AU765013B2 - Tuberculosis vaccines - Google Patents

Description

P/00/01128/5/91 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: TUBERCULOSIS VACCINES The following statement is a full description of this invention, including the best method of performing it known to us TUBERCULOSIS VACCINES This specification is concerned with a method of assaying for activated peptide-specific T-cells and its use in identifying human CD8' T cell epitopes of the M. tuberculosis protein ESAT-6. It is a development of the known ELISPOT assay, which is reviewed in current protocols in Immunology, Unit 6.19, pages 6.19.1-8.

The filter immunoplaque assay, otherwise called the enzyme-linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody-secreting B cells. At the time it was developed, the technique provided a rapid and versatile alternative to conventional plaque-forming cell assays.

Recent modifications have improved the sensitivity of the ELISPOT assay such that cells producing as few as 100 molecules of specific protein per second can be detected. These assays take advantage of the relatively high concentration of a given protein (such as a cytokine) in the environment immediately surrounding the proteinsecreting cell. These cell products are captured and detected using high-affinity antibodies.

The ELISPOT assay utilises two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one 20 polyvalent antiserum. ELISPOT generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a "footprint" of the original cytokine-producing cell. Spots are permanent and can be quantitated visually, microscopically, or electronically.

The ELISPOT assay involves five specific steps: coating a purified cytokine-specific antibody to a nitrocellulose-backed microtitre plate; blocking the plate to prevent nonspecific absorption of any other i -2proteins, incubating the cytokine-secreting cells at several different dilutions; adding a labelled second anti-cytokine antibody; and detecting the antibody-cytokine complex.

The technique has been used to develop an assay for peptide-specific T-cells that have been pre-sensitised in vivo to a particular peptide.

There is disclosed herein a method of assaying for peptide-specific T-cells, which method comprises providing a fluid containing T-cells, adding a peptide to the fluid, incubating the fluid to cause cytokine release, and detecting the released cytokine. Preferably the method comprises providing the fluid containing T-cells in contact with a surface carrying an immobilised first antibody to the cytokine, adding the peptide to the fluid, incubating the resulting fluid mixture under conditions to cause any peptide-specific T-cells that have been pre-sensitised in vivo to the peptide to secrete the cytokine, and detecting any secreted cytokine bound to the immobilised first antibody.

The cells are preferably peripheral blood mononuclear cells (PMBC).They may suitably be taken from a patient known to be suffering, or to have suffered, from an infection with an intracellular pathogen, e.g. a 20 virus. It is a preferred feature of the method that fresh cells are used, because cells cultured in vitro may develop altered characteristics thus reducing the diagnostic value of the assay. The purpose of the assay is to identify or quantitate peptide-specific T-cells e.g. CD8+ or CD4+ cells that have been activated or pre-sensitised in vivo to a particular peptide. These S 25 are unrestimulated T-cells, i.e. cells capable of immediate effector function without the need to effect division/differentiation by in vitro culture. When the peotide in question is presented to such cells, the cells secrete various cytokines, of which any one may be selected for the purposes of this assay. Preferably the cytokine selected is interferon-y (IFN-y).

-3- The secreted cytokine can be detected by any of a variety of methods known in the literature. Preferably the assay method involves providing a surface carrying an immobilised first antibody to the IFN-y or other cytokine. A fluid containing the PBMC or other fresh cells is placed in contact with that immobilised antibody. About 30% of the PBMC are CD8+ cells. In the PBMC of a patient who has recovered from a previous influenza virus infection, about 1 CD8+ cell in 10' 106 is a memory cell that has been pre-sensitised to a specific epitope associated with the influenza virus.

The peptide employed may be a known epitope for a well characterised viral infection; or may be a candidate epitope possibly associated with a less well characterised viral infection. The resulting fluid mixture is incubated under conditions to stimulate any peptide-specific T-cells that may have been pre-sensitised to that particular virus-derived peptide in vivo. The peptide needs to be of a length, e.g. 7 15 and particularly 8- 12 or 8 amino acid residues long, that is recognised by CD8+ cells. It is supposed that the generality of the CD8+ cells (and other PBMC) present the peptide to the small minority of CD8+ cells that may have been pre-sensitised to 20 the peptide. If such activated or pre-sensitised peptide-specific T-cells are present in the test fluid, they respond by secreting IFN-y or other cytokine which then becomes bound to the immobilised antibody.

It is preferred that the peptide be added in uncombined form to the fresh cells. While it is possible to add cultured cells that have been 25 pulsed with the peptide, this is not necessary when using defined peptide epitopes. The peptides should be added in an amount sufficient to generate an observable signal; a preferred concentration range in the fluid is 0.01 up to 100 pM particularly 0.5 5.0 gM.

Incubation should be continued for a time sufficient to permit CD8+ cells that have been pre-sensitised in vivo to the particular peptide -4chosen to secrete the IFN-y or other cytokine. The incubation should not continue for so long that quiescent CD8+ cells have time to differentiate and become activated by the peptide and start to secrete cytokines. This suggests an incubation time of 4 24 hours, more particularly 6 16 hours.

It is an advantage of the method that the incubation part of the test can be performed in a single working day or overnight, and without the use of sterile conditions required for cell culture in vitro.

During the incubation, any IFN-y or other cytokine secreted by CD8+ cells becomes bound to the first antibody immobilised on the surface. After incubation, the surface may be washed to remove unbound material. For detection, preferably a labelled second antibody to the cytokine is used. When this is applied to the surface it becomes bound to any cytokine present. The second antibody should preferably recognise a different epitope from the first antibody. One or both of the first and second antibodies should preferably be monoclonal. The label may be any that is conventionally used in the field, including radioisotopes, enzymes to generate colour or chemiluminescence, fluorescent groups or groups for detection by mass spectrometry or refractive index by surface plasmon resonance). It is convenient but not necessary to use a labelled antibody, any reagent that binds specifically to the cytokine could be labelled and used. Detection and perhaps quantitation of the label is effected by means well known in the field and appropriate to the nature of the label used.

The assay may conveniently be carried out in a multiwell 25 plate. Each well of the plate has a surface carrying a bound first antibody.

To each well is added a fluid containing an appropriate number, e.g.

S- 101 106 of cells. Different peptides and/or controls are added to individual wells of the plate. Cells that secrete a cytokine during incubation show up as spots (spot forming cells or SFCs).and the number or density of these in each well can readily be determined.

The assay technique has a number of advantages over prior known techniques:a) It is quicker and more convenient; the duration of the assay is only 6 hours and thus does not require sterile conditions or technique.

Current methods of enumerating precursor effector T cells require in vitro culture with the specific antigen and autologous feeder cells in a limiting dilution assay (LDA). The method is laborious and time consuming.

b) It requires minimal technical equipment and is suitable for field conditions in the tropics and developing countries as well as routine o diagnostic laboratories. The LDA, by contrast, requires many peripheral blood lymphocytes, a source of gamma irradiation to inactivate the feeder cells and sterile conditions, as the cells need to be cultured for 1-2 weeks.

c) It is safe and non-radioactive. In the LDA, however, the cultured cells are assayed in a cytotoxic T cell assay (CTL) assay using the radioactive isotope chromium-51.

d) It is an immediate ex vivo assay. As such it measures effector cells in their natural state without the introduction of unknown biases that occur as cells proliferate in vitro culture with antigen and exogenous cytokines.

20 e) The assay is performed over only 6 hours; as such it measures peptide-specific effector cells directly, without requiring these cells to proliferate in vitro. The short duration of the assay also eliminates the possibility that the cells may be becoming activated in vitro; it therefore measures effector function that is present in vivo. LDAs require the cells to proliferate many fold; however many effector cells do not proliferate in these conditions and therefore the result of the LDA is often an underestimate of the true number of circulating effectors.

The assay technique is expected to be of value in a number of different ways:i) For research into the mechanisms involved in peptide 6 presentation and recognition and activation. Through the experimental work described in outline below, the inventors have gained insight into the phenotype and effector function of antigen-specific T-cells freshly isolated from peripheral blood.

ii) For qualifying peptide-specific effectors in HIV-infected individuals direct from peripheral blood.

iii) For monitoring the progress of, or resistance to, a chronic infectious disease, for example in response to a drug or therapeutic vaccine. This is expected to be particularly useful for HIV, Hepatitis B and Hepatitis C.

iv) For identifying peptides that may be implicated in a disease state (Epitope mapping), an important preliminary step in the design of a new vaccine. This is expected to be of interest for tuberculosis, malaria and HIV.

v) For monitoring the extent to which a patient, who has suffered from a particular disease such as influenza, may be resistant to future infections.

vi) For monitoring induction and maintenance of CD8+ and CD4+ antigenspecific T cells following immunisation with experimental preventative vaccines, e.g. malaria.

Using the ELISPOT assay technique herein disclosed, it has been found that CD8+ CTLS have a protective role in M. tuberculosis infection in humans as 20 described in more details in the following example.

The invention therefore relates to the identification of CD8+ T Cell epitopes in the ESAT-6 protein of Mycobacterium tuberculosis and the use of peptides Scontaining such epitopes in the provision of vaccines.

In one aspect of the invention there is provided a peptide selected from AMASTEGNV and LQNLARTI.

In another aspect of the invention, there is provided a vaccine wherein the peptide selected from AMASTEGNV and LQNLARTI is used as a human CD8' T cell-activating agent.

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In a further aspect of the invention there is provided the use of a human CD8 T cell epitope of ESAT-6 of M. tuberculosis in the manufacture of a vaccine for administration to a human patient to activate a CD8' T cell response against M. tuberculosis infection, the CD8' T cell epitope being present in a peptide.

In yet a further aspect of the invention there is provided a method of activating a CD8 T cell response in a human patient against M. tuberculosis infection comprising administering to the patient a CD8 T cell epitope of ESAT-6 of M. tuberculosis, the CD8' T cell epitope being present in a peptide.

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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EXAMPLE

Identification of CD8 Epitopes in Secreted Antigens of M. Tuberculosis Mounting evidence points to a protective role for CD8+ cytotoxic T lymphocytes in Mycobacterium tuberculosis infection, but M. tuberculosis-specific CD8+ T cells have not hitherto been identified in man. Using a reverse immunogenetic approach, the inventors have synthesised an array of candidate HLA Class I restricted peptides from two immunodominant antigens of M. tuberculosis, ESAT-6 and antigen The inventors have screened 75 subjects representing a broad clinical ooooi b I -8spectrum of M. tuberculosis infection. Peripheral blood lymphocytes were stimulated in vitro with the peptides and then tested for cytotolytic activity in a 5 'Cr release assay and for single cell interferon-y release in an ELISPOT assay. The inventors have identified several octamer and nonamer epitopes from ESAT-6 and antigen 85 in patients and exposed contacts.

Certain epitopes are recognised by CD8+ lymphocytes in an MHC Class I restricted manner; others are recognised preferentially by CD4+ T cells.

The sequences of ESAT-6 and antigen 85A, B and C were scanned with allele-specific peptide motifs for the HLA class I types -A2, B7, -B8, -B35, -B52 and -B53 all of which were present in the study population.

For ESAT-6, sequences congruent with the peptide motifs for HLA-A2, -B8 and -B52 were identified; these peptides were synthesised and are displayed in Table 2. No sequences congruent with HLA-B7, and -B53 were present in ESAT-6 and thus no peptides were synthesised for these HLA class I alleles. Peptides were sorted into pools that were used for in vitro restimulation of donor PBMC. Peptides found to be CD8+ epitopes are shown in boldface. Similarly, 42 peptides were synthesised based on the sequences of antigens 85A and 85C. No CD8+ epitopes S: 20 were identified amongst these and the peptides are not shown.

Table 2 HLA class I allele Peptide motif Peptide Sequence Position HLA-A2 -L//M--VIL/I ES8 GIEAAASAI 10-18 ES9 AIQGNVTSI 17 LLDEGKQSL 28 36 ES11 ELNNALQNL 64-72 ES13 AMASTEGNV 82 HLA-B8 ES7 EGKQSLTKL 31 39 HLA-B52 -Q-IN ES12 LQNLARTI 69-76 ELISPOT assay for IFN-y.

96-well PVDF-backed plates pre-coated with the anti-IFN-y mAb 1-DIK at 15 pg/ml were washed with RPMI and blocked with R10 for 1-h at room temperature. In one experiment, 500,000 freshly isolated uncultured PBMC were used per well. In another experiment short term 10 cell lines (STCL) or CD8+ cytotoxic T lymphocytes (CTL) or clones were washed x 2 in RPMI, resuspended in R10, and dispersed at known cell input number/well in duplicate wells. Responses were considered significant if a minimum of 10 SFCs were present per well and additionally this number was at least twice that in control wells. Peptide was added 15 directly to the supernatant at a final concentration of 2 p.1 (free peptide).

SPlates were incubated for 12 hrs at 37°C, 5% CO,. After washing x 6 with phosphate buffered saline 0.05% Tween-20 to remove cells, plates were i incubated for 3 hrs with the second biotinylated anti-IFN-y mAb 7-B6-1e* biotin at 1 pg/ml. A further wash as above was followed by incubation with a 1:1000 dilution of streptavidin-alkaline phosphatase conjugate for 2 hrs.

After another wash, chromogenic alkaline phosphatase substrate was 1 11 added to the wells and 30 mins. later plates were washed with tap water.

After drying, spot forming cells (SFC) were counted under x magnification.

STCL were generated by the method described in Nature 346 (1990) 183-7. CD8+ T-cell clones were generated by standard methods.

Identification of ESAT-6 specific effector T cells direct from peripheral blood.

Two CD8+ epitopes in ESAT-6 were identified. The T cells from donor NPH54, who had tuberculous mediastinal lymphadenitis recognised peptides corresponding to both of these epitopes. Uncultured PBMC isolated at the time of diagnosis from NPH54, who has HLA-B52 and HLA-A2.01, secreted IFN-y in response to an ESAT-6-derived peptide pool for these class I alleles in an ex vivo ELISPOT assay. The mean number of IFN-y spot-forming cells (SFCs) enumerated from 5 x 105 PBMC in duplicate wells was 19 for the ESAT-6 peptides compared to 2 in the control wells with no peptide. A subsequent assay tested freshly isolated PBMC against each of the individual peptides within the responding pools; IFN-y SFCs were detected in response to peptides ES12 and ES13, whose S 20 sequences are congruent with the HLA-B52 and HLA-A2.01 peptide motifs respectively. The frequency of ES12- and ES13-specific IFN-y SFCs is of the same order of magnitude as SFCs for HLA-A2.01-restricted influenza matrix epitope M1 58-66. Unrestimulated PBMC from a second donor, *O *O NPH97, with tuberculous osteomyelitis, also recognised the ES12 peptide.

25 This patient also has HLA-B52 and -A2.01 and the magnitude of the ES12-specific response was similar to the response to the HLA-A2-restricted influenza matrix epitope. Single cell IFN-y release by freshly isolated T cells in these short 12 hr ex vivo assays, employing no stimulus other than cognate peptide, indicates that these cells are highly likely to be circulating activated effector T cells.

Claims (4)

1. Use of a human CD8' T cell epitope of ESAT-6 of M. tuberculosis in the manufacture of a vaccine for administration to a human patient to activate a CD8' T cell response against M. tuberculosis infection, said CD8 T cell epitope being present in a peptide.

2. A method of activating a CD8 T cell response in a human patient against M. tuberculosis infection comprising administering to the patient a CD8' T cell epitope of ESAT-6 of M. tuberculosis, said CD8' T cell epitope being present in a peptide.

3. A use or method as claimed in claim 1 or claim 2 wherein said peptide is selected from AMASTEGNV and LQNLARTI.

4. A peptide selected from AMASTEGNV and LQNLARTI. A vaccine wherein a peptide as claimed in claim 4 is provided as a human CD8' T cell-activating agent. DATED this 27th day of June 2003 ISIS INNOVATION LIMITED WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA 0 KJS/ALJ/MEH o o 5