, 0.2% SDS, 400 mM LiCl.
  • RNA was eluted from the column at 40°C with 10 mM
  • Tris-HCl pH 7.5, 1 mM EDTA, 0.2% SDS.
  • T. cruzi RNAs were purified from total T. cruzi RNA using the technique of
  • the filter was washed 10 times with 1XSSC, 0.1% SDS at 60oC, 3 times with 0.002 M EDTA at 60oC, and once with water at room temperature.
  • RNA specifically hybridized mRNA is eluted from the filter by boiling the filter in a small volume of water for two minutes, quick-freezing the solution, then ethanol precipitating the RNA.
  • the purified RNA is resuspended in water, then translated in an in vitro translation system (such as rabbit reticulocyte).
  • a 1:10 to 1:50 dilution of individual serum was prepared using the 10 mM Tris-HCl, pH7.5, 1% Nonidet P-40 (NP 40), 1 mM N-alpha-p-tosyl-L-Lysine
  • Inactivator Units KIU/ml aprotinin.
  • the diluted serum was mixed with an equal volume of cell-free translation reaction mixture, and incubated overnight at 4°C.
  • 10 ⁇ l of 10% protein-A-Sepharose (Pharmacia, Piscataway, NJ) was added and gently mixed for 1 hour at 4oC.
  • the immune complexes were washed and analyzed on SDS-polyacrylamide gels as described in Dragon et al., Mol. and Biochem., Parasitology, 16:213-229, 1985.
  • cDNA was synthesized by methods known to those of ordinary skill in the art. Briefly, 2 ⁇ g of epimastigote or trypomastigote A+ mRNA was transcribed by the action of AMV reverse transcriptase as described by Ullrich et al., Science, 196:1313-1319, (1977) and Gubler, Gene, 25:263-269, (1983). Transcription was initiated at the 3' polyadenylated end of the mRNA using oligo(dt) as a primer. The second strand was copied using DNA polymerase I and RNAse H
  • RNA-DNA duplex was extracted once with phenol chloroform and then precipitated with ethanol. The pelleted material was then resuspended in 100 microliter reaction mixture containing the
  • RNAase H 100 units/ml Pharmacia Molecular Biology Division, Piscataway, NJ
  • DNA Polymerase I --Klenow fragment 50 units/ml Boehringer
  • the sample was then re-extracted with phenol: chloroform and ethanol precipitated.
  • a pUC18 vector was used for the cloning step.
  • the vector had been digested with Sacl and Smal. Smal provided the blunt end site necessary for ligation of the 3' end of the cDNA.
  • the ligation reaction was performed using 40 ng of vector DNA and 50 ng of cDNA. Ligation was done overnight at 12°C in a ligase buffer of 50 mM Tris-HCl (pH 7.8), 10 mM MgC12, 20 mM dithiothreitol, 1.0 mM rATP using one unit of T4 DNA ligase.
  • the recombinant DNA molecules were then introduced into E. coli K-12 strain JM83 by
  • the transformed bacteria were spread on agar plates containing the antibiotic ampicillin at a concentration of 50 micrograms/ml. Since the plasmid pUC18 contains the ampicillin resistance gene, only those bacteria which acquired a recombinant plasmid survived. These bacteria each grew and divided to form a bacterial colony. Each cell in the colony is a descendant of the original parental cell and contains the same recombinant plasmid. Using hybridization - selection/translation and immunoprecipitation
  • the cDNA clone was used as a probe to screen the T. cruzi Sau3a partial genomic library as described by Maniatis et al.
  • a lambda phage designated FG21 was identified which contained multiple copies of the 70 kD gene.
  • a 2.4 kb Smal fragment was sub-cloned into pUC9 from FG 21. This subclone called pEG22 contained one full length copy of the 70 kD gene.
  • the DNA sequence of PEG22 was determined.
  • FG21 was sequenced and used to construct an expression plasmid to allow production of the 70 kd antigen in E. coli.
  • the transformed E. coli are grown in liquid culture containing 50 micrograms per ml of ampicillin to enhance plasmid ability. Cultures are harvested at an OD of 2.0 measured at 550 nm. The cells are then pelleted and washed and lysed by freeze/thaw and sonication. A detergent extraction solubilizes most of the remaining polypeptides. The 70 kd expressed product, however, remains insoluble and is harvested by centrifugation. This insoluble "cement” is denatured in urea and subsequently diluted at a high pH and the pH is then adjusted back to neutral. During the renaturation process the antigen refolds and achieves that immunologically active conformation. The details of this procedure used are identical to those used to restore enzyme activity to recombinant chymosin as described by McCaman et al., J. Biotech., 12:117-191, (1985).
  • the resulting DNA solution was extracted with cesium chloride saturated isopropanol, to remove ethidium bromide, and
  • Plasmid pEG22, described in Example 1 is purified from E. coli by methods in the art, and labeled with 32 p by nick translation using DNA polymerase I.
  • pEG22 is used as a probe as follows:
  • Mycoplasma genomic DNA was digested with EcoRI under the following conditions at 37oC for 2 hours.
  • endonuclease fragment of approximately 6 kB in length and thus include the antigen's gene.
  • fraction 4 was shown to contain the DNA fragment of interest.
  • Plasmid DNA preparations which hybridize to the DNA probe are subjected to EcoRI digest analysis to show that each plasmid contains the same size insert fragment, and most likely the same gene.
  • a plasmid is selected for DNA sequence analysis which shows greater than 50% identity to pEG22.
  • Mycoplasma hyopneumoniae P-57223 was done using 200 units of EcoRI in a total volume of 1 ml and 250 ⁇ l aliquots were removed at 6 min, 25 min, 42 min and 63 min.
  • the four preparative samples of partially digested Mycoplasma DNA were then combined (200 ⁇ g) and loaded onto an exponential sucrose gradient.
  • the gradient was centrifuged in a Sorvall AH627 rotor at 26 k rpm for 21 hrs at 15oC.
  • the gradient was then slowly fractioned from the bottom by collecting 15 drop fractions (90 fractions total). 20 ⁇ l of each fraction was then run on a 1% agarose gel as described above. Fractions containing DNA fragments smaller than 18 kbp and larger than 15 kbp were pooled (fractions 32-40) and dialyzed against TE (10 mM Tris.HCl pH 7.5, 1 mM EDTA pH 8.0) to remove the sucrose. The DNA (3.5ml) was then precipitated with ethanol and resuspended to about 15 ⁇ l (1 mg/ml) under vacuum and stored at -20oC.
  • EcoRI Arms of bacteriophage lambda-Dash were obtained from Vector Cloning Systems (StrataGene) and were ligated at a concentration of 200 ⁇ g/ml to
  • Mycoplasma target DNA at a concentration of 25 ⁇ g/ml in a total volume of 10 ⁇ l using T4 ligase (Boehringer GmbH) at a concentration of 100 units/ml.
  • the ligation reaction was incubated at room temperature for 2 hours. 4 ⁇ l of the ligation was then packaged into lambda particles using the in vitro packaging kit Gigapack (StrataGene). The phage was then titered on E. coli strain P2392 (StrataGene) and found to be 7.75 x 10 5 pfu/ml (3.1 x 10 5 pfu/ug of lambda-Dash).
  • the library is screened using the plasmid previously obtained which shows greater than 50% homology to pEG22, by the previously 'described
  • DNA from positive recombinants is prepared, digested with EcoRI, analyzed by gel
  • Plasmid pWHA148 is prepared by inserting a synthetic oligonucleotide into the Hind III site of pUC18. The amino terminal coding sequence of the
  • X-complementing peptide of B-galactosidase is shown in Figure 4, and contains 8 additional restriction sites over the parent pUC18.
  • the oligonucleotide insert into pUC18 is shown in Figure 4 between the Sphl and Hind III sites.
  • N-terminal portion of pEG22 is used by Southern analysis to hybridize to the 0.6kb
  • AccI-AsuII restriction fragment of pMYCOl ⁇ DNA sequence analysis of the 0.6 kb fragment identifies that start codon of the homologous gene.
  • restriction fragment extends clockwise within the 0.4 kb AsuII - Clal, 1.2 kb Clal - Clal, and 1.4 kb Clal- Hindlll fragments, and ends short of the Hindlll site.
  • DNA sequence analysis shows that pMYC016 contains a 74.5 kD protein homologous to the 70 kD T. cruzi heat shock antigen.
  • Plasmid pMYC016 DNA ( Figure 3) was digested with Accl, treated with Mung Bean nuclease to remove the single stranded Accl tails, re-ligated to delete the 1.9 kb Accl fragment in front of the 74.5 kD antigen gene and transformed into E. coli strain JM83.
  • One transformant was named pMYCO29; its DNA was
  • pMYCO29 was subjected to DNA sequence analysis which showed that a spontaneous deletion had occured at the ligation juncture, where two bases were deleted and the Pstl site was retained, as shown below (only a portion of the 5' to 3' strands are represented).
  • Plasmid pMYCO29 is a low level expression plasmid.
  • pMYC029 DNA was digested with Pstl and EcoRI, the Pstl - EccRI fragment containing the entire 74.5 kD coding sequence was purified, ligated to the Pstl and EcoRI digested vector pUC9, and transformed into E. coli strain JM83.
  • One transformant was named pMYC031
  • TGA codons encode the amino acid tryptophan in mycoplasma but normally terminate peptide chain elongation in E. coli and that the trpT176 gene, a mutant tryptophan t-RNA which
  • Plasmid pCAM101 was purchased from James Curran (University of Colorado) as a convenient source of the trpT176 gene and is shown in Figure 8.
  • DNA from pCAM101 was digested with EcoRI, the
  • a W3110 (pMYCO32) transformant was selected, grown in L-broth, lysated as previously described, and a portion subjected to polyacrylamide gel
  • a W3110 (pMYCO32) transformant from Example 2 was selected, grown in M-9 minimal medium in a 14 liter Chemap fermenter to a cell density of 110 O.D. 600, and 120 g (wet weight) of cells were harvested from 500 ml by centrifugation.
  • a suspension was prepared consisting of 2.3 g of cells per 10 ml of PBS containing 12 mM EDTA, 0.5 mg/ml lysozyme. The suspension was incubated at 25 oC for 15 minutes.
  • Example 3 The 70 kD Hsp Analog from Mycoplasma Gallisepticum.
  • the DNA-containing aqueous phase was dialyzed against 4 liters of 10 mM Tris-HCl, 5 mM EDTA twice, and 10 mM Tris-HCl, 1 mM EDTA once. From each strain, 60 ⁇ g of DNA was recovered, an amount sufficient for restriction analyses. Southern blot analyses, and library
  • oligonucleotide probes were selected for use as oligonucleotide probes. These were synthesized as follows: COD1159 Ile-Ile-Asn-Glu-Pro-Thr
  • Plasmid DNA from pMYC087, containing the gene for M. hyopneumoniae was labeled using the Boeringer Mannheim nonradioactive Southern hybridization kit (Genius kit) and used to probe a Southern transfer of EcoRI and Hindlll
  • the probe detected bands of the expected size in the M.
  • gallisepticum genomic DNA 1 ⁇ g from both the R strain and the F-K8 I 0 strain, was digested to completion with the bacterial restriction endonuclease Hindlll and separated on 3.25% polyacrylamide gels. DNA from four gel slices containing restriction digest fragments between 2 and 5 kb was electroeluted. An aliquot of DNA electroeluted from each of the four gel slices was subjected to agarose gel electrophoresis, transfered to a nitrocellulose membrane by Southern transfer and probed with 32 P-labeled COD1159 to identify the fraction which contains the 3.3kb hybridizing Hindlll band. In this way, a positive DNA fraction was identified. This positive DNA fraction was then ligated into Hind III digested pUC9 and transformed into E. coli DH5a.
  • Plasmid pMGA4 contains a positive R-strain insert and has been deposited with the American Type Culture Collection on ____________________ 1989 with the
  • FIG. 10 The sequence of of the M. gallispeticum Hsp70 DNA and the derived amino acid sequence is provided in Figure 11.
  • DNA from pCAM101 was digested with EcoRI, a 0.3 kb EcoRI fragment including trpT176 was purified, ligated to EcoRI digested pUC9, transformed into E.
  • Plasmid pMGA4 DNA was digested with Hindlll and Bglll, ligated to Hindlll and BamHI digested pWHA160, digested with BamHI and Bglll, and transformed into E. coli strain DH5a.
  • One transformant was named pMGA10.
  • the MGA10 transformant was grown in L-broth at 37oC, and the cells harvested by centrifugation and frozen. The cell pellet from 4 ml of culture was resuspended in 100 ⁇ l of a solution consisting of 0.5 mg/ml hen egg-white lysozyme dissolved in 25 mM Tris pH 8.0 10 mM EDTA; and incubated at 25°C for 10 minutes.
  • the purified M. gallisepticum protein is concentrated by lyophilization and resuspended to a final concentration of 0.5-2.0 mg/ml in 0.1% SDS.
  • the immunizing antigen is formulated in one volume of protein concentrate to three volumes of oil carrier consisting of 5% Arlacel, 94% Drakeol 6-VR and 1% Tween 80.
  • the dose of the antigen employed is 100 ⁇ g/dose.
  • Chicken receive the formulated vaccine by subcutaneous injection. A booster vaccination by the same route is done two weeks later.

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Abstract

Cette invention concerne des vaccins et des diagnostics, et plus particulièrement des vaccins et des diagnostics qui utilisent des protéines et/ou des fragments et/ou des dérivés de celles-ci et présentant une homologie à des protéines de thermochoc de Trypanosoma cruzi
PCT/US1989/003955 1988-09-12 1989-09-12 Vaccin de diagnostic utilisant des proteines homologues a des proteines de thermochoc de trypanosoma cruzi WO1990002564A1 (fr)

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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5348945A (en) * 1990-04-06 1994-09-20 Wake Forest University Method of treatment with hsp70
US5750119A (en) * 1994-01-13 1998-05-12 Mount Sinai School Of Medicine Of The City University Of New York Immunotherapeutic stress protein-peptide complexes against cancer
US5756662A (en) * 1995-03-14 1998-05-26 Corixa Corporation Compounds and methods for the detection of T. cruzi infection
US5830464A (en) * 1997-02-07 1998-11-03 Fordham University Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy
US5837251A (en) * 1995-09-13 1998-11-17 Fordham University Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment and prevention of neoplastic diseases
US5871742A (en) * 1993-03-31 1999-02-16 Nippon Zeon Co., Ltd Recombinant Avipox virus encoding polypeptide of mycoplasma gallisepticum, and utilized a live vaccine
US5916572A (en) * 1995-11-14 1999-06-29 Corixa Corporation Compounds and methods for the detection and prevention of T. cruzi infection
US5919620A (en) * 1995-06-07 1999-07-06 Biochem Vaccines Inc. Heat shock protein HSP72 of Streptococcus pneumoniae
US5935576A (en) * 1995-09-13 1999-08-10 Fordham University Compositions and methods for the treatment and prevention of neoplastic diseases using heat shock proteins complexed with exogenous antigens
US5948646A (en) * 1997-12-11 1999-09-07 Fordham University Methods for preparation of vaccines against cancer comprising heat shock protein-peptide complexes
US5961979A (en) * 1994-03-16 1999-10-05 Mount Sinai School Of Medicine Of The City University Of New York Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens
US5985270A (en) * 1995-09-13 1999-11-16 Fordham University Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes
US5997873A (en) * 1994-01-13 1999-12-07 Mount Sinai School Of Medicine Of The City University Of New York Method of preparation of heat shock protein 70-peptide complexes
US6001361A (en) * 1996-08-29 1999-12-14 Genesis Research & Development Corporation Limited Mycobacterium vaccae antigens
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
US6017540A (en) * 1997-02-07 2000-01-25 Fordham University Prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with heat shock/stress protein-peptide complexes
US6054135A (en) * 1996-11-14 2000-04-25 Corixa Compounds and methods for the detection and prevention of T. cruzi infection
WO2001013944A2 (fr) * 1999-08-19 2001-03-01 Immunobiology Limited Vaccins contre des agents infectieux
US6228372B1 (en) 1997-04-15 2001-05-08 Corixa Corporation Compounds and methods for the detection and prevention of T. cruzi infection
WO2001097827A1 (fr) * 2000-06-16 2001-12-27 Argonex Pharmaceuticals Peptides mhc surexprimes sur des cellules cancereuses de la prostate et methodes d'utilisation
US6419933B1 (en) 1995-11-14 2002-07-16 Corixa Corporation Compounds and methods for the detection and prevention of T.cruzi infection
US6716591B1 (en) * 1993-07-30 2004-04-06 Yale University B. burgdorferi polypeptides
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US6855802B1 (en) * 1998-04-22 2005-02-15 Institut Gustave Roussy Mutated peptide compounds, derived from hsp70, useful in cancer immunotherapy
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
WO2009086398A2 (fr) * 2007-12-27 2009-07-09 Abbott Laboratories Anticorps anti t. cruzi et procédés d'utilisation
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
US8475785B2 (en) 2008-03-03 2013-07-02 The University Of Miami Allogeneic cancer cell-based immunotherapy
US8685384B2 (en) 1998-02-20 2014-04-01 University Of Miami Recombinant cancer cell secreting modified heat shock protein-antigenic peptide complex
US8968720B2 (en) 2008-03-20 2015-03-03 University Of Miami Heat shock protein GP96 vaccination and methods of using same
US10046047B2 (en) 2015-02-06 2018-08-14 Heat Biologics, Inc. Vector co-expressing vaccine and costimulatory molecules
CN113215170A (zh) * 2021-05-30 2021-08-06 吉林大学 一种sHSPs重组侵入型植物乳杆菌活载体DNA疫苗
US11548930B2 (en) 2017-04-04 2023-01-10 Heat Biologics, Inc. Intratumoral vaccination
US11666649B2 (en) 2016-10-11 2023-06-06 University Of Miami Vectors and vaccine cells for immunity against Zika virus

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US5348945A (en) * 1990-04-06 1994-09-20 Wake Forest University Method of treatment with hsp70
US5871742A (en) * 1993-03-31 1999-02-16 Nippon Zeon Co., Ltd Recombinant Avipox virus encoding polypeptide of mycoplasma gallisepticum, and utilized a live vaccine
US6716591B1 (en) * 1993-07-30 2004-04-06 Yale University B. burgdorferi polypeptides
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US6468540B1 (en) 1994-01-13 2002-10-22 Mount Sinai School Of Medicine Of New York University Immunotherapeutic stress protein-peptide complexes against cancer
US6610659B1 (en) 1994-01-13 2003-08-26 Mount Sinai School Of Medicine Of New York University Use of heat shock protein 70 preparations in vaccination against cancer and infectious disease
US5750119A (en) * 1994-01-13 1998-05-12 Mount Sinai School Of Medicine Of The City University Of New York Immunotherapeutic stress protein-peptide complexes against cancer
US6017544A (en) * 1994-01-13 2000-01-25 Mount Sinai School Of Medicine Of The University Of New York Composition comprising immunogenic stress protein-peptide complexes against cancer and a cytokine
US5997873A (en) * 1994-01-13 1999-12-07 Mount Sinai School Of Medicine Of The City University Of New York Method of preparation of heat shock protein 70-peptide complexes
US6455503B1 (en) 1994-03-16 2002-09-24 Mount Sinai School Of Medicine Of New York University Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens
US6048530A (en) * 1994-03-16 2000-04-11 Mount Sinai School Of Medicine Of New York University Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens
US5961979A (en) * 1994-03-16 1999-10-05 Mount Sinai School Of Medicine Of The City University Of New York Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens
US5942403A (en) * 1995-03-14 1999-08-24 Corixa Corporation Compounds and methods for the detection of t. cruzi infection
US5756662A (en) * 1995-03-14 1998-05-26 Corixa Corporation Compounds and methods for the detection of T. cruzi infection
US5919620A (en) * 1995-06-07 1999-07-06 Biochem Vaccines Inc. Heat shock protein HSP72 of Streptococcus pneumoniae
US7601359B1 (en) 1995-09-13 2009-10-13 Fordham University Compositions and methods for the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases with heat shock/stress proteins
US6187312B1 (en) 1995-09-13 2001-02-13 Fordham University Compositions and methods using complexes of heat shock protein 90 and antigenic molecules for the treatment and prevention of infectious diseases
US5935576A (en) * 1995-09-13 1999-08-10 Fordham University Compositions and methods for the treatment and prevention of neoplastic diseases using heat shock proteins complexed with exogenous antigens
US6410028B1 (en) 1995-09-13 2002-06-25 Fordham University Therapeutic and prophylactic methods using heat shock proteins
US6136315A (en) * 1995-09-13 2000-10-24 Fordham University Compositions and methods using complexes of heat shock protein 70 and antigenic molecules for the treatment and prevention of neoplastic diseases
US6139841A (en) * 1995-09-13 2000-10-31 Fordham University Compositions and methods using complexes of heat shock protein 70 and antigenic molecules for the treatment and prevention of infectious diseases
US6143299A (en) * 1995-09-13 2000-11-07 Fordham University Compositions and methods using complexes of heat shock protein gp96 and antigenic molecules for the treatment and prevention of infectious diseases
US6156302A (en) * 1995-09-13 2000-12-05 Fordham University Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes
US6162436A (en) * 1995-09-13 2000-12-19 Fordham University Compositions and methods using complexes of heat shock protein 90 and antigenic molecules for the treatment and prevention of neoplastic diseases
US6030618A (en) * 1995-09-13 2000-02-29 Fordham University Therapeutic and prophylactic methods using heat shock proteins
US5985270A (en) * 1995-09-13 1999-11-16 Fordham University Adoptive immunotherapy using macrophages sensitized with heat shock protein-epitope complexes
US6447781B1 (en) 1995-09-13 2002-09-10 Fordham University Therapeutic and prophylactic methods using heat shock proteins
US5837251A (en) * 1995-09-13 1998-11-17 Fordham University Compositions and methods using complexes of heat shock proteins and antigenic molecules for the treatment and prevention of neoplastic diseases
US6461615B1 (en) 1995-09-13 2002-10-08 Fordham University Therapeutic and prophylactic methods using heat shock proteins
US5916572A (en) * 1995-11-14 1999-06-29 Corixa Corporation Compounds and methods for the detection and prevention of T. cruzi infection
US6419933B1 (en) 1995-11-14 2002-07-16 Corixa Corporation Compounds and methods for the detection and prevention of T.cruzi infection
US6001361A (en) * 1996-08-29 1999-12-14 Genesis Research & Development Corporation Limited Mycobacterium vaccae antigens
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
US6054135A (en) * 1996-11-14 2000-04-25 Corixa Compounds and methods for the detection and prevention of T. cruzi infection
US6399069B1 (en) 1997-02-07 2002-06-04 Fordham University Prevention of infectious diseases with hsp70-peptide complexes
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US6387374B1 (en) 1997-02-07 2002-05-14 Fordham University Treatment of primary and metastatic neoplastic diseases with hsp90-peptide complexes
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US5830464A (en) * 1997-02-07 1998-11-03 Fordham University Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy
US6322790B1 (en) 1997-02-07 2001-11-27 Fordham University Compositions and methods for eliciting an immune response using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy
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US6436404B1 (en) 1997-02-07 2002-08-20 Fordham University Prevention of primary and metastatic neoplastic diseases with GP96-peptide complexes
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US6410027B1 (en) 1997-12-11 2002-06-25 Fordham University Methods for preparation of vaccines against cancer
US5948646A (en) * 1997-12-11 1999-09-07 Fordham University Methods for preparation of vaccines against cancer comprising heat shock protein-peptide complexes
US6406700B1 (en) 1997-12-11 2002-06-18 Fordham University Methods for preparation of vaccines against cancer
US8685384B2 (en) 1998-02-20 2014-04-01 University Of Miami Recombinant cancer cell secreting modified heat shock protein-antigenic peptide complex
US6855802B1 (en) * 1998-04-22 2005-02-15 Institut Gustave Roussy Mutated peptide compounds, derived from hsp70, useful in cancer immunotherapy
WO2001013944A2 (fr) * 1999-08-19 2001-03-01 Immunobiology Limited Vaccins contre des agents infectieux
WO2001013944A3 (fr) * 1999-08-19 2001-09-20 Immunobiology Ltd Vaccins contre des agents infectieux
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
WO2001097827A1 (fr) * 2000-06-16 2001-12-27 Argonex Pharmaceuticals Peptides mhc surexprimes sur des cellules cancereuses de la prostate et methodes d'utilisation
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
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US9482667B2 (en) 2007-12-27 2016-11-01 Abbott Laboratories Anti-T. cruzi antibodies and methods of use
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JP2016176944A (ja) * 2007-12-27 2016-10-06 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ 抗Ｔ．ｃｒｕｚｉ抗体及び使用方法
US9238064B2 (en) 2008-03-03 2016-01-19 University Of Miami Allogeneic cancer cell-based immunotherapy
US8475785B2 (en) 2008-03-03 2013-07-02 The University Of Miami Allogeneic cancer cell-based immunotherapy
US8968720B2 (en) 2008-03-20 2015-03-03 University Of Miami Heat shock protein GP96 vaccination and methods of using same
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US11548930B2 (en) 2017-04-04 2023-01-10 Heat Biologics, Inc. Intratumoral vaccination
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