[go: up one dir, main page]

WO1995018148A1 - Epitopes de cellules t - Google Patents

Epitopes de cellules t Download PDF

Info

Publication number
WO1995018148A1
WO1995018148A1 PCT/US1993/011703 US9311703W WO9518148A1 WO 1995018148 A1 WO1995018148 A1 WO 1995018148A1 US 9311703 W US9311703 W US 9311703W WO 9518148 A1 WO9518148 A1 WO 9518148A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
peptide
peptides
cell
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1993/011703
Other languages
English (en)
Inventor
H. Mario Geysen
Stuart J. Rodda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chiron Mimotopes Pty Ltd
Original Assignee
Chiron Mimotopes Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Mimotopes Pty Ltd filed Critical Chiron Mimotopes Pty Ltd
Priority to EP94912144A priority Critical patent/EP0737205A1/fr
Priority to PCT/US1993/011703 priority patent/WO1995018148A1/fr
Priority to JP7517991A priority patent/JPH09510437A/ja
Priority to AU64413/94A priority patent/AU693437B2/en
Publication of WO1995018148A1 publication Critical patent/WO1995018148A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to the fields of molecular biology and immunology. More specifically, this invention relates to methods for determining T-cell epitopes and the specific peptide epitopes determined thereby.
  • T-cell determinants have for the most part been previously studied using clones of T- cells.
  • the cells of such a clone are homogeneous and by their very nature are not representative of T-cells in the general population.
  • generalization of discoveries made with these clones to the population as a whole is not possible.
  • a knowledge of the epitopes that stimulate T-cells in the general population would be invaluable in designing new diagnostic and therapeutic agents. For example, detection of an abno ⁇ nally high proportion of T-cells to a particular antigen would allow earlier diagnosis of exposure to the antigen than standard tests requiring the production of specific antibodies.
  • T-cell determinants for each individual must be identified before treatment may commence.
  • One fundamental problem in determining T-cell epitopes in the general population is obtaining sufficient lymphocytes to carry out systematic studies. Furthermore, in every sample there will be an unknown and unknowable number of antigen-specific memory T- cells.
  • Fig. 1 depicts the sequence of HIN SF2 gpl20 (31-509), used in Example 3.
  • One aspect of the invention is a method for identifying T cell determinants, which method comprises reacting each of a plurality of pools with an immunological agent, the agent being immunologically reactive with the antigen, each pool comprising a plurality of overlapping peptide sequences of the antigen, assessing the strength of reaction between the agent and each of the pools, selecting one or more pools giving the strongest reaction with the agent, preparing a plurality of sub-pools, each sub-pool comprising one or a plurality of peptide sequences selected from one of the selected pools, reacting each of the sub-pools with the agent, and assessing the strength of reaction between the agent and each of the sub- pools.
  • the agent is preferably a population or sample of peripheral blood mononuclear cells (PMBCs), where the strength of reaction is gauged by determining the amount of T cell activation that results.
  • PMBCs peripheral blood mononuclear cells
  • Another aspect of the invention is a method for detecting the exposure of a subject to an antigen or pathogen by determining the response of a T cell-containing sample.
  • Another aspect of the invention is a method for detecting the exposure of a subject to an antigen or pathogen by detecting binding of a T cell epitope peptide to a T cell antigen receptor.
  • Another aspect of the invention is an assay kit for detecting the exposure of a subject to an antigen or pathogen, comprising a T cell epitope, preferably in combination with means for detection of a mitogenic response or surface binding.
  • Another aspect of the invention is an improved vaccine composition which comprises a T cell epitope peptide in combination with B cell epitope peptide.
  • Another aspect of the invention is a method for inducing immunity in a bird or mammal, by administering a composition comprising a T cell epitope and a B cell epitope specific for a pathogen.
  • Another aspect of the invention is a method to increase the number of T cells capable of responding to a pathogen ex vivo, by contacting T cells obtained from a subject with a T ' cell determinant, culturing the reactive T cells to increase the number of T cells capable of responding to the antigen, and administering said T cells to the subject.
  • T cell epitope and “T cell determinant” refer to peptides or regions within a longer protein which bind T cell antigen receptors in conjunction with mammalian MHC proteins. Preferably, T cell epitopes are characteristic of a pathogen or malignancy.
  • a "T cell epitope peptide” is a peptide of about 6 to about 20 amino acids, preferably about 8 to about 15 amino acids, which primarily consists of a T cell determinant.
  • T cell epitopes are prepared from the primary sequence of antigens.
  • a T cell epitope "having a sequence derived from” an antigen is a peptide which comprises a sequence of amino acids found con ⁇ secutively within the antigen's primary sequence.
  • B cell epitope and B cell determinant refer to antigens which are immunoreactive with an antibody or B cell surface antigen receptor (membrane-bound antibody).
  • B cell epitopes/determinants need not be proteins or peptides, but may include lipids, carbohydrates, and other molecules.
  • vaccine refers to a composition or formulation suitable for administration to a mammal or bird, and capable of inducing an immune response in the mammal or bird.
  • Vaccines of the invention will include at least one T cell epitope and at least one B cell epitope, which need not be derived from the same antigen or pathogen.
  • the B cell antigen may be presented as a whole protein or large fragment, or as whole killed pathogen, if desired.
  • the T cell epitope is preferably included as a T cell epitope peptide.
  • label and “detectable label” as used herein refer to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a protein or peptide. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32 P and 125 I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity.
  • horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • HRP may serve as an enzyme or as an antigen for a MAb. Further, one may combine various labels for desired effect.
  • MAbs and avidin also require labels in the practice of this invention: thus, one might label a peptide with biotin, and detect its presence with avidin labeled with 125 I, or with an anti-bio- tin MAb labeled with HRP.
  • Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
  • biological sample refers to any sample obtained from a bird or mammal which contains live T cells, preferably peripheral blood mononuclear cells. Suitable biological samples are typically derived from blood, but may be derived from any biological tissue or fluid (e.g. , biopsy specimens, lymph, pus, saliva, semen, and the like) where T cells may be found.
  • tissue or fluid e.g. , biopsy specimens, lymph, pus, saliva, semen, and the like
  • a “kit” within the scope of this invention includes at least one T cell epitope peptide, and printed instructions for performing an assay.
  • "Printed instructions” may be written or printed on paper or other media, or committed to electromc media such as magnetic tape, computer-readable disks or tape, CD-ROM, and the like.
  • Kits also preferably include means for detecting positive responses, for example labels for the T cell epitope peptides, 3 H-T, or similar means. Kits may also include culture dishes, culture reagents, and other such supporting materials.
  • pharmaceutically acceptable carrier means refers to compounds and com ⁇ positions which may be administered to mammals and/or birds without undue toxicity.
  • Exemplary pharmaceutically acceptable salts include mineral acid salts such as hydro- chlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydro- chlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • effective amount refers to the amount of T cell epitope peptide and B cell antigen required to effect an immune response in a subject.
  • the precise effective amount will vary from subject to subject, depending on age, species, size, weight, and general health, but will generally correspond to the amount effective for traditional vaccines.
  • This invention involves the combination of various test compounds in a plurality of pools, and testing the pools for immunoreactivity with T cells.
  • the pools are tested for activity, and selected active pools resynthesized as a plurality of sub-pools, generally derived from the sequences of the first pool.
  • the plurality of sub-pools is then assayed, and active sub-pools selected for resynthesis as a plurality of sub-sub-pools.
  • This process is reiterated as many times as desired, preferably until the sub-pools contain only a single species of peptide, thus identifying the active peptides. It is essential to minimize the number of assays performed, as one must use peripheral blood lymphocytes (PBLs) to obtain an accurate reading. This process minimizes the number of assays which must be performed, thus making possible T cell epitope determination for an individual.
  • PBLs peripheral blood lymphocytes
  • the size of the pool will depend on the number of active test compounds expected to be discovered. Obviously, the major benefit of the invention will be realized where the number of active test compounds is a small proportion of the compounds to be screened. To minimize the amount of test material required for the assays, the number of test compounds in each pool should be chosen so that the majority of pools will be inactive.
  • T cell epitopes are primarily or solely composed of peptides. Protein antigens are digested by antigen-presenting cells (APCs), and fragments of the antigens presented on the APC surface in the context of a major histocompatibility complex (MHC) protein. Thus, T cell epitopes are always linear fragments of the native antigen: there are no discontinuous epitopes, as in the case of B cell epitopes. Accordingly, T cell epitopes may be determined by preparing a series of peptides which span the amino acid sequence of the antigen. The peptides are selected to overlap, so that determinants are not missed due to straddling the junction between two test peptides.
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • the peptides used are 13mers, one should prepare overlapping 13mers that overlap by 12 residues (i.e. , that are offset by one residue, e.g. , ⁇ XXXXXXXXXXX 13 , 2 XXXXXXXXXX 14 , etc.). If longer peptides are used, the offset may be increased accordingly.
  • one employs 15mers one may synthesize a series of peptides in which each is offset from the next by two or three residues (e.g. , XXXXXXXXXXXXXX 15 , 3 XXXXXXXXXXXXXXXXXXX 17 , etc.).
  • the peptides may be synthesized by any convenient method.
  • a presently preferred method is the synthetic scheme disclosed in WO90/09395, in which peptides are synthesized bound to a plurality of plastic pins via a cleavable linkage.
  • the peptides are cleaved from the support and are combined into pools for testing.
  • the peptides are preferably grouped into pools on the basis of their sequences, i.e. , the first ten sequential peptides in pool 1 , the next ten peptides in pool 2, etc.
  • other grouping strategies may also be useful. For example, one may wish to group peptides derived from similar sections of a protein having internal repeats (e.g.
  • pool 1 may contain the first several peptides from the beginning of each immunoglobulin fold in an antibody).
  • T cells are obtained from a subject, and are contacted with the peptide pools.
  • T cell epitopes specific to a given pathogen one should select subjects who have been exposed to the pathogen, either as the result of infection or vaccination. Subjects who have recovered from infection may provide the best (most protective/diagnostic) epitopes.
  • the T cells may be either fresh or frozen, but are not cultured or cloned prior to assay in order to preserve the natural distribution of T cells having different antigen recep ⁇ tors.
  • the T cells are preferably obtained from peripheral blood as PBMCs, and are separ ⁇ ated from erythrocytes and polymorphonuclear cells by centrifiigation or fluorescence- activated cell sorting.
  • the T cells and peptide pools are placed together in a plurality of wells or culture dishes, and the response determined by determining binding directly (e.g. , by fluorescence of a labeled peptide) or through mitogenicity (typically measured by 3 H-T uptake after culture).
  • the cells are preferably cultured in autologous serum, in the absence of xenogeneic or pooled serum, to reduce background responses.
  • T cells which recognize one or more of the peptides respond by mitosis, clonally expanding the number of specific T cells recognizing the peptide determinant.
  • T cell epitopes may be employed as reagents in T cell-based immunoassays. Such assays are advantageous over antibody-based assays because they do not require that the subject have already mounted a detectable antibody response to the antigen.
  • the T cell (T H ) response to an antigen necessarily precedes the B cell (antibody) response: thus, the T cell response may be detected earlier than the antibody response.
  • Diagnostic assays may be performed with a much smaller sample, because it is not necessary to scan the entire length of the antigen once the epitopes have been determined. It is likely that some antigens will exhibit different epitopes in different individuals, based on the heterogeneity of the MHC proteins which present the antigen to the immune system.
  • T cell epitope peptides may include a number of T cell epitope peptides in each assay. For example, one may screen a subject's PBMCs against a pool comprising all known HTV SF2 gpl20 T cell epitopes. Response to any peptide in the pool may be counted as a positive response. The response may be detected by a variety of methods. The presently preferred method is to culture PBMCs in contact with the peptide pools, followed by pulsing with 3 H- T to determine T cell proliferation. One may also detect mitogenic effects by other means, for example, by monitoring the increase in interleukin-2 mRNA using PCR, and the like. Alternatively, one may label the peptides and detect binding directly to the T cell antigen receptor. For example, the peptides may be labeled with fluorescein.
  • Example 1 (M. bovis Determinants) A) In this assay, the stimulation of T cells is detected by their proliferation after exposure to the test compounds. It will be appreciated that other assay methods can be used without violating the essence of the invention.
  • Autologous serum was obtained after defibrination of 50 to 100 mL freshly-drawn human venous blood by gentle agitation with 3-5 g of sterile acid-washed glass balls (5-8 mm diameter) for at least 10 min. Serum was collected from above PBMC bands after density interface centrifugation. Autologous plasma was collected from above the PBMC band after density interface centrifugation of anticoagulated whole blood.
  • Plasma was converted into serum by the addition of 10 mM CaCl 2 and 1 IU/mL (final) of human thrombin (CSL) to plasma prewarmed to 37°C, followed by vigorous agitation for 5-10 min. The mixture was allowed to stand at room temp, for 60 min and the supernatant serum was collected by centrifugation at an RCF of 20000 at 4°C for 20 min. Sera or plasmas were heat-inactivated at 56°C for 45 min in a water bath.
  • CSL human thrombin
  • PBMC peripheral blood mononuclear cells
  • the 152 peptides were synthesized in quadruplicate on plastic rods according to the methods disclosed in WO90/09395.
  • the base-labile moiety Lys-Pro was incorporated at the carboxy terminal of each peptide, and the amino terminal amine group was acetylated.
  • the peptides were cleaved from the rods into 150 ⁇ L of 0.1 M bicarbonate buffer at pH 8.2, and the solutions of each different peptide were combined into pools.
  • Pool #1 consisted of the 11 peptides ⁇ DLVGPGGCA- EYA to U YAAANPTGPASV (where the superscript indicates the residue within the MPB 70 sequence above). Pools were made by mixing 200 ⁇ L volumes of each of the peptide prep ⁇ arations. To each peptide pool was added 1.8 mL of RPMI-1640 cell culture medium. Each assay was carried out in 48 replicates. Lymphocytes were separated from whole blood (50 mL) and resuspended in RPMI complete medium at a cell concentration of approximately 1.07 x 10 6 cells/mL.
  • a threshold point was calculated based on the probability that no assay result would exceed the threshold by chance (on a two-tailed test). This procedure was repeated until all assay results below the threshold were included in the "background” estimate and change was made in the estimate of the threshold. Thus, when the data were submitted to this procedure, the lowest 615 assay results (of 768 total) yielded a mean of 3170 cpm (standard deviation 1420) and gave a threshold estimate of 7700 cpm. Thus, from this assay, we would not expect any assay result to return a value greater than 7700 cpm if there was no specific proliferation of the lymphocytes. In this assay, 153 results returned values greater than this. Pools 1 and 2 returned 33 and 40 positive results (out of 48 assays), respectively, as shown in Table 1 : TABLE 1: Positive Results per Pool
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs are prepared and preserved from 45 mL of whole blood. The cells should not be exposed to bovine or other xerosera. Replicate numbers ( > 24) are necessary to assure reas ⁇ onable confidence in the value calculated. The number of cells seeded per well depends on the expected frequency of responding T cells in the preparation.
  • Fresh heparinized whole blood (45 mL) is partitioned in three 50 mL conical centrifuge tubes, then diluted with 10 mL of PBS warmed to room temperature.
  • the diluted blood is carefully underlaid with 10 mL Ficoll, taking care not to mix the interface, then centrifuged in a swinging bucket rotor at 400 X g for 20 min at 20 °C with the brake off, between 18-22°C.
  • the serum is aspirated from above the lymphocyte band and the cells removed from the interface, taking care to avoid removing material from the Ficoll layer (which contains granulocytes). Each band should be collected in about 5 mL.
  • the PBMC from the three tubes are combined into a 50 mL centrifuge tube and diluted with 4 volumes of PBS, .then centrifuged at 60-100 X g for 8-10 min at 20 °C with the brake on.
  • the supernatants ' are decanted, and the cell pellets resuspended in 10 mL PBS.
  • the cells are transferred to a 15 mL tube and centrifuge as before.
  • the PBMC are washed one more time with 10 mL PBS. Cells are resuspended in 10 mL RPMI CM-1 % PHS and counted in a hemacytometer using trypan blue exclusion to estimate viability.
  • Assay plates are prepared before thawing the PBMC. Use 96-well U-bottom plates for cultures seeded with > 20,000 cells/well; 96-well V-bottom plates for cultures seeded with ⁇ 20,000 cells/well. Antigens are diluted in lymphocyte basal medium to 10 X the final concentration, and are added in 20 ⁇ L to the wells. The PHA is added 48 hours before the assay is completed. Basal medium alone is added to the "cells alone" wells. Vials of frozen PBMC are retrieved from the liquid N 2 and kept on dry ice until they ready to be thawed. Six to eight samples can be processed at a time.
  • Conical centrifuge tubes (15 mL) and an equal number of 60 mm tissue culture dishes labeled with the sample ID are set up.
  • Lymphocyte wash medium (5 mL) is added to each of the tubes and dishes, and vials corresponding to the samples to be processed placed in a 37° C water bath. The vials are removed from the water bath before each sample is completely thawed, the outside rinsed with ethanol, and the contents placed in it's respective dish.
  • One mL of medium from the 15 mL tube is used to rinse the vial.
  • the cell suspension is transferred from the thawing dish to the centrifuge tube, and the dish rinsed with the medium used to rinse the vial.
  • the PBMC are pelleted in a tabletop centrifuge at 900 x g for 8 min, and the pellets washed twice with 10 mL of wash medium, then resuspended in 5 mL of lymphocyte culture medium. The cells are counted after the second wash and the volume of medium and cells needed to set up the plate calculated. To seed 96 wells with 180 ⁇ L of cell suspension/well, 18.5 mL of cell suspension is needed. The number of cells seeded per well depends on the expected responder frequency. If the expected frequency is low (e.g. 1 responder in 500,000 PBMC), 10 5 cells/well is a good starting point. For expected frequencies of 1 in 20,000 to 1 in 10,000 about 20,000 cells/well should be used.
  • the object is to achieve roughly half of the antigen-containing wells scored as positive.
  • the washed cells are diluted such that the number of cells/ well will be delivered in 180 ⁇ L to the U-bottom plates.
  • V-bottom plates are used for high-frequency assays, the cells are resuspended at 1-3 x 10 5 cells/mL and the appropriate volume added. As little as 20 ⁇ L of cells can be seeded in V-bottom wells. However, in this configuration, the cells should be diluted in medium containing anti ⁇ gen rather than adding the antigen to the wells prior to thawing the PBMC.
  • the plates are incubated in a humidified 7% CO 2 incubator. Two days before the pulse, PHA is added to the appropriate wells in 20 ⁇ L of basal medium. Early on day 6, 20 ⁇ L of 3 H-thymidine diluted to 25 mCi/mL in basal medium is added to each well using a multichannel pipettor, and the plates returned to the incubator. After six hours, the plates are harvested with a Cambridge 2800 harvester using program 0. Alternatively, the plates can be frozen and harvested at a later date. The filter mats are dried for several hours or overnight before sealing them into their bags with 10 mL of scintillation fluid. The filters are counted in the ⁇ -plate counter using the appropriate protocol. Antigen Preparation:
  • Peptides 13mers were prepared in quadruplicate on plastic rods according to the methods disclosed in WO99/09395, as described in the Example above, based on the amino acid sequence of the He ⁇ es simplex virus 2 antigen gD2: Lys Tyr Ala Leu Ala Asp Pro Ser Leu Lys
  • pool 1 contained 12 peptides
  • pools 2-11 contained 10 peptides
  • pool 12 contained 8 peptides. Pools were made by mixing 200 ⁇ L volumes of each of the peptide preparations. To each peptide pool was added 1.8 mL of RPMI- 1640 cell culture medium. Each assay was carried out in 48 replicates. In the assay, 180 ⁇ L cell suspension was added to 20 ⁇ L of peptide pool in microculture plates. Results:
  • Each pool was scored for strength of response (none, weak, moderate, high) for each donor. Pools 1-3 and 7-12 exhibited weak to zero response. Pools 4-6 exhibited at least moderate responses for 7, 6, and 5 of the donors, respectively. For three of the donors, samples were retested against individual peptides from the responding pools to determine which peptides were responsible for the observed activity.
  • Donor A responded to peptides in the following regions: 19 GKNLPVLDQL, 97 IAWYRMGDNCAIP ⁇ N, 161 AGTYLRLV, and 202 LTSKAYQQG.
  • Donor C responded to peptides in the following regions: 136 CPIRTQPRWSYYDSF, and 17 ⁇ DWTEITQFILE.
  • Donor D responded to peptides in the following regions: 133 PRWPRWSYYDSFS-
  • Samples from Donor D were additionally tested for CD8+ T cell epitopes. The following CD8 epitope was determined: 307 APAAPSNPG. Interestingly, the CD8 epitope occurs in a region which is different in HSV-1 and HSV-2, and may account for the fact that immune responses to HSV are type-specific.
  • Example 3 Human Immunodeficiency Virus Determinants
  • Eight subjects were selected from participants in a clinical study to examine a recom- binant HIV vaccine based on HIV SF2 gpl20.
  • the subjects were vaccinated with HIV SF2 gpl20 in an adjuvant formulation on day 0, and boosted at one month and six months. Some subjects received placebos. Samples were obtained and treated as described in Example 2 above, both prior to vaccination and two weeks following the last boost.
  • Peptides were prepared as described in Example 2 above.
  • the peptides were 15mers, offset by two residues, spanning the length of HIN SF2 gpl20 (excluding the 30 residue signal sequence).
  • the g l20 sequence is shown in Figure 1.
  • the peptides were grouped in pools of 13 peptides, for a total of 18 pools. The results are shown in Table 3.
  • pools 3 and 11 probably contain the T cell determinants for HIV SF2 gpl20, based on the increase in response between pre-vaccination and post vaccination tests.
  • Pools 3 (peptides 27-39) and 11 (peptides 121-133) were analyzed for five subjects to determine which peptides were responsible for the observed activity. Pools for which responses were obtained both before and after vaccination probably indicate cross- reactive with other pathogens. The results are shown in Table 4.
  • “Incomplete” culture medium composition was RPMI- 1640 supplemented with L-glutamine (2 mM), sodium bicarbonate (2 g/L), gentamicin (50 ⁇ g/mL) and HEPES (5 mM).
  • “Complete” medium was incomplete medium supplemented with 10% human serum.
  • Autologous serum was obtained after defibrination of 50 to 100 mL freshly-drawn human venous blood by gentle agitation with 3-5 g of sterile acid-washed glass balls (5-8 mm diameter) for at least 10 min. Serum was collected from above PBMC bands after density interface centrifugation. Autologous plasma was collected from above the PBMC band after density interface centrifugation of anticoagulated whole blood.
  • Plasma was converted into serum by the addition of 10 mM calcium chloride and 1 IU/mL (final) of human thrombin (CSL) to plasma prewarmed to 37 °C, followed by vigorous agitation for 5-10 min. The mixture was allowed to stand at room temperature for 60 min and the supernatant serum was collected by centrifugation at an RCF of 20000 at 4°C for 20 min. Sera or plasmas were heat-inactivated at 56°C for 45 min in a water bath.
  • CSL human thrombin
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • screened buffy coats from blood anticoagulated with CPD kindly supplied by the Red Cross Blood Bank, Melbourne, Victoria, were used.
  • Blood was diluted slightly to between 1 : 1 and 2.5: 1 (50% to 72 % (v/v) whole blood) with incomplete medium and underlay ed with Ficoll/Paque (Pharmacia LKB, Uppsala, Sweden) in a 50 mL polypropylene centrifuge tube in a final ratio of 2: 1 diluted blood: Ficoll/Paque.
  • a band of PBMC was isolated at the interface by centrifugation at an RCF of 450 for 25 min.
  • Tetanus Toxoid (TT), and Purified Protein Derivative (PPD) from BCG cultures were supplied by CSL, Melbourne, Australia, and were used at 0.1-10 Lf/mL and 0.1-10 ⁇ g/mL respectively.
  • TT was dialysed to remove thiomersal preservative before use.
  • Betapropriolactone-inactivated zonally purified A/Shanghai/ 11/87 (H3N2) influenza virus suspension was also supplied by CSL.
  • Concanavalin A (Sigma, St. Louis, MO) was used at 2-10 ⁇ g/mL.
  • Recombinant He ⁇ es Simplex virus type 2 glycoprotein B was kindly supplied by Chiron Co ⁇ . (Emeryville, CA).
  • Single micromolar-scale resin peptides were made on a Milligen 9050 synthesizer (Milligen/Biosearch, Burlington, MA) and purified by reverse-phase HPLC. Multiple peptides were made on polyethylene pins using the Multiple Peptide Synthesis System (Chiron Mimotopes, Clayton, Australia). Solution phase peptides were generated by cleavage from the pin at neutral pH of a lysine-proline ester linker to form a diketopiperazine (cyclic dipeptide) moiety at the carboxy terminus of each peptide.
  • PBMC lymphocyte proliferation assay
  • Tests were performed in as many replicates as the cell yield and size of the blood donation from each donor allowed. A minimum of 12 replicates per experimental group was generally used. Results are expressed as: the mean ⁇ SD of the cpm of the replicates; as stimulation indices (mean cpm of stimulated cultures/mean cpm of Cells Alone controls); or the replicates were scored as positive (responding) or negative (nonresponding) based on a cutoff value for cpm of the inco ⁇ orated 3 H-TdR. For the calculation of the cutoff, data from all experimental groups was pooled for analysis. B. Results
  • RPMI- 1640 medium Seven lots of RPMI- 1640 medium, and two other base media (MEM and DMEM), were used to make up "complete medium” without antibiotics, supplemented with identical additives including 10% (v/v) of a single batch of pooled human serum. They were then tested in proliferation assays with PBMC from one donor, in response to antigenic stimulation with PPD (Table 1). The characteristics sought were congruous, low background levels with strong antigen-specific proliferation. RPMI- 1640 media tested included five different brands (three liquid and two powdered) and different batches of two brands. We also tested MEM and DMEM. A serum-free medium, "Monomed” , was tested without sup ⁇ plementation with serum.
  • Negative control cultures of PBMC from some donors were found to give unacceptably high frequencies of "spontaneous" proliferation in medium containing the antibiotic combination penicillin/streptomycin but not in medium containing gentamicin. With some donors, responses to antigen were also heightened in the presence of these antibiotics.
  • gentamicin kanamycin, neomycin/polymyxin, and ciprofloxacin. Gentamicin was chosen due to its low toxicity and absence of stimulatory effect on controls, as well as its stability during culture.
  • serum is a vital component of media for proliferation assays on PBMC.
  • Human AB sera are widely used for the pu ⁇ ose.
  • the selection of reliable sera also requires a screening process, as illustrated by the following data.
  • ACD-anticoagulated human plasmas from blood group AB donors were converted to serum and each separate donation tested as a medium supplement in antigen- and mitogen-driven proliferation of three lots of human PBMC.
  • Serum was graded as suitable for use on two criteria. Firstly, each lot of serum should provide a low, even background with all three test PBMC. Secondly, the serum should provide good growth ( > 3 x mean of background wells) of both the mitogen and antigen stimulated cultures of at least two of the three lots of PBMC.
  • Autologous serum from defibrinated blood was found to be at least as good a medium supplement as screened pooled serum. In most cases media containing autologous serum gave higher stimulation indices than media containing pooled serum. This was particularly so for antigen-driven proliferation rather than mitogen-driven proliferation.
  • the practical advantage of using autologous serum is that it requires no prescreening, is very easily obtained in sufficient quantity for the experiment at hand, and requires no additional processing to remove anticoagulants or fibrinogen. Autologous serum from a previous bleed of the same donor can also be used.
  • the incubation time at which maximal proliferation occurs is a factor which should be evaluated in any proliferation assay.
  • Use of a long incubation time prior to pulse labeling of stimulated PBMC could give the impression that low doses of antigen are more effective than high doses, due to the "overgrowth" of cultures under optimal stimulation conditions.
  • the experimental conditions should not only avoid nonspecific stimulation, but also provide an environment in which specifically stimulated cells can proliferate optimally.
  • Serum is the most important single factor in the success of proliferation assays.
  • bovine serum and other heterologous sera enhance background proliferation in human PBMC assays to levels which can obscure antigen-specific responses, whereas selected human sera do not.
  • foetal bovine serum has also been found to be inhibitory to human PBMC proliferation in whole blood culture systems.
  • PBMC and serum can be recovered from the same whole blood sample in high yield by defibrination and dilution of blood with RPMI- 1640 medium, followed by density interface centrifugation as described. This process provides more autologous serum than required for a 10% (v/v) final concentration in culture medium at the cell densities used.
  • autologous serum can be heat-inactivated at 56 °C for 45 min without loss of growth-supporting qualities.
  • the incubation time and the number of PBMC added per well are interrelated factors which must be adjusted together. If input cell concentration is too high, proliferation at long incubation times can be dramatically reduced, possibly by exhaustion of the nutrient or buffering capacity of the medium, or by inhibition of cells due to high cell densities. Use of too few cells per well requires very high numbers of wells to allow detection of antigen-specific precursors at biologically significant frequencies (e.g. > 1 per million PBMC). Short incubation times (4 days) give sensitive detection of positives with low and consistent backgrounds.
  • the optimal time for incubation of antigen-driven PBMC proliferation assays using the methods described is 4 days, whereas 2 to 3 days is optimal for mitogen-driven cultures.
  • Cell numbers per well and well shape are also interrelated factors in antigen-driven PBMC proliferation.
  • the use of round-bottom wells to improve cell-cell contact allowed the use of reduced cell numbers in proliferation experiments.
  • Use of V-bottom wells, although useful for improving sensitivity, should be avoided if high cell numbers (> 100,000 cells/well) are to be used, as responses can be inhibited under these conditions.
  • Exogenous thymidine is quickly assimilated into the intracellular pool and used for DNA synthesis during the S phase of the cell cycle. Trace-labeling can be used to measure rates of DNA synthesis, provided the thymidine is of low specific activity, ⁇ 2 Ci/mmol. However, use of low specific activity thymidine can lead to addition of excess total thymidine, changing the conditions to flood labeling. Use of high specific activity thymidine (40-80 Ci/mmol) results in cytotoxicity, probably due to radiological damage.
  • Plates may be placed at 4°C overnight or frozen at -20 °C for several days without apparent loss or breakdown of the DNA. Plates may be then processed for harvest of DNA and counting of thymidine as time permits.
  • P399 (Ac-QEIYMQHTYPIS-b-dkp, tt 257-268), P442 (H-EQDPSGATTKSAM- LTNIJIFGPGPVLNKNEV-OH, tt 141-171), P443 (H-SVDDALINSTKIYSY- FPSVISKVNQGAQGIL-OH, tt 581-611), P444 (H-DTQSKNILMQYIKANSKFI- GITELKKLESKI-OH, tt 821-851), P445 (H-IEYNDMFNNFTNSFWLRVPKVS- ASHLEQYGT-OH, tt 941-971), P459 (Ac-VRDHDDFT ⁇ ESSQKT- ⁇ H2, tt 616-631) and P480 (H-FNNFTVSFWLRVPKVSASHLE-OH, tt 947-967) were prepared by solid phase peptide synthesis using an Applied Biosystems 430A
  • PBMC peripheral blood mononuclear cells
  • Peptide-stimulated proliferation assays using 2 X 10 5 PBMC per well were performed in 96-well round bottom tissue culture grade mictrotitre plates (Nunc, Roskilde, Denmark). Antigens were added in 20 ⁇ L of 0.1 M sodium bicarbonate to give a final volume of 200 ⁇ L per well. Because PBMC often exhibited a low frequency of T cells specific for particular determinants, all assays were carried out using at least 16 replicates per test. PBMC were incubated at 37°C in 5 % CO 2 in humidified air. After 138+2 h, proliferation ' was detected by pulsing with 0.5 Ci tritiated (methyl- 3 H) thymidine, 3 H-TdR, (40-60).
  • the restimulation assay was that of E.J. Hensen et al , Human Immunol (1984) J_0:95 modified for use with peptides.
  • TT 1.0 LF/mL
  • P399 1.0 g/mL
  • no antigen 1.0 g/mL
  • cells were washed to remove any residual peptide or antigen, and resuspended in the same volume of fresh complete medium.
  • aliquots of 100 ⁇ L were pulse-labeled using 3 H-TdR.
  • tt 941-971 homologous with a reported universally immunogenic T cell determinant (tt 947-967) was chosen because the pooled 12mers were not stimulatory for any of the donors tested. This was therefore a test of the dependence, on peptide length, of stimulation by peptides from a known stimulatory sequence.
  • PBMC were stimulated with pooled 12mers or with the corresponding 31mer peptide containing all the residues of the individual peptide pool.
  • the PBMC determinant scanning method was developed in studies with tt. Initially, a panel of 12mer peptides homologous with the tt sequence as likely T cell determinants was chosen. One peptide, P399, was found to give proliferation with PBMC of ten out of seventeen TT-responsive donors tested and was therefore chosen for a specificity test. To see if proliferative responses observed in individual peptide-stimulated PBMC cultures were due to peptide-specific T cells, and to see if these cells were also able to respond to the whole antigen from which the peptide was derived, a sensitization-restimulation assay was carried out.
  • PBMC peripheral blood mononuclear cells
  • P399 1.0 ⁇ g/mL
  • TT 1.0 Lf/ml
  • Unstimulated control cells were incubated without added antigen under the same conditions. After 6 days in culture, proliferation of antigen-stimulated cultures was demonstrated. All three groups of cells were washed to remove lymphokines and any antigen still present in the medium. On day 9, proliferation had subsided and the PBMC were washed again.
  • Each culture was then divided into six treatment groups and restim- ulated (P399 at 10, 1.0 and 0.1 ⁇ g/mL, TT at 1.0 and 0.1 Lf/ml, and No Antigen control) in triplicate cultures. To ensure that sufficient APC were available, 10 5 gamma-irradiated autologous PBMC were added to each culture. Proliferation was measured three days after restimulation.
  • peptide-stimulated PBMC cultures are able to be restimulated in a dose-dependent fashion by both the sensitizing peptide P399, and the whole antigen TT.
  • Control incubated PBMC not previously stimulated with either antigen showed little proliferation, in the short time frame and with the low number of input unirradiated PBMC, to either the peptide or the whole antigen.
  • peptide-stimulated cultures could be reproducibly restimulated by the same peptide and were not responding to undefined components of the culture medium. They were also restimulated by whole TT showing that they were specific for the antigen from which the peptide was derived.
  • the TT-sensitized PBMC were restimulated by both P399 and TT.
  • the cells responding to TT must have included a high proportion of P399-responsive cells also.
  • PBMC polyclonal T cells
  • Antigen processing and determinant formation by APC has been shown to vary even for donors with the same restriction element or recombinant mice of identical MHC haplotype, which may be due to a requirement for specific protease(s) for generation of particular determinants.
  • Example 5 can be applied to any antigen of known sequence to which human or animal subjects have a measurable T helper response.
  • tt tetanus toxin
  • PBMC from donors shown to respond to tetanus toxoid (TT) in vitro were screened against peptide pools to locate all determinants in the sequence. Pools were used to keep testing to a manageable, realistic scale. PBMC were chosen as the source of responsive T helper cells because they have the advantage of providing a repertoire unbiased by in vitro selection of the best growing or most prevalent clones.
  • the concentration of each peptide used in the final culture was 0.3 ⁇ M, which was less than the estimated peptide concentration required to approach optimum stimulation (estimated to be about 1 ⁇ M).
  • the choice of final peptide concentration was constrained by the need to keep the volume of peptide solution to ⁇ 10% of the culture medium to avoid dilution and possible toxicity effects, and also by the peptide concentration (60 ⁇ M) of the stock solutions. It should be emphasized that determinants of less than 12 residues in length will be present in 2, 3 or more overlapping peptides in the pool, and therefore the effective con ⁇ centration of shorter determinants will be higher than that of longer determinants.
  • PBMC from nine HLA-typed donors known to respond to TT in vitro were initially scanned for their ability to respond to each of the 66 peptide pools. Results are only shown for pools which stimulated a significantly higher number of wells than the cells alone control (p ⁇ 0.05). Where only one well in a test group showed proliferation, even where this was significantly higher (p ⁇ 0.05) than the cells alone control, we chose to treat this as not significant in the sense of representing a determinant region of tt. Therefore, such single positive wells were not counted in the summary of donors responding to that pool.
  • the individual peptides within four stimulatory pools were tested to identify the individual peptide(s) responsible for proliferative responses incurred by the pool. This test is termed a "decode”.
  • Single peptides were tested at 1 ⁇ M, approximately three times the concentration of individual peptides used in the pool. The pu ⁇ ose of this was to use a concentration close to the effective concentration which occurs when more than one peptide within a pool contains a determinant. That is, because the length of determinants found with T helper cell clones has been eight or nine amino acids, stimulatory sequences of this length would be present in four or five different overlapping peptides within the pool, making the effective concentration 1.2 ⁇ M or 1.5 ⁇ M respectively.
  • T helper cell determinants were decoded. This enabled us to find out whether published T helper cell determinants could be precisely identified using this method.
  • Peptides within pool 30 contain sequence YSYFPSVI (tt 593-600), the determinant for a human tt-specific T helper cell clone.
  • Pool 42 spans sequence QYIKANSKFIGITEL (tt 830-844), reported to contain a "universally immunogenic" DR-restricted determinant.
  • Decoding of pool 30 showed that five overlapping 12mers with start residues 589 to 593 were stimulatory for at least one of the four donors. These 12mers all contain the sequence YSYFPSVI, identical to the published determinant. In this case, single positive wells were regarded as meaningful because low frequency positive responses appeared to be clustered, i.e. , occurred with peptides related by having a high degree of overlap (shared sequence).
  • Decoding of pool 42 showed eight successive 12mers, with start residues 826 to 833, capable of stimulating PBMC of at least two of the four donors. All these peptides overlap the core of five residues, KANSK, within the reported determinant, tt 830-844.
  • T helper cell determinants within this region were centred on sequences IVPYIGPA (tt 642-649) and KQGYEGNFI (tt 654-662) respectively.
  • peptide P459 16mer which encompassed the "envelope" sequence of the stimulatory peptides from pool 32 (residues 616 to 631 ; referred to as peptide P459).
  • P459 was tested at two concentrations; high (10 ⁇ M or 5 ⁇ M) and low (1 ⁇ M), using 8 to 32 replicates per concentration depending on the number of PBMC available. The results deomonstrated that PBMC from the four donors who had responded to the pooled peptides also responded to P459. Of the 11 donors randomly selected, 10 responded to at least one concentration of P459, suggesting that these responses were probably not restricted to a single HLA Class H allotype.
  • MHC class H allotype were identified. Two of these regions correspond to published T helper cell determinants whereas three have not previously been reported.
  • the peptide pooling strategy for T cell determinant identification has major advantages over the use of protein cleavage fragments or use of long synthetic peptides with small overlaps. All overlapping peptides of a length within the range of naturally processed peptides (13 to 18 residues) can be synthesized without stretching the resources of most research groups. With the pooling/decoding approach, the task of testing all these short peptides of an antigen on PBMC of individual donors is achievable.
  • the "decoding" of adjacent peptide pools showing significant stimulation of PBMC can distinguish between two possibilities.
  • the stimulatory sequence(s) in successive positive pools of overlapping peptides could occur at the boundaries of the pools. Both pools may then contain the same stimulatory sequence, despite the impression that two determinants are involved (donors B and D responding to pools 31 and 32).
  • donors responding to adjacent pools may be responding to independent, unrelated determinants separated by nonstimulatory sequences (donor B responding to pools 30 and 31).
  • a test of these alternatives would be to use peptides of the minimum length needed to bind to MHC Class H molecules and stimulate T helper cells. This would enable the effect of small differences in recognised sequences on the measured frequency of specific clonal progeny in PBMC to be seen.
  • T helper cell clones can be stimulated by b-dkp bearing peptides of 8, 9 or 10 residues, suggesting that peptides containing 12 residues of the antigen sequence have more than the required amount of sequence needed to allow MHC class H binding and recognition by the helper T cell receptor.
  • the finding that the amino terminus of the peptide is an important and consistent part of the peptide that binds to MHC Class H antigens suggests that peptides differing in amino-terminal position by only one residue would activate different populations of T helper cells. If this is the case, then testing smaller numbers of longer peptides could result in failure to detect some determinants, since peptides with the required N-terminal residues may not be present in the pool.
  • APC play a critical role in antigen-stimulated PBMC proliferation assays.
  • Short synthetic peptides can be efficiently presented by a range of APC, including B cells, monocytes, dendritic cells and possibly other APC. It is likely that efficient uptake and presentation of 12mer peptides by APC in PBMC occurs in our experimental conditions, because addition of adherent cells from autologous PBMC does not cause an increase in the frequency of positive responses. It is known that short peptides can be taken up directly by MHC class ⁇ mol ⁇ ecules without being processed, but the relative significance of this pathway versus an intracellular pathway for peptides interacting with APC in PBMC is unknown at this time. For longer peptides, however, inefficient detection of precursor T cells may be occurring, since certain pools of 12mers were stimulatory for PBMC in contrast to 31mer peptides spanning the same sequences as the stimulatory pools.
  • T helper cell determinants of an antigen could be identified by an unbiased method, leading to a greater understanding of many aspects of the immune system, including the basis of determinant selection and the factors in peptide sequences underlying MHC Class H restriction. This may enable accurate prediction of T helper cell determinants from primary sequence data alone. Knowledge of the T cell determinants of an antigen will allow design of reagents for immunization or immunosuppression. Peptides containing T cell determinants may be able to be used alone or in combination with whole antigens to increase the immunogenicity of vaccines. A peptide which helps the formation of IgG rather than IgE may alleviate an allergy. Alternatively, a determinant responsible for an autoimmune disease could be modified to lead to tolerance and therefore alleviation of the disease.
  • T cell determinants may also be used as effective substitutes for whole antigens in diagnostic proliferation assays. Testing of the pool of peptides containing five human tetanus toxin T helper cell determinants showed that all donors tested recognized at least one determinant within the pool. This pool of peptides thus represents a reproducible T cell stimulatory antigen mixture which may help the standardization of T cell proliferation tests worldwide.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Pulmonology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Afin de détecter des épitopes de cellules T, on analyse des amas de peptides dérivés de la séquence d'antigène complète en l'absence de sérum xénogénique, puis on détermine l'effet mitogénique des peptides.
PCT/US1993/011703 1993-12-28 1993-12-28 Epitopes de cellules t Ceased WO1995018148A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP94912144A EP0737205A1 (fr) 1993-12-28 1993-12-28 Epitopes de cellules t
PCT/US1993/011703 WO1995018148A1 (fr) 1993-12-28 1993-12-28 Epitopes de cellules t
JP7517991A JPH09510437A (ja) 1993-12-28 1993-12-28 Ｔ細胞エピトープ
AU64413/94A AU693437B2 (en) 1993-12-28 1993-12-28 T-cell epitopes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1993/011703 WO1995018148A1 (fr) 1993-12-28 1993-12-28 Epitopes de cellules t

Publications (1)

Publication Number Publication Date
WO1995018148A1 true WO1995018148A1 (fr) 1995-07-06

Family

ID=22237260

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/011703 Ceased WO1995018148A1 (fr) 1993-12-28 1993-12-28 Epitopes de cellules t

Country Status (4)

Country Link
EP (1) EP0737205A1 (fr)
JP (1) JPH09510437A (fr)
AU (1) AU693437B2 (fr)
WO (1) WO1995018148A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018680A1 (fr) * 2002-07-15 2004-03-04 Institute Of Gene And Brain Science Procede pour cribler un antigene tumoral
WO2003099860A3 (fr) * 2002-05-24 2004-09-30 Dev Des Antigenes Soc D Et Composition immunogene et sequences peptidiques pour la prevention et le traitement d'une infection a hsv
EP2263686A1 (fr) * 2002-07-18 2010-12-22 University of Washington Compositions pharmaceutiques comportant des fragments de protéine du virus de l'herpès simplex d'acitvité immunologique
US9522962B2 (en) * 2010-03-15 2016-12-20 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Peptides, conjugates and method for increasing immunogenicity of a vaccine
US9555099B2 (en) 2012-05-16 2017-01-31 Immune Design Corp. Vaccines for HSV-2
US9579376B2 (en) 2009-04-03 2017-02-28 University Of Washington Antigenic peptide of HSV-2 and methods for using same
US9671411B2 (en) 2005-12-22 2017-06-06 Baxter International Inc. Monocyte activation test better able to detect non-endotoxin pyrogenic contaminants in medical products

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2016414B1 (fr) * 2006-05-05 2015-09-02 Opexa Therapeutics Vaccin à lymphocytes t

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989002277A2 (fr) * 1987-08-28 1989-03-23 Board Of Regents, The University Of Texas System Prophylaxie et therapie du syndrome immunodeficitaire acquis
US4943628A (en) * 1988-06-13 1990-07-24 Ortho Pharmaceutical Corporation HIV peptide-inducted T cell stimulation
EP0429816A1 (fr) * 1989-10-31 1991-06-05 F. Hoffmann-La Roche Ag Composition de vaccin à base d'un épitope non-immunosuppressif de cellules T
WO1992021377A1 (fr) * 1991-06-03 1992-12-10 Syntello Inc. Peptides a utiliser pour induire l'activation de lymphocytes t contre le vih-1

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989002277A2 (fr) * 1987-08-28 1989-03-23 Board Of Regents, The University Of Texas System Prophylaxie et therapie du syndrome immunodeficitaire acquis
US4943628A (en) * 1988-06-13 1990-07-24 Ortho Pharmaceutical Corporation HIV peptide-inducted T cell stimulation
EP0429816A1 (fr) * 1989-10-31 1991-06-05 F. Hoffmann-La Roche Ag Composition de vaccin à base d'un épitope non-immunosuppressif de cellules T
WO1992021377A1 (fr) * 1991-06-03 1992-12-10 Syntello Inc. Peptides a utiliser pour induire l'activation de lymphocytes t contre le vih-1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MODROW, S. ET AL.: "Use of synthetic oligopeptides in identification and characterization of immunological functions in the amino acid sequence of the envelope protein of HIV-1", J. ACQUIRED IMMUNE DEFIC. SYNDR., vol. 2, no. 1, 1989, pages 21 - 27 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003099860A3 (fr) * 2002-05-24 2004-09-30 Dev Des Antigenes Soc D Et Composition immunogene et sequences peptidiques pour la prevention et le traitement d'une infection a hsv
WO2004018680A1 (fr) * 2002-07-15 2004-03-04 Institute Of Gene And Brain Science Procede pour cribler un antigene tumoral
EP2263686A1 (fr) * 2002-07-18 2010-12-22 University of Washington Compositions pharmaceutiques comportant des fragments de protéine du virus de l'herpès simplex d'acitvité immunologique
US8197824B2 (en) 2002-07-18 2012-06-12 University Of Washington Rapid, efficient purification of HSV-specific T-lymphocytes and HSV antigens identified via same
US9138473B2 (en) 2002-07-18 2015-09-22 University Of Washington Rapid, efficient purification of HSV-specific T-lymphocytes and HSV antigens identified via same
US9675688B2 (en) 2002-07-18 2017-06-13 University Of Washington Rapid, efficient purification of HSV-specific T-lymphocytes and HSV antigens identified via same
US9671411B2 (en) 2005-12-22 2017-06-06 Baxter International Inc. Monocyte activation test better able to detect non-endotoxin pyrogenic contaminants in medical products
US9579376B2 (en) 2009-04-03 2017-02-28 University Of Washington Antigenic peptide of HSV-2 and methods for using same
US9522962B2 (en) * 2010-03-15 2016-12-20 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Peptides, conjugates and method for increasing immunogenicity of a vaccine
US9555099B2 (en) 2012-05-16 2017-01-31 Immune Design Corp. Vaccines for HSV-2
US9895435B2 (en) 2012-05-16 2018-02-20 Immune Design Corp. Vaccines for HSV-2
US10391164B2 (en) 2012-05-16 2019-08-27 Immune Design Corp. Vaccines for HSV-2

Also Published As

Publication number Publication date
JPH09510437A (ja) 1997-10-21
EP0737205A1 (fr) 1996-10-16
AU6441394A (en) 1995-07-17
AU693437B2 (en) 1998-07-02

Similar Documents

Publication Publication Date Title
JP2573815B2 (ja) マイコバクテリウム感染検出合成ポリペプチド
US7157091B1 (en) MAGE-A1 peptides presented by HLA class II molecules
US20100015169A1 (en) Tumor Antigens
US5888512A (en) Lymphocyte activity regulation by HLA peptides
CA2881594A1 (fr) Composition de vaccin contre le cancer
NO302175B1 (no) Fremgangsmåte for fremstilling av peptider, anvendelse av disse in vitro, diagnostisk sett, samt paratopiske molekyler som immunreagerer med peptidene
CA1340907C (fr) Antigene synthetique provoquant une reaction anti-vih
US20100324829A1 (en) Method of selecting hla-dp4 ligands and the applications thereof
US4596707A (en) Babesia parasite antigen useful in vaccine and diagnostic reagent
Zafiropoulos et al. Induction of antigen-specific isotype switching by in vitro immunization of human naive B lymphocytes
JPH10501682A (ja) 細胞毒性ｔ細胞エピトープ
AU693437B2 (en) T-cell epitopes
US5081226A (en) Synthetic peptides sharing sequence homology with the HIV envelope protein
WO1993021220A1 (fr) Peptides synthetiques utilises dans un vaccin contre l'infection par les streptocoques et pour le diagnostic de cette infection
US20030165479A1 (en) Methods for isolating proteins expressed by dendritic cells
Vyakarnam et al. T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals
CA2180287A1 (fr) Epitopes de cellules t
JP2004535369A (ja) 試薬およびワクチンとして使用するための免疫原性ｈｉｖペプチド
HUT77262A (hu) Új immunokontraceptív peptidek
Lehner et al. Identification of T-and B-cell epitopes in synthetic peptides derived from a Streptococcus mutans protein and characterization of their antigenicity and immunogenicity
Mishra et al. Human T-helper cell responses to a synthetic peptide derived from the hepatitis B surface antigen
Walker et al. Mapping major and minor T-cell epitopes in vitro and their immunogenic or tolerogenic effect in vivo in non-human primates
WO2005023295A2 (fr) Peptides immunodominants traites naturellement derives de la proteine a des porines de neisseria meningitidis et leur utilisation
JP2007291112A (ja) 腫瘍抗原
Zhou Immunogenicity and requirement for antigen processing of asexual-stage malarial T cell epitopes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB GE HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
LE32 Later election for international application filed prior to expiration of 19th month from priority date or according to rule 32.2 (b)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1994912144

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2180287

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 1994912144

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1994912144

Country of ref document: EP