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WO2001070764A2 - Epitopes - Google Patents

Epitopes Download PDF

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Publication number
WO2001070764A2
WO2001070764A2 PCT/GB2001/001205 GB0101205W WO0170764A2 WO 2001070764 A2 WO2001070764 A2 WO 2001070764A2 GB 0101205 W GB0101205 W GB 0101205W WO 0170764 A2 WO0170764 A2 WO 0170764A2
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WIPO (PCT)
Prior art keywords
cells
cell
polypeptide
sequence
peptide
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PCT/GB2001/001205
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English (en)
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WO2001070764A3 (fr
Inventor
Michel Robert Klein
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Glaxo Group Limited
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Publication date
Application filed by Glaxo Group Limited filed Critical Glaxo Group Limited
Priority to AU2001240897A priority Critical patent/AU2001240897A1/en
Publication of WO2001070764A2 publication Critical patent/WO2001070764A2/fr
Publication of WO2001070764A3 publication Critical patent/WO2001070764A3/fr

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/45—Bacterial antigens
    • A61K40/4524—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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

Definitions

  • the invention relates to prophylactic or therapeutic vaccination against mycobacterial infection and to polynucleotides and polypeptides which can be used for such vaccination.
  • Mycobacterium tuberculosis the causative agent of human tuberculosis, is re- emerging as a major public health risk world- wide.
  • the World Health Organisation has estimated that about one third of the world's population is infected with
  • HLA-B*35 restricted CD8 T cell epitopes in M.tuberculosis proteins We identified a number of peptides that carried the HLF-B*3501 peptide-binding motif. In vitro binding assays revealed five peptides that bound very strongly to HLA-B*3501, and bound to HLA-B*5301 too. One of the peptides caused CD8 T cells to kill M. tuberculosis-infected macrophages and induced intracellular IFN ⁇ and TNF ⁇ production in activated (i.e. CD69*) CD8 cells. This peptide is composed of amino acids 201 to 209 of Signal peptidase-I (EPYLDPATM; Spase-I 20 ⁇ -209 ).
  • EPYLDPATM Signal peptidase-I
  • - Xi is E, X 2 is Y, X is L, X is D, X 5 is P, X 6 is A, X is T, X 8 is V, L, I or M and X 9 is absent;
  • - Xi is L, X is G, X 3 is T, X 4 is A, X 5 and X 6 are each V, X 7 is P, X 8 is V, L, I or M and X 9 is absent,
  • - Xi is T, X 2 is A, X 3 is N, X 4 is Q, X 5 is A, X 6 is I, X 7 is S, X 8 is V, L, I or M and X 9 is absent; or
  • X 2 is W
  • X 3 is Q
  • X 4 is P
  • X 5 is A
  • X 6 is F
  • X 8 is F, Y or W and X is absent or F, Y or W; or an epitope sequence which is an analogue of (I) and which can be recognised by a
  • CD8 T cell that recognises (I);
  • an expression vector comprising a polynucleotide encoding a said polypeptide (a) operably linked to a regulatory sequence capable of providing for expression of the said polypeptide (a); for use in the manufacture of a medicament for vaccinating prophylactically or therapeutically against infection by a mycobacterium.
  • polypeptide (a) and expression vector (b) are capable of stimulating a
  • the invention further provides a vaccine composition which comprises a polypeptide or expression vector as defined above and an adjuvant or delivery system capable of stimulating a CD8 T cell response.
  • a method of vaccinating a pre-selected host against a mycobacterial infection comprising administering to the host an effective amount of a polypeptide, expression vector or vaccine composition as defined above; specific novel polypeptides, expression vectors and cells, suitable for use in vaccinating against infection by a mycobacterium; a product which selectively binds a T cell receptor that recognises an epitope sequence as defined above such as the epitope sequence of formula (I), which product comprises an HLA molecule, or a fragment thereof, comprising a peptide with the sequence of the epitope sequence in its peptide binding groove; a method of detecting in a population of T cells the presence or absence of CD8 T cells that recognise an epitope sequence as defined above such as the epitope sequence of formula (I), said method comprising:
  • FIG. 1 Screening of Spase-I complex-derived peptides for binding to HLA-B*3501.
  • RMA-S B*3501 cells were pulsed with 50 ⁇ M unlabeled competitor peptide and 1 ⁇ M of FL-labeled reference peptide as described in the Materials & Methods section of Example 1.
  • Results are expressed as % inhibition of the maximum signal of the FL-labeled reference peptide.
  • the vertical dotted line identifies peptides that inhibited the maximum signal by >75%.
  • FIG. 1 Titration of HLA-B*3501 binding peptides.
  • RMA-S B*3501 cells were pulsed with a fixed concentration of 1 ⁇ M of FL-labeled reference peptide to yield a maximum signal.
  • Increasing amounts of unlabeled competitor peptides were added to reduce the signal (Van der Burg et al, Hum. Immunol. 44, 189-198, 1995).
  • Results are expressed as % inhibition of the maximum signal of the FL-labeled reference peptide.
  • the concentration at which the maximum signal was inhibited by 50% corresponds to the 1C 50 -value.
  • Figure 4 Killing of peptide pulsed- and M. tuberculosis-infected autologous macrophages. Short-term cultures of PBMC stimulated with peptide SPase-I 201-209 were tested in standard 51 Chromium-release assays. CTL activity is expressed as % specific lysis (mean+SD). Effector cells were tested at E/T ratio of 50:1 on control targets (white bars), and at E/T ratio of 5:1 (dotted bars) and 50:1 (black bars) on peptide-pulsed macrophages, and at E/T ratio of 50:1 on M. tuberculosis-infected macrophages (hatched bars). Figure 5.
  • HLA-B*3501 restricted IFN ⁇ production by CD8 + CD69 + T cells in response to mycobacteria-derived peptides Short-term cultures of PBMC stimulated with peptide SPase-l 201- 09 were co-cuftivated for 6 h with RMA-S B*3501 cells pulsed with a control peptide, HIV- 1 SF 2 Nef7 -80 (4T6R) (panel a) or the specific peptide (panel b). Cytokine secretion was blocked by Brefeldin A. Surface markers CD8 and CD69 and intracellular IFN ⁇ and TNF ⁇ were stained following fixation and permeabilization of cells. Dot plots show log fluorescence intensity.
  • CD8 + bright T cells were gated in the fiuorescence-3 versus side scatter light plots.
  • Quadrant markers for CD69, IFN ⁇ and TNF ⁇ were set using the proper isotype control reagents. Frequencies of positive cells are given as % of the gated CD8 + T cells.
  • SEQ ID NO: 1 shows the amino acid sequence of SPase-I o ⁇ -2 o 9
  • SEQ ID NO: 2 shows the amino acid sequence of 38kDa-LP ⁇ 75-183
  • SEQ ID NO: 3 shows the amino acid sequence of 38kDa-LP 2 2-3 oo
  • SEQ ID NO: 4 shows the amino acid sequence of MPT53 134- ⁇ 43
  • SEQ ID NO: 5 shows the amino acid sequence of MPT53 134- ⁇ 2
  • SEQ ID NO: 6 shows the DNA and amino acid sequences of SPase-I
  • SEQ ID NO: 7 shows the amino acid sequence of SPase-I
  • SEQ ID NO: 8 shows the DNA and amino acid sequences of 38kDa-LP
  • SEQ ID NO: 9 shows the amino acid sequence of 38kDa-LP
  • SEQ ID NO: 10 shows the DNA and amino acid sequences of MPT 3
  • SEQ ID NO: 11 shows the amino acid sequence of MPT53.
  • polypeptides which consist essentially of an epitope sequence of formula (I):
  • - is E
  • X 2 is Y
  • X 3 is L
  • X4 is D
  • X 5 is P
  • X 6 is A
  • X 7 is T
  • X 8 is a hydrophobic amino acid, typically V, L, I or M and X 9 is absent;
  • - X is L, X 2 is G, X 3 is T, X 4 is A, X 5 and X 6 are each V, X 7 is P, X 8 is a hydrophobic amino acid, typically V, L, I or M and " X 9 is absent;
  • - Xi is T, X 2 is A, X 3 is N, X 4 is Q, X 5 is A, X 6 is I, X 7 is S, X 8 is a hydrophobic amino acid, typically V, L, T or M, and X 9 is absent; or
  • and X 7 are each V, X 2 is W, X 3 is Q, » is P, X 5 is A, X 6 is F, X 8 is an aromatic amino acid, typically F, Y or W, and X 9 is absent or an aromatic amino acid, typically F, Y or W.
  • the polypeptides can consist essentially of an epitope sequence which is an analogue of (I) and which can be recognised by a CD8 T cell that recognises (I).
  • the invention is also concerned with expression vectors which comprise a polynucleotide encoding such polypeptides and in which the polynucleotide is operably linked to a regulatory sequence capable of providing for expression of the polypeptide.
  • the polypeptides and expression vectors are useful for vaccinating a host against infection by a mycobacterium. Such a vaccination may be prophylactic (of a host which does not have a mycobacterial infection) or therapeutic (of a host that does have a mycobacterial infection).
  • the host which is vaccinated is generally a mammal, such as a human or animal, typically one which can be naturally or artificially infected by a mycobacterium.
  • the host may be a primate, cow or badger.
  • the host may be at risk of a mycobacterial infection, typically because it is resident in a location in which mycobacterial infection is endemic.
  • the host may be susceptible to mycobacterial infection due to malnutrition or infection by other pathogens, such as HIV.
  • the mycobacterium against which the host is vaccinated expresses the protein from which the epitope is derived, i.e. SPase-I, 38kDa LP or MPT 53.
  • the mycobacterium against which the host is vaccinated expresses another protein which contains the epitope sequence.
  • the protein may therefore be a homologue of the amino acid sequence of SPase-I, 38 kDa LP or MPT 53 shown as SEQ ID NOS: 6 to 11.
  • the mycobacterium is typically pathogenic and capable of infecting mammals, such as those mammals discussed above.
  • the mycobacterium is typically M.tuberculosis, M.marinum, M.kansasii, M. bovis or M.avium.
  • the vaccination stimulates a CD 8 T cell response to the epitope (which may comprise responses to different epitopes of the invention as defined by (I) or the analogue of (I)).
  • the vaccination may or may not lead to a CD8 T cell response to one or more other epitopes in SPase-1, ⁇ 38 kDa LP or MPT 53, or to one or more epitopes in other mycobacterial proteins.
  • the vaccination may or may not lead to an antibody response that recognises an epitope in SPase-I, 38 kDa LP or MPT 53 or in 1, 2, 3 or more other mycobacterial proteins.
  • the epitope sequence may have the sequence of formula (I).
  • the epitope has the sequence EPYLDPATM (SEQ ID NO: 1, SPase-I 20 ⁇ -209 ),
  • the epitope may have a sequence which is an analogue of (I) which is recognised by a T cell that recognises (I).
  • T cell recognition includes the binding of the T cell receptor to the analogue, and typically also antigen-specific functional activation of the T cell.
  • An analogue of (I) is typically an analogue of any of SEQ ID NOS: 1 to 5.
  • a peptide with the sequence of the analogue is capable of inhibiting the binding of a peptide of sequence (I) (referred to as peptide (I) herein) to a T cell receptor.
  • a peptide of sequence (I) referred to as peptide (I) herein
  • the binding of the analogue peptide to the T cell receptor is a specific binding.
  • an MHC class I molecule such as HLA-B*3501.
  • T cell receptor used binds specifically to the peptide (I).
  • T cells with such receptors can be produced by stimulating antigen naive T cells in vitro or in vivo with peptide (I), which is generally presented to the T cell by an appropriate HLA molecule.
  • Antigen-specific functional activation of the T cell by the analogue peptide may be measured using suitable technique discussed herein.
  • the analogue peptide causes such activation when it is presented to the T cell associated with an MHC class I molecule, such as HLA-B*3501 (for example on the surface of a cell).
  • the analogue peptide (or analogue sequence within a larger peptide) is typically capable of stimulating a CD8 ⁇ T cell response directed to peptide (I), for example when administered to a human or animal (such as in any of the forms or with any of the adjuvants mentioned herein).
  • a response may be protective against tuberculosis in an animal model or of therapeutic benefit in a human patient.
  • the analogue peptide typically has a shape, size, flexibility or electronic configuration which is substantially similar to peptide (I). It is typically a derivative of the peptide (I).
  • the analogue peptide may also be able to bind other specific binding agents that bind the peptide (I).
  • an agent may be HLA-B*3501.
  • the analogue peptide typically binds to antibodies specific for peptide (I), and thus inhibits binding of peptide (I) to such an antibody.
  • the analogue peptide is either a peptide or non-peptide or may comprise both peptide and non-peptide portions. Such a peptide or peptide portion may have homology with the peptide (I) (e.g. homology with any of SEQ ID NOS: 1 to 5).
  • the analogue sequence may comprise 1, 2, 3, 4 or more non-natural amino acids, for example amino acids with a side chain different from natural amino acids.
  • the non-natural amino acid will have an N terminus and/or a C terminus.
  • the non-natural amino acid may be an L- or a D-amino acid.
  • the analogue sequence is a peptide sequence which comprises one or more modifications.
  • the modification may be any of those mentioned herein which can be present on the polypeptide of the invention.
  • the modification can be present on any of the amino acids of the analogue sequence, such as any of the amino acids responsible for binding the MHC molecule or which contact the T cell receptor during recognition by a T cell.
  • the analogue sequence is typically designed or selected by computational means and then synthesised using methods known in the art. Alternatively the analogue can be selected from a library of compounds.
  • the library from which the analogue sequence is selected is typically a library comprising peptides, such as peptides which have an HLA-B*3501 binding motif.
  • the library may be a combinatorial library or a microorganism display library, such as a phage display library.
  • the library of compounds may be expressed in the display library in the form of being bound to a MHC class I molecule, such as HLA-B*3501.
  • An analogue peptide or sequence can be selected from the library based on any of the characteristics mentioned above, such as the ability to mimic the binding characteristics of peptide (I), for example the ability to bind a T cell receptor, HLA- B*3501 or antibody which recognises peptide (I).
  • the analogue may be selected based on the ability to cause antigen specific functional activity of a T cell that recognises peptide (I) (for example using any of the suitable techniques mentioned herein).
  • the polypeptide is generally 8 to 2000 amino acids in length, such as 9 to 1000, 10 to 500, 11 to 200, 12 to 100 or 15 to 50 amino acids in length. Typically the polypeptide has a length of up to 50 amino acids.
  • the polypeptide is typically a non-naturally occurring protein, such as a fusion protein comprising sequence from the same or different proteins.
  • a preferred fusion protein comprises a derivative of the sequence of SPase-I, 38kDa-LP or MPT 53 discussed below.
  • the polypeptide may comprise a sequence which is a derivative of the sequence of SPase-I, 38kDa-LP or MPT 53 (e.g. of the sequence shown in SEQ ID NOS: 6 and 7, SEQ ID NOS: 8 and 9 or SEQ ID NOS: 10 and 11).
  • sequence is typically not a derivative of the sequence of SPase-I, 38kDa-LP or MPT53 (derivative being defined according to any manner mentioned herein).
  • the derivative sequence will typically comprise the epitope sequence.
  • Such a derivative may be fragment of SPase-I, 38kDa-LP or MPT53.
  • the fragment only contains SPase-I, 38kDa-LP or MPT53 sequence that lies close to the epitope sequence. In one embodiment the fragment only comprises the epitope sequence.
  • the derivative of the SPase-I, 38kDa-LP or MPT53 sequence may be homologous to the whole SPase-I, 38KDa-LP or MPT53 sequence or any of the fragments thereof mentioned above.
  • the polypeptide typically comprises 1, 2, 3, 4, or from 5 to 10, or more copies of the epitope sequence, which may be the same or different.
  • a linker sequence may or may not separate the epitope sequences and or there may or may not be additional (non-epitope) sequence at the N terminal or C terminal of the peptide.
  • the peptide comprises 1, 2, 3 or more linkers.
  • the linkers are typically 1, 2, 3, 4 or more amino acids in length.
  • 1, 2 or more, or all of the epitope sequences may be contiguous with each other or separated from each other.
  • the polypeptide may also comprise sequence which enhances the immunogenicity of the epitope sequence. Such sequences are discussed below.
  • the polypeptide may also comprise 1, 2, 3, 4 or from 5 to 10, or more, other epitope sequences, such as other CD8 T cell epitope sequences (which are recognised by different T cells), CD4 T cell epitopes or antibody epitopes.
  • other epitope sequences such as other CD8 T cell epitope sequences (which are recognised by different T cells), CD4 T cell epitopes or antibody epitopes.
  • the other epitope(s) may be mycobacterial epitopes (such as epitopes from mycobacterial proteins other than the protein from which the epitope sequence is derived).
  • the other epitope(s) may be of another pathogen, such as a viral pathogen (e.g. HIV).
  • the other epitope(s) may be of Clostridium (e.g. from the Clostridium tetani neurotoxin fragment C), hepatitis B (e.g. from core or surface protein).
  • the other epitope(s) may be artificial or consensus epitope (such as PADRE), for example as described in del Guercio et al (1997) Proc. Natl. Acad. Sci. USA 93, 11786-91.
  • the polypeptide is modified, for example by a natural post-translational modification (e.g. glycosylation) or an artificial modification.
  • the peptide may comprise the modifications that occur when it is expressed in a eukaryotic (e.g. human) or prokaryotic (e.g. E. coli) cell.
  • the peptide lacks glycosylation.
  • the modification may provide a chemical moiety (typically by substitution of a hydrogen, e.g. the hydrogen of a C-H bond), such as an amino, acetyl, hydroxy or halogen (e.g. fluorine) group or carbohydrate group.
  • the modification is present on the N or C terminus.
  • the polypeptide is typically capable of being processed by the class I antigen presenting pathway of a cell to provide a peptide (peptide (I) or the analogue peptide) on the surface of the cell bound a MHC class I molecule. Typically such a cell is able to present the peptide to a T cell.
  • the polypeptide may be produced synthetically or expressed in a recombinant system.
  • Solid phase or solution phase synthesis methods may be used.
  • solid phase synthesis the amino acid sequence of the desired peptide is built up sequentially from the C terminal amino acid which is bound to an insoluble resin. When the desired peptide has been produced it is cleaved from the resin.
  • solution phase synthesis the desired peptide is again built up from the C terminal amino acid. The carboxy group of this amino acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.
  • each amino acid added to the reaction system typically has a protected amino group and an activated carboxy group. Functional side chains are also protected.
  • the expression vector capable of expressing the polypeptide is a polynucleotide, typically DNA or RNA, and is typically single or double stranded.
  • the polynucleotide may be linear or circular (e.g. a plasmid).
  • RNA it is capable of being directly translated to provide the polypeptide (and typically does not contain the sequences mentioned below which aid transcription).
  • the vector comprises a sequence which encodes the polypeptide, which is typically operably linked to a control sequence capable of providing for expression of the coding sequence.
  • the vector comprises 5' and 3' to the coding sequence sequences which aid expression, such as aiding transcription and/or translation of the coding sequence.
  • the vector comprises a promoter, enhancer, transcription terminator, polyadenylation signal, polyA tail, intron, translation initiation codon or translation stop codon.
  • the expression vector is capable of expressing the polypeptide.
  • the vector is capable of expressing the polypeptide in a cell of the host (using the host cell transcription and translation mechanisms).
  • the expression vector is within a cellular vector (as discussed below), and therefore is capable of expressing the polypeptide in the cellular vector (using the transcription and translation mechanisms of the cellular vector).
  • the vector may also be capable of expressing a substance which enhances the immunogenicity of the polypeptide (such as any such substance mentioned herein).
  • the vector comprises 2 or more coding sequences, and may be capable of expressing 2 or more different polypeptides of the invention.
  • the vector may be associated with (e.g. within) a moiety which aids delivery or expression of the vector. Preferably such a moiety aids delivery of the polypeptide expressed by the vector to the class I processing and presentation pathway.
  • a moiety may be a virus or cell.
  • the vector is present in a virus vector (i.e. a viral vector), such as a virus which is capable of stimulating a CD8 T cell response (e.g. a vaccinia virus).
  • the virus is typically one whose wild-type is capable of infecting the host.
  • the cell in which the vector may be present may be a prokaryotic (e.g. bacterium) or eukaryotic cell. It is typically the cell of a pathogen which is capable of infecting the host. Such a pathogen may be virulent in its wild-type form.
  • the cell is of a mycobacterium (such as any of those mentioned herein including M.bovis bacillus Calmette-Guerin).
  • the cell may be of the species of the host or may be a cell which has previously been extracted from the host (and optionally cultured and/or replicated in vitro). Such a cell may be any type of cell mentioned herein, and is typically a professional antigen presenting cell.
  • a recombinant replicable polynucleotide vector which comprises a sequence that encodes the polypeptide of the invention may also be mentioned. In one embodiment this is the same as the expression vector of the invention.
  • the vector may be replicated in a compatible cell.
  • the invention provides a method of making vectors of the invention by introducing a polynucleotide sequence that encodes the polypeptide of the invention into a replicable vector, introducing the vector into a compatible cell, and growing the cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the cell. Suitable cells are described below.
  • the sequence which encodes the polypeptide is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the cell.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Such vectors may be transformed into a suitable cell to provide for expression of a polypeptide of the invention.
  • a polypeptide according to the invention can be obtained by cultivating a cell transformed or transfected with a vector as described above under conditions to provide for expression of the polypeptide, and recovering the expressed polypeptide.
  • the vector may be for example, plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for the expression of the coding sequence and optionally a regulator of the promoter.
  • the vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Promoters and other expression regulation signals may be selected to be compatible with the cell for which the vector is designed.
  • Cells transformed (or transfected) with the vector will be chosen to be compatible with the said vector and preferably will be bacterial such as E. coli. Alternatively they may be cells of a human or animal cell line such as CHO or COS cells, or yeast or insect cells. The cells may also be cells of a non-human animal such as a sheep or rabbit or plant cells.
  • polypeptide may comprise a sequence which enhances the immunogenicity of the epitope.
  • the polypeptide or expression vector may also be associated with a substance (which may be a peptide or non-peptide substance) or may be in a form which is capable of enhancing the immunogenicity of the epitope. Generally this will enhance the speed and/or magnitude of the CD8 T cell response to the epitope, and thus generally after vaccination a larger number of CD8 T cells specific for the epitope will be present in the host (for example in the peripheral blood).
  • the substance may act as adjuvant or may target the peptide to antigen presenting cells (APCs) or to compartments in the antigen processing pathway, for example acting as a carrier protein.
  • the sequence may stimulate a T helper response, such as a response that favours a CD8 T cell response, and thus may comprise a T helper (e.g. Thl) cell epitope.
  • Preferred substances which enhance immunogenicity include sequence from the hepatitis B core antigen, sequence from a stress protein or sequence from Clostridium tetani neurotoxin fragment C.
  • the stress protein is typically a bacterial (e.g. mycobacterial) heat shock protein (HSP) or a protein which has homology with such a protein, such as mycobacterial or E. coli proteins of the HSP 60 and HSP 70 families (e.g. HSP 65 or HSP 71 of mycobacteria) or mammalian homologue (e.g. gp96 of mice or humans, Anthony et al (1999) Vaccine 17, 373-83).
  • HSP heat shock protein
  • mammalian homologue e.g. gp96 of mice or humans, Anthony et al (1999) Vaccine 17, 373-83.
  • the substance may cause the polypeptide or vector to adopt a particulate form.
  • the substance may be a virus or virus-like particle (such as a yeast Ty particle, e.g. as in Allsopp et al (1996) Eur. J. Immunol. 26, 1951-9).
  • the substance may be a cytokine, such as a cytokine which stimulates a MHC class I restricted T cell response or favourable MHC class II restricted T cell response (e.g. IL-2, IL-7, IL-12, IFN- ⁇ or GMCSF).
  • the substance may be, for example, CFA, a muramyl dipeptide (e.g.
  • lipid A monophosphoryl lipid A
  • lipopolysaccharide e.g. from B. abortus
  • liposomes SAF-1
  • a saponin e.g. Quil A
  • keyhole limpet hemocyanin beta 2-microglobulin
  • mannan e.g. oxidised mannan
  • acrylic based microbead e.g. oil in water or water in oil
  • soybean emulsion e.g as in Hioe et al (1996) Vaccine 14, 412-8).
  • a cell may incorporate the polypeptide or vector.
  • the polypeptide In the case of the polypeptide, it is typically presented on the surface of the cell.
  • Such a cell is generally an antigen presenting cell such as a dendritic cell.
  • the cell can thus be loaded with the polypeptide.
  • dendritic cells that may be achieved as described in Ah et al, Transplantation 69, 221-226, 2000.
  • the polypeptide or vector may be administered to a host in the form of such a cell.
  • the cell has previously been obtained from the same individual, i.e. is an autologous cell.
  • the particular route of administration used may aid the stimulating of a CD8 T cell response, and thus the polypeptide vector may be provided in a form suitable for administering by such a route. Delivery by an intramuscular route or by biolistic means is preferred.
  • a low dose of antigen favours the development of a CD8 T cell response.
  • a suitable low dose of the polypeptide or vector may be administered.
  • the polypeptide or vector may thus be in an amount and concentration that is suitable for administering to provide an appropriate low dose.
  • the vector is administered: in the form of "naked DNA”.
  • the product of the invention selectively binds a T cell receptor of the T cell of the invention, typically in a reversible manner. Such a product is generally able to inhibit the binding of epitope peptide (e.g. bound to an MHC molecule) to the T cell receptor.
  • the product comprises an HLA molecule, or a fragment thereof, comprising a peptide with the sequence of the epitope sequence in its peptide binding groove.
  • the HLA molecule is generally a MHC class I molecule (e.g. HLA- B*3501).
  • Such molecules comprise an ⁇ chain and a ⁇ chain.
  • the fragment may comprise only the extracellular domain of the HLA molecule.
  • the fragment may or may not be capable of binding a cell membrane.
  • the HLA molecule an ⁇ chain and a ⁇ chain which are not naturally occurring chains, but will typically have homology with naturally occurring chains (such as those of HLA- B*3501).
  • 2, 3, 4 or more products are linked together in a multimer.
  • the products in the multimer may be linked by a covalent bond or by non-covalent means.
  • the products are linked by a streptavidin-biotin interaction, and thus typically the products comprise a biotin portion (typically chemically linked to or in a fusion protein with the product) which allows the products to be linked together by streptavidin.
  • the multimer generally has a higher binding affinity to the T cell receptor of the invention than the product.
  • the multimer may also comprise a detectable label, such as a radioactive or a light detectable (e.g. fluorescent) label. The label may allow the multimer to be sorted by flow cytometry (e.g.
  • the multimer when the multimer is bound to a T cell receptor which is present on a T cell of the invention).
  • the multimer may be a soluble multimer or may be capable of associating with a cell membrane.
  • the multimer is attached to a solid support, such as a microtitre plate.
  • the invention provides a method of detecting the presence or absence of CD8 T cells that recognise the epitope sequence (which may be in the form of the product discussed above).
  • the determination of whether the T cells recognise the epitope sequence is done by detecting a change in the state of the T cells in the presence of the epitope sequence or determining whether the T cells bind the epitope sequence.
  • the change in state is generally caused by antigen specific functional activity of the T cell after the T cell receptor binds the epitope sequence.
  • the epitope sequence is bound to an MHC class I molecule, which is typically present on the surface of an APC (antigen presenting cell) and typically presented by an appropriate HLA molecule.
  • the change in state of the T cell may be the start of or increase in the expression of a substance in the T cells and/or secretion of a substance from the T cell, such as a cytokine (e.g. IFN- ⁇ , IL-2 or TNF- ⁇ ). Determination of IFN- ⁇ expression or secretion is particularly preferred.
  • the substance can typically be detected by allowing it to bind to a specific binding agent and then measuring the presence of the specific binding agent/substance complex.
  • the specific binding agent is typically an antibody, such as polyclonal or monoclonal antibodies. Antibodies to cytokines are commercially available, or can be made using standard techniques.
  • the specific binding agent is immobilised on a solid support (and thus the method may based on the ELISPOT assay to detect secretion of the substance).
  • the solid support can optionally be washed to remove material which is not specifically bound to the agent.
  • the agent/substance complex may be detected by using a second binding agent which will bind the complex.
  • the second agent binds the substance at a site which is different from the site which binds the first agent.
  • the second agent is preferably an antibody and is labelled directly or indirectly by a detectable label.
  • the second agent may be detected by a third agent which is typically labelled directly or indirectly by a detectable label.
  • the second agent may comprise a biotin moiety, allowing detection by a third agent which comprises a streptavidin moiety and typically alkaline phosphatase as a detectable label.
  • the change in state of the T cell which can be measured may be the increase in the uptake of substances by the T cell, such as the uptake of thymidine.
  • the change in state may be an increase in the size of the T cells, or proliferation of the T cells, or a change in cell surface markers on the T cell.
  • the change in state may be the killing (by the T cell) of a cell which presents the epitope sequence.
  • the determination of whether the T cells recognise the peptide may be carried out using a CTL assay.
  • the T cells which are contacted in the method are taken from the host in a blood sample, although other types of samples which contain T cells can be used.
  • the sample may be added directly to the assay or may be processed first.
  • the processing may comprise diluting of the sample, for example with culture medium or buffer.
  • the sample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold.
  • the processing may comprise separation of components of the sample.
  • MCs mononuclear cells
  • the MCs will include the T cells and APCs.
  • the APCs present in the separated MCs can present the peptide to the T cells.
  • T cells in one embodiment only CD8 T cells
  • PBMCs peripheral blood mononuclear cells
  • MCs and T cells can be separated from the sample using techniques known in the art.
  • the T cells used in the assay can be in the form of unprocessed or diluted samples, or are freshly isolated T cells (such as in the form of freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method. However, more typically the T cells are cultured before use, for example in the presence of the epitope sequence, and generally also exogenous growth promoting cytokines. During culturing the epitope sequence is typically present on the surface of APCs, such as the APC used in the method. Pre-culturing of the T cells may lead to an increase in the sensitivity of the method.
  • the APC which is typically used in the method is from the same host as the T cell or from a different host.
  • the APC can be an non-professional APC, but is typically a professional APC, such as any of the APCs mentioned herein.
  • the APC maybe an artificial APC.
  • the APC is a cell which is capable of presenting the peptide to a T cell. It is typically separated from the same sample as the T cell and is typically co-purified with the T cell. Thus the APC may be present in MCs or
  • the APC is typically a freshly isolated ex vivo cell or a cultured cell. It may be in the form of a cell line, such as a short term or immortalised cell line. The APC may express empty MHC class I molecules on its surface.
  • polypeptide per se is added directly to an assay comprising T cells and APCs. This may be taken up directly onto the surface of the APCs (particularly if the APCs express empty MHC class I molecules on their surface).
  • polypeptide is provided to the APC in the absence of the T cell. The APC is then provided to the T cell, typically after being allowed to present the epitope sequence on its surface.
  • the duration for which the epitope sequence is contacted with the T cells will vary depending on the method used for determining recognition of the peptide.
  • concentration of T cells used is 10 3 /ml to 10 9 /ml, preferably 10 5 /ml to 10 7 /ml.
  • concentration is typically from 0.1 to 1000 ⁇ g/ml, preferably 10 to lOO ⁇ g/ml.
  • length of time for which the T cells are incubated in the assay is from 4 to 24 hours, preferably 6 to 16 hours, and in one embodiment for at least 40, 120 or 180 hours.
  • the determination of the recognition of the epitope sequence by the T cells may be done by measuring the binding of the epitope sequence to the T cells.
  • T cells which bind the peptide can be counted and sorted based on this binding, for example using a FACS machine.
  • the presence of T cells which recognise the peptide will be deemed to occur if the frequency of cells identified using the peptide is above a 'control' value (i.e. above the frequency of antigen naive T cells which recognise the epitope sequence).
  • the frequency of antigen- experienced T cells specific for a particular epitope during a disease state would be expected to be between 5 and 60% of the total CD8 T cells.
  • the invention provides a method of diagnosis of a mycobacterial infection or of testing the effectiveness of a vaccination against a mycobacterial infection. It may not be possible to distinguish between the host having a CD8 T cell response to the epitope due to mycobacterial infection or due to vaccination. A host which is found not to have a CD8 T cell response (or a level of CD8 T cell response which is not sufficient to provide protective/sterilising immunity) to the epitope may be selected for vaccination to stimulate such a T cell response (e.g. by the vaccination method mentioned herein). In the method of diagnosis or testing the effectiveness of a vaccination of the invention the presence or absence of the CD8 T cell response is typically determined by the method of identifying a CD8 T cell response discussed above.
  • the invention also provides a CD8 T cell that recognises the epitope sequence, i.e. the TCR of the T cell is able to bind a peptide with the epitope sequence (typically when the peptide is bound to an appropriate HLA molecule and presented on a cell).
  • the T cell is able to undergo antigen specific functional activation upon binding the epitope sequence.
  • the T cell typically produces IFN- ⁇ , and typically does not produce IL-4, IL-10 or TGF- ⁇ .
  • the T cell r may be cyto toxic and typically contains cytotoxic granules comprising perform, granzymes and granulysin.
  • the T cell is cytotoxic towards autologous macrophages infected with a mycobacterium (such as any mycobacterium mentioned herein).
  • the T cell is not immunosuppressive.
  • the T cell has previously been extracted from the same host and has been replicated in vitro.
  • Such replication may comprise culturing in the presence of the epitope sequence (typically presented on the surface of a cell).
  • the cell may be used in a method of treating a mycobacterial infection.
  • the invention provides a T cell receptor (TCR), or fragment thereof, which is capable of binding the epitope sequence.
  • TCR may have any of the properties of the TCR present on the T cell of the invention.
  • the epitope sequence is generally bound to an MHC molecule during binding with the TCR.
  • the polypeptide, expression vector, cell (including T cell), product and TCR (or fragment thereof) of the invention may be in substantially purified form. They may be in substantially isolated form, in which case they will generally comprise at least 80% e.g. at least 90, 95, 97 or 99% of the polypeptide, polynucleotide, cells or dry mass in the preparation.
  • the polypeptide, expression vector, product or TCR (or fragment thereof) is typically substantially free of other cellular components, or free of other mycobacterial components, or free of (other) polypeptide or polynucleotide.
  • the polypeptide, expression vector, cell or product may be used in any of the above forms in any aspect of the invention mentioned herein.
  • the polypeptide and expression vector discussed above in any form or in association with any other agent is included in the termed "vaccination agent" below.
  • An effective non-toxic amount of such a vaccination agent may be given to a preselected host, such as a human or non-human.
  • the vaccination agent may be administered prophylactically to a host who does not have a mycobacterial infection in order to prevent the individual developing a mycobacterial infection.
  • the vaccination agent may be administered therapeutically to a host who has a mycobacterial infection, in order to treat the infection (including ameliorating the symptoms of the disease).
  • the T cells of the invention may be administered in an effective non-toxic amount to a patient in need thereof, such as a patient with a mycobacterial infection.
  • the invention provides the vaccination agent and the T cells for use in a method of treating the human or animal body by therapy, and for use in the manufacture of a medicament for the treatment of a mycobacterial infection.
  • the vaccination agent or the T cells may be given to the host in one or more administrations. Typically after the initial administration a 'booster' can be given. Typically the host is given 1, 2, 3 or more separate administrations, each of which is separated by at least 12 hours, 1 day, 2, days, 7 days, 14 days, 1 month or more.
  • the vaccination agent or T cells may be in the form of a pharmaceutical composition which comprises the vaccination agent or T cells and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the administration is (and thus the composition is formulated for) parenteral, intravenous, intramuscular, subcutaneous, transdermal, intradermal, oral, intranasal, intravaginal, or intrarectal administration.
  • a preferred method of delivering the vaccination agent is by biolistic means (a particle-mediated method).
  • the agent is typically coated onto carrier particles using a variety of techniques known in the art.
  • Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for (e.g. intracellular) delivery from a gene gun device. The optimum carrier particle size will, of course, depend on the desired target (e.g. diameter of the target cells).
  • tungsten, gold, platinum and iridium carrier particles can be used.
  • Tungsten and gold particles are preferred.
  • Tungsten particles are readily available in average sizes of 0.5 to 2.0 ⁇ m in diameter. Such particles have optimal density for use in particle acceleration delivery methods, and allow highly efficient coating with DNA.
  • Gold particles or microcrystalline gold e.g., gold powder A1570, available from Engelhard Corp., East Newark, NJ
  • gold powder A1570 available from Engelhard Corp., East Newark, NJ
  • provide uniformity in size available from Alpha Chemicals in particle sizes of 1-3 ⁇ m, or available from Degussa, South Plainfield, NJ in a range of particle sizes including 0.95 ⁇ m
  • Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.5-5 ⁇ m.
  • a number of methods are known and have been described for coating or precipitating agents onto particles (e.g. combining a predetermined amount of gold or tungsten with plasmid DNA, CaCl 2 and spermidine).
  • the resulting solution is vortexed continually during the coating procedure to ensure uniformity of the reaction mixture.
  • the coated particles can be transferred to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular gene gun instruments.
  • particles coated with the agent are delivered using particle-mediated delivery techniques.
  • Suitable particle acceleration devices are known in the art. Typically such a device employs an explosive, electric or gaseous discharge to propel coated particles towards the target.
  • the coated particles can themselves be releasably attached to a movable carrier sheet, or removably attached to a surface along which a gas stream passes, lifting the particles from the surface and accelerating them toward the target.
  • An example of a gaseous discharge device is described in U.S. Patent No. 5,204,253.
  • An explosive-type device is described in U.S. Patent No. 4,945,050.
  • One example of an electric discharge-type particle acceleration apparatus is the ACCELL M instrument (Geniva, Madison, Wl), which instrument is described in U.S. Patent No. 5,120,657. Another electric discharge apparatus suitable for use herein is described in U.S. Patent No. 5,149,655.
  • a single administration may include more than one gene gun dose, e.g. two to six gene gun doses.
  • the dose of vaccination agent administered may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient.
  • a suitable dose may however be from 10 ⁇ g to lOg, for example from 100 ⁇ g to lg of the vaccination agent or T cells. These values may represent the total amount administered in the complete treatment regimen or may represent each separate administration in the regimen.
  • the polypeptides of the invention may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with - inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl
  • transfection agents may also be administered to enhance the uptake of the expression vector by cells.
  • suitable transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam and transfectam ).
  • the vaccination agent is in the form of a viral vector the amount of virus administered is in the range of from 10 4 to 10 12 pfu, preferably from 10 7 to 10 10 pfu (for example for adeno viral vectors), more preferably about 10 8 pfu for. herpes viral vectors.
  • a pox virus vector may also be used (e.g. vaccinia virus), typically at any of the above dosages.
  • When injected typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered.
  • cells are admimstered typically 10 to 10 cells, preferably 10 to 10 cells are admimstered.
  • Homologous sequences are mentioned herein. Such sequences typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology with the relevant sequence, for example over a region of at least 15, 20, 40, 100 more contiguous amino acids (of the homologous sequence). Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
  • HSPs high scoring sequence pair
  • Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
  • One measure orsimilarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the homologous sequence typically differs from the relevant sequence by at least (or by no more than) 2, 5, 10, 15, 20 more mutations (which may be substitutions, deletions or insertions). These mutations may be measured across any of the regions mentioned above in relation to calculating homology.
  • the substitutions are preferably "conservative". These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • this typically differs from the epitope sequence (such as SEQ ID NO:l or 2) by at least (or no more than) 1, 2, 3, 4 or more mutations (which may be insertions, deletion or substitution (e.g. conservative substitutions)).
  • Homologous sequences mentioned herein may be encoded by a polynucleotide which hybridises to a polynucleotide that encodes the relevant polypeptide, typically hybridising selectively at a level significantly above background.
  • Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and 0.003M sodium citrate at from about 50°C to about 60°C).
  • medium to high stringency for example 0.03M sodium chloride and 0.003M sodium citrate at from about 50°C to about 60°C.
  • suitable conditions include 0.2 x SSC at 60°C.
  • suitable conditions include 2 x SSC at 60°C.
  • Healthy BCG vaccinated blood donors were recruited from the Banjul Blood Bank, Royal Victoria Hospital. Blood samples were obtained only after informed consent was given and only after a BCG scar was identified as proof of vaccination. HLA class-I genotyping was kindly performed by Dr. A. Jaye (MRC Laboratories, Fajara, Gambia).
  • tuberculosis H37Rv was analyzed: probable Signal peptidase-I (Rv2903c). Peptides were selected for this study when they ranked in the top-5 of best binding peptides of both algorithms and when a Proline (P) at position 2 was present as potential anchor residue. Based on the presence of an aromatic (F, Y, W) or hydrophobic (V, L, I, M) anchor residue at the C-terminus 8-, 9-, and 10-mer peptides were selected to determine HLA-binding properties.
  • Lyophilized 8-mer to 10-mer peptides were purchased from Research Genetics, Inc. (Huntsville, AL, USA). Peptides were synthesized using Fmoc- chemistry and analyzed with HPLC and mass spectrometry. Upon arrival peptides were dissolved in 400 ⁇ l dimethyl-sulfoxide (DMSO) and diluted in 0.9% NaCl to a concentration of 10 mM. Peptide stocks were aliquotted, stored at -70°C and once in use kept at 4°C. Peptides known to bind to HLA-B*35 were used as positive controls, including HIV-1 SF2 Nef 72-80 (4T6R) (FPVTPRVPL) and Epstein-Barr virus
  • HLA-A3 restricted-CD 8 T cell epitope was selected from Influenza A virus Nucleoprotein NP 265- 273 (ILRGSVAHK) (DiBrino et al. J. Immunol. 151, 5930-5935, 1993). Fluorescein (FL)-labeled reference peptides were synthesized as Lysine- derivatives of positive control peptides and were also obtained from Research Genetics, Inc.
  • Binding assays were performed essentially as described (Schonbach et al, 1995; Takamiya et al, Int. Immunol. 6, 255-261, 1994; Van der Burg et al, Hum. Immunol. 44, 189-198, 1995). Briefly, Tap-deficient RMA-S cells expressing human ⁇ 2-microglobulin and HLA-B*3501 (generously provided by Prof. Dr. M.
  • Binding peptides which reduced the maximum signal of the FL-labeled reference peptide by >75%, were subsequently tested in eight serial 2-fold dilutions to determine the concentration at which the maximum signal was inhibited by 50% (IC 50 ). The latter was used as a direct measure of peptide binding (Van der Burg et al, 1995). Alternatively, RMA-S cells were pulsed with 50 ⁇ M peptide for 16 h at 26°C followed by 3 h at 37°C. In case of stabilization assays to determine the half- life of peptide-HLA class-I complexes on the cell surface (Van der Burg, J.
  • HLA class-I surface expression was measured immediately to determine the level of HLA class-I surface expression. The latter was used as an indirect measure of peptide binding, since HLA class-I surface expression rapidly disappears at 37°C in case peptides do not significantly bind (Schonbach et al, 1995; Takamiya et ⁇ /, 1994).
  • Antigen-specific CD8+ T cells were expanded from PBMC as previously has been described (Lalvani et al, J. Immunol. Methods 210, 65-77, 1997). Briefly, PBMC were pulsed with 50 ⁇ M of peptide for 1 hour at room temperature. Cells were cultured in RPMI-1640 supplemented with 10% autologous plasma, antibiotics and 5 ng/ml recombinant human IL-7 (R&D Systems Europe Ltd., Abingdon, UK). On day 3 and every 3-4 days thereafter complete medium was added containing 10 U/ml rlL- 2 (generously provided by Dr. R.
  • effector cells were tested at different E/T ratios on Cr-labeled target cells.
  • Target cells were autologous monocyte-derived-macrophages pulsed with peptide or acutely infected with M.tuberculosis H37Ra, or autologous EBV- transformed B-LCL pulsed with peptide.
  • Supematants were harvested after 4-6 hours and mixed with OptiPhase SuperMix scintillant (Wallac Scintilation Products, Fisher
  • Chromium release was measured using a 1450- MicroBeta Trilux liquid scintillation counter (Wallac, Turku, Finland) and expressed as specific lysis (%) using the following formula: [(experimental release-spontaneous release) ⁇ (maximum release-spontaneous release)] x 100%.
  • Activated CD8 + T cells from short-term bulk cultures were analyzed for intracellular IFN ⁇ production in response to mycobacteria-derived peptides essentially as previously has been described (Kern et al, Nat. Med. 4, 975-978, 1998). Briefly, short-term bulk cultures were harvested and 2 x 10 6 cells were incubated for 4-6 hours at 37°C/5%C0 2 with 10 6 target cells pulsed with peptide.
  • Target cells were autologous EBV-transformed B-LCL or RMA-S B*3501 transfected cells. During the last 4 hour Brefeldin A (Sigma Chemical Co., Poole, UK) was added (final concentration lO ⁇ g/ml). Cells were harvested and washed once with PBS/0.3% (w/v; BSA. Becton Dickinson Immunocytometry Systems (BDIS) (San Jose, CA, USA) reagents and protocols were used to perform intracellular staining of cytokines. Briefly, cells were incubated with FACSTM Lysing Solution (BDIS) and FACSTM Permeabilizing Solution (BDIS) according to manufacturer's instructions. Subsequently, cells were stained using the following antibodies:
  • CaliBRYFETM beads and FACSCompTM software were used to optimize instrument settings prior to use. Data was acquired and analyzed using CellQuestTM software. Data is displayed as two-color dot plots to determine the level of cytokine expression in activated CD8+ T cells.
  • M.tuberculosis H37Rv-derived sequences of Signal peptidase-I were searched for HLA-B*35 peptide binding motifs. Peptides were selected when they ranked among the 5 best binding peptides according to the Epimatrix and BIMAS algorithms and when they contained a Proline on position 2 as anchor residue. The peptides were first tested for their ability to stabilize HLA-B*3501 molecules at a fixed and saturating concentration of 50 ⁇ m ( Figure 1). The positive control peptides HIV-1 SF 2 Nef (aa. 72-80)-4T6R and EBV TEGU (aa. 1974-1982) gave strong and reproducible inhibition of the maximum signal of the FL-labeled reference peptide. Only peptide SPase-I 201-2 Q 9 inhibited >75% and was considered for further testing.
  • Peptide SPase-I 2 o ⁇ -2 0 9 was subsequently tested in eight serial 2-fold dilutions to determine the concentration at which it inhibited the signal of 1 ⁇ M FL-labeled reference peptide by 50% (IC 50 ). Titration of the peptides resulted in reproducible dose-response curves, with an IC 5 0 value of 1.6 to 2.1 ⁇ M ( Figure 2).
  • the IC 5 o-value of HLA-B*3501 binding peptide HIV-1 SF2 Nef (aa. 72-80)-4T6R was between 0.6 - 1.3 ⁇ M.
  • PBMC from a BCG vaccinated healthy donor were stimulated with peptide SPase-I 201 - 209 -
  • Short-term bulk cultures were tested for their ability to kill peptide pulsed-target cells and M.tuberculosis-infected autologous macrophages.
  • the peptide was able to induce cell-mediated cytotoxicity against peptide-pulsed macrophages as well as against M.tuberculosis-infected macrophages (Figure 4).
  • Bulk cultures were also tested for IFN ⁇ and TNF ⁇ production by activated CD8 + T cells ( Figure 5).
  • Peptide SPase-I 2 o ⁇ -2 09 was able to induce intracellular IFN ⁇ and TNF ⁇ production in activated (i.e.
  • CD69* CD8 cells.
  • the number of activated CD8+ T cells producing IFN ⁇ was 22.5% when using autologous cells pulsed with peptide SPase-I 20 ⁇ -2 0 9, whereas only 0.1% of cells were positive when tested on target cells pulsed with control peptide HIV-1 SF2 Nef7 2-8 o (4T6R) ( Figure 5).
  • TNF ⁇ we observed in response to peptide SPase-I 20 1- 2 0 9 about 14.4% activated CD8 T cells with control targets only 0.3%. Comparable results were obtained when we used the autologous B-LCL as targets (not shown).
  • HLA-B*07, -B*3501, -B*5l, -C*w0401 and H-2L d peptide-binding motifs see http://bimas.dert.nih.gov/molbio/hla_bind/) (Parker et al, J. Immunol. 152, 163-175, 1994).
  • predictions of potential HLA-B*07 and -B*35 restricted CTL epitopes were done using the EpiMatrix software in collaboration with Dr. A.S. de Groot (TB/HIV Research Lab, Brown University, Buffalo, RI, USA) (De Groot et al, AIDS Res. Hum.
  • Retroviruses _3, 529-531, 1997 Peptides were selected based on the following criteria: a) 10-mer sequences with COVER value ⁇ 50% for significant binding to HLA-B*35 according to EpiMatrix calculations, b) corresponding 9-mer sequences ranking in the top- 10 of best binding peptides according to BIMAS algorithm, and c) a Proline (?) as anchor residue at position-2 according to the HLA-B7 supermotif (Sidney et al, Immunol. Today 17, 261-266, 1996a; Sidney et al. J. Immunol. 157, 3480-3490, 1996b).
  • Lyophilised 8 to 10-mer peptides were purchased from Research Genetics, Inc. (Huntsville, AL, USA). Peptides were synthesised using Fmoc-chemistry and analysed with HPLC and mass spectrometry. Upon arrival peptides were dissolved in 40 ⁇ l dimethyl-sulfoxide (DMSO) and diluted in 0.9% NaCl to a concentration of 10 mM. Peptide stocks were aliquotted, stored at -70°C and once in use kept at 4°C. Peptides known to bind to HLA-B*35 were used as positive controls, including HIV- 1 S F 2 Nef (aa.
  • HLA-A3 restricted-CD 8 T cell epitope was selected from Influenza A virus Nucleoprotein (aa. 265-273) ILRGSVAHK (DiBrino et al, J. Immunol. L51, 5930- 5935, 1993). Fluorescein (FL)-labeled reference peptides were synthesised as Cys- and Lys-derivatives of positive control peptides and were also obtained from Research Genetics, Inc.
  • Binding peptides which reduced the maximum signal of the FL- labeled reference peptide by >75%, were subsequently tested in eight serial 2-fold dilutions to determine the concentration at which the maximum signal was inhibited by 50% (IC 50 ). The latter was used as an indirect measure of peptide binding (Van der Burg et al, 1995).
  • RMA-S cells were pulsed with 50 ⁇ M peptide for 16 h at 26°C followed by 3 h at 37°C. In case of stabilization assays to determine the half-life of peptide-HLA class-I complexes on the cell surface (Van der Burg, J.
  • Peptides were selected from M.tuberculosis-de ⁇ ved antigens, based on a significant binding score to HLA-B*3501 using the EpiMatrix software (COVER value ⁇ 50%), ranking among the best 10 binders according to the BIMAS algorithm and with a Proline at position 2 as anchor residue. The latter was being done with the hope to select for peptides and also able to bind to other members of the HLA-B7 superfamily (Sidney et al, 1996a; Sidney et al, 1996 b).
  • the peptides were first tested for their ability to bind and stabilise HL A- B*3501 molecules at a fixed and saturating concentration of 50 ⁇ M. IC 50 values were determined. The stability of the peptides complexed to HLA-B*3501 was assessed by detecting the level of HLA class-I surface expression over a 2 to 9 h period, and the half-life (TV2) was determined. Peptides were also tested for their ability to bind and stabilise HLA-B* 3501 molecules. The results are shown in the Table below:

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des épitopes de lymphocytes CD8 T, dérivés à partir de ∫i⊃Mycobacterium tuberculosis∫/i⊃ et correspondant à la formule (I): X1PX2X3X4X5X6X7X8X9 dans laquelle: X1 représente E, X2 représente Y, X3 représente L, X4 représente D, X5 représente P, X6 représente A, X7 représente T, X8 représente V, L, I ou M et X9 est absent; X1 représente L, X2 représente G, X3 représente T, X4 représente A, X5 et X6 représentent chacun V, X7 représente P, X8 représente V, L, I ou M et X9 est absent, X1 représente T, X2 représente A, X3 représente N, X4 représente Q, X5 représente A, X6 représente I, X7 représente S, X8 représente V, L, I ou M et X9 est absent; ou X1 et X7 représentent chacun V, X2 représente W, X3 représente Q, X4 représente P, X5 représente A, X6 représente F, X8 représente F, Y ou W et X9 est absent ou F, Y ou W.
PCT/GB2001/001205 2000-03-20 2001-03-20 Epitopes WO2001070764A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001240897A AU2001240897A1 (en) 2000-03-20 2001-03-20 Epitopes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0006692.8 2000-03-20
GBGB0006692.8A GB0006692D0 (en) 2000-03-20 2000-03-20 Epitopes

Publications (2)

Publication Number Publication Date
WO2001070764A2 true WO2001070764A2 (fr) 2001-09-27
WO2001070764A3 WO2001070764A3 (fr) 2001-12-20

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/001205 WO2001070764A2 (fr) 2000-03-20 2001-03-20 Epitopes

Country Status (3)

Country Link
AU (1) AU2001240897A1 (fr)
GB (1) GB0006692D0 (fr)
WO (1) WO2001070764A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9624456D0 (en) * 1996-11-25 1997-01-15 Isis Innovation Assay method
KR100692227B1 (ko) * 1998-04-07 2007-03-09 코릭사 코포레이션 마이코박테리움 튜베르쿨로시스 항원의 융합 단백질 및이의 용도

Also Published As

Publication number Publication date
WO2001070764A3 (fr) 2001-12-20
AU2001240897A1 (en) 2001-10-03
GB0006692D0 (en) 2000-05-10

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