WO2023152510A1 - Methods for assessing the status of the immune system - Google Patents

Methods for assessing the status of the immune system Download PDF

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Publication number
WO2023152510A1
WO2023152510A1 PCT/GB2023/050312 GB2023050312W WO2023152510A1 WO 2023152510 A1 WO2023152510 A1 WO 2023152510A1 GB 2023050312 W GB2023050312 W GB 2023050312W WO 2023152510 A1 WO2023152510 A1 WO 2023152510A1
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peptides
cells
cytokine
sample
antibody
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PCT/GB2023/050312
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French (fr)
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Tomasz Evan GEORGE
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Virax Biolabs (UK) Limited
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Publication of WO2023152510A1 publication Critical patent/WO2023152510A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the methods are useful for testing samples from subjects who may have been exposed to a virus; are concerned about being exposed to a virus; and may be used to determine if a subject has inherent protection from a virus.
  • Viral infection elicits a response in the immune system to fight the infection. This consists of a short term and longer term immune response.
  • T-cells reactive to a virus can be present in the blood long after recovery, whereas antibodies to the virus may only be present for months after disease recovery.
  • T-cells to the SARS virus have been found in survivors 17 years after the original infection and this kind of long-term protection may be expected for other viruses and including other coronaviruses such as SARS-CoV-2.
  • test tests that determine T cell responsiveness may be useful for determining inherent protection from viruses in a subject who has avoided, or at least believes they have avoided, viral infection.
  • Tests that determine T cell responsiveness are also useful for determining the degree of long-term protection a subject may have after recovering from a viral infection. Protection from re-infection or serious disease may be conferred by T cells which may be present long after antibodies have disappeared from the blood. Thus examining T-cell responses can be particularly useful for assessing the degree of long term protection from a viral threat.
  • Tests examining T-cell responsiveness to viruses are known in the art.
  • MyriadRBM market a trueculture ® test (for research use only) which examines T-Cell activation in response to peptides from the SARS-CoV-2 Spike protein.
  • MyriadRBM employs ELISA to measure cytokines produced by the T Cells.
  • Miltenyi market a SARS-CoV-2 T cell Analysis Kit that examines T-Cell activation in response to a mixture of peptides generated from across the SARS- CoV-2 genome (from their Peptivator® Peptide Pools) and employ flow cyometry to detect cytokine production from the activated T Cells.
  • a method of determining T cell activation in response to exposure to viral peptides comprising:
  • the methods according to the invention are useful for determining inherent protection from viruses in a subject who has avoided, or at least believes they have avoided, viral infection.
  • the methods are also useful for determining the degree of long-term protection a subject may have after recovering from a viral infection.
  • the methods are particularly useful for determining the degree of long-term protection present in a subject after recovery from COVID-19 or for assessing whether or not a subject has inherent protection from COVID-19 (e.g. following vaccination).
  • the assay used according to step (d) of the first aspect of the invention may be any assay that determines antigen specific (i.e. peptide induced) cytokine production by cells (e.g. peripheral blood cells).
  • Example of assays that may be used include ELISA and ELISPOT. It is most preferred that cells that are CD3 + and which express the at least one cytokine are detected by employing flow cytometry.
  • the use of ELISA, ELISPOT and particularly flow cytometry has the advantage that the methods of the invention may employ laboratory equipment that is easy to use and standard in both the developed and developing world. This increases the scope of the test beyond wealthy nations.
  • Peptides encoded by ORF 1 and ORF 9 of the SARS-CoV-2 genome are advantageously antigenic viral proteins and provide a way of stimulating T-cells to respond as if they have come in contact with the virus in question, but without having to use viral particles within a test.
  • the peptides also enable the methods of the invention to establish whether or not innate immunity to a specific viral threat or antigen is built up through: a) adaptive immunity after exposure to the virus or antigen in the past; b) adaptive immunity after exposure to a similar virus or antigen (providing a cross- reactive immune response to the virus or antigen in question); and c) adaptive immunity through vaccination to the virus in question or a similar virus.
  • the methods provide an indication as to whether or not the immune system of the subject can generate a T-cell response if infected with the specific virus or antigen in question, and also provides an indication as to how well the body would respond if infected in the future.
  • the methods of the invention may also give an indication as to what disease protection strategies are required for an individual who is concerned about catching a specific virus (vaccination, social distancing, mask wearing, isolation/shielding, and additional pharmaceutical or nutraceutical strategies to boost the immune function where required). This provides additional information about immune function for, in particular, the vaccine hesitant to influence their vaccination decisions.
  • Data generated by the methods of the invention may be used to help governments and healthcare groups prioritize vaccination more effectively by targeting those who are identified by the methods of the invention of having lower levels of adaptive immunity.
  • Data generated by the methods of the invention may be used to establish a database of T-cell responses in individuals which allows for the prediction of future immune responses based on individuals with a similar immune profile. For instance, if an individual has been infected with, and recovered from Sars-Cov-1 in the past, it may be possible to predict that they will effectively mount a T-cell response to Sars-Cov-2. Peptide Selection
  • the peptides may be derived from any virus (e.g. a torovirus) although it is preferred that the peptides are derived from Beta-Coronaviruses. It is more preferred that the peptides are derived from SARS-CoV, HCoV-OC43, HCov-HKUl, MERS-CoV or SARS-CoV-2. It is most preferred that the peptides are derived from SARS-CoV-2.
  • viruses e.g. a torovirus
  • Beta-Coronaviruses It is more preferred that the peptides are derived from SARS-CoV, HCoV-OC43, HCov-HKUl, MERS-CoV or SARS-CoV-2. It is most preferred that the peptides are derived from SARS-CoV-2.
  • Coronaviruses including SARS-CoV-2, has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins.
  • the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope.
  • the Spike protein is encoded by Open Reading Frame (ORF) 1 of the viral genome whereas the E, M and N proteins are encoded by ORF4, ORF5 and ORF9 respectively.
  • Peptide mixes used according to the invention should include peptides derived from ORF1 and ORF 9.
  • peptides are derived from regions of ORF1 and ORF 9 which the inventor has associated with (a) being conserved between Sars-Cov-2 variants and also other viruses (particularly coronaviruses); and (b) elicit T-cell and B-cell mediated immune responses after prior infection with Sars-Cov-2 and other related coronaviruses or with no prior Sars-Cov-2 infection.
  • ORF 1 region shows conservation between both coronaviruses and toroviruses. Peptides derived from ORF1 are therefore useful because they show cross reactivity to T-cells after infection with different viruses.
  • peptides used according to the invention are encoded by these conserved regions because infection with many different viruses, particularly different coronaviruses, can produce a T-cell response to the peptides contained within these regions. Subsequent infection with a different virus that also contains peptide sequences that are the same or similar, can elicit a rapid immune response that is mediated by memory T-cells that were produced to the initial infection.
  • Methods according to the invention may be used to identify T cells that are: (i) reactive due to previous exposure to Sars-Cov-2; (ii) which are reactive due to exposure to another virus (e.g. another coronavirus) which have the same or similar conserved regions; (iii) which are reactive following vaccination; or (iv) are primed/reactive for an unknown reason which may represent natural immunity.
  • another virus e.g. another coronavirus
  • the peptides include peptides encoded by ORF 1 or ORF 9 of the SARS- CoV-2 genome and also peptides encoded by ORF2 (encoding the Spike Protein).
  • Peptides used according to the invention may be of any size that is suitable for activating a T cell.
  • a peptide may be 5 - 50 amino acids in length, is preferably 5- 25 amino acids in length and more preferably 6 -18 amino acids in length.
  • a mixture of peptides or a peptide pool is used according to the invention.
  • This mixture may comprise between 10 and 100 different peptides (with at least one peptide from ORF 1 and ORF 9 and preferably at least one peptide from ORF2); more preferably the mixture comprises between 15 and 50 peptides (with at least one peptide from ORF 1 and ORF 9 and preferably at least one peptide from ORF2); and more preferably the mixture comprises between 20 and 40 peptides (with all of the peptides derived from ORF 1, ORF 9 and ORF2). It is most preferred that the mixture comprises about 30 peptides (e.g. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 peptides) and with all of the peptides derived from ORF 1, ORF 9 and ORF2.
  • a most preferred pool of peptides is listed in Table 1.
  • Peptide according to the invention activate T cells such that they start producing cytokines. It is preferred that the peptides induce T cells to produce at least one of Tumour Necrosis Factor Alpha (TNFa), Interleukin 2 (IL-2) and Interferon Gamma (IFN-y). It is more preferred that the T cells are induced to produce at least two of these cytokines and most preferred that the T cell produce all three.
  • peptides according to the invention are chosen with one or more positive antigenicity characteristics, as determined by the three assessment methods outlined below:
  • SAS A Solvent Accessible Surface Areas
  • EDTSurf Euclidean Distance Transform Surface
  • SVMTriP score predicts antigenic epitopes, surface regions of protein that are preferentially recognised by antibodies or T-cells for receptor binding.
  • Support Vector Machine SVM was used by combining the Tri-peptide similarity and Propensity scores (SVMTriP). SVMTriP scores of >0.90 were preferred for peptide selection.
  • Peptides may also be selected on the basis of Kolaskar and Tongaonkar Antigenicity Analysis. These analyses provides a useful tool to predict antigenic determinants on proteins by analysing the occurrence of hydrophobic residues Cys, Leu and Vai. Peptides from ORF 1 or ORF9 comprising hydrophobic residues Cys, Leu and Vai are preferred peptides fro use according to the invention.
  • peptides according to the invention include peptides derived from ORF 1 and ORF 9 of the SARS-CoV-2 genome and have the characteristics identified in at least one of (1) - (3) above; preferably two out of three of (1) - (3) above; and most preferably each of (1) - (3) above.
  • Samples used according to the invention should be derived from a mammal and preferably from a Human.
  • the sample may be any tissue sample that contains T cells.
  • the sample is blood or a fraction thereof (e.g. a buffy coat).
  • the sample is whole blood.
  • a suitable amount of blood should be used. Given that T cell numbers are relatively low in blood it is preferred that samples comprise at least one milliliter of blood.
  • Whole blood is preferably treated with heparin, according to conventional techniques, to prevent coagulation.
  • T cells in samples are preferably stimulated by adding a suitable amount of the peptides according to the first aspect of the invention and incubating for a suitable length of time.
  • Sufficient peptides should be added to the sample to stimulate cytokine production from activated T cells. It will be appreciated that individual laboratories may need to optimize the amount used and this will at least in part depend upon the specific peptides that are used according to the invention. By way of example, the inventor has found, when using the peptide mix specified in Table 1, that an end concentration of about 1 pg/mL of total peptides in a whole blood sample is suitable.
  • Stimulated samples are preferably incubated at 37°C and for 1-24 hours, preferably 1-12 hours, more preferably 2-8 hour and in one embodiment for about 6 hours.
  • the secretion of cytokines from a cell can be disadvantageous when employing certain assays according to the invention and particularly when flow cytometry is used.
  • a cytokine secretion inhibitor is included that retains a higher concentration of the cytokine in the T cells. It is therefore preferred that a secretion inhibitor is used during stimulation of the samples in order that the amount of detectable cytokine is maximized.
  • a preferred secretion inhibitor for use according to the invention is Brefaldin A. It is preferred that Brefaldin A is used at a concentration of about 1 pg/mL in a whole blood sample.
  • a sample is first incubated with the peptides for about an hour.
  • a sufficient amount of a secretion inhibitor is then added to the sample and the sample is incubated at 37°C and for a further 2-7 hour and in one embodiment for about a further 5 hours.
  • the T cells should be isolated from the sample.
  • red blood cells should be lysed and the white cells fixed and permeabilized.
  • fixatives may be used.
  • the fixative is formalin.
  • a preferred procedure for fixing cells is described in 2.5 of Example 2.
  • Cells may be permeabilized prior to staining using conventional methods.
  • cells may be permeabilized with Phosphate buffered saline containing 10% saponin (which may be added to a sample to 0.1% end concentration).
  • Phosphate buffered saline containing 10% saponin which may be added to a sample to 0.1% end concentration.
  • a preferred procedure for permeabilizing cells is described in 2.6 of Example 2.
  • Step (c) of the first aspect of the invention requires the fixed and permeabilized cells to be stained with a labelled anti-CD3 antibody and a labelled anti-cytokine antibody against at least one cytokine.
  • CD3 Cluster of Differentiation 3
  • CD3 is a protein complex and T cell co-receptor that is involved in activating both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) and the CD3( ⁇ chain to generate an activation signal in T lymphocytes. The TCR, CD3( ⁇ , and the other CD3 molecules together constitute the TCR complex.
  • the inventor has found that the labelled anti- CD3 antibodies represent an ideal way of identifying T cells in a sample.
  • the anti-CD3 antibody may be raised against antigens derived from the CD3y chain, CD36 chain, CD3s chain or TCR.
  • a preferred anti-CD3 antibody is raised against CD3s.
  • Antibodies are preferably monoclonal antibodies and a most preferred antibody is clone UCHT1 from ThermoFisher. It is preferred that a label is selected that is suitable for cell sorting by flow cytometry. It is preferred that the label for anti-CD3 antibodies is Fluorescein isothiocyanate (FITC). FITC may be conjugated to the antibody via a succinimidyl ester.
  • Anti-cytokine antibodies used according to the invention should be raised against at least one cytokine selected from TNFa, IL-2 and IFN-y.
  • the detection of cytokine positive T Cells is optimized by evaluation of at least two cytokines by using anti-cytokine antibodies raised against at least two cytokines selected from TNFa, IL-2 and IFN-y. It is most preferred that anti-cytokine antibodies used according to the invention are raised against each of TNFa, IL-2 and IFN-y.
  • the detection of multiple cytokines may be achieved by evaluation of all cytokines in the same fluorescence channel. This may be achieved by all cytokine antibodies being conjugated with the same fluorochrome. This strongly increases the sensitivity of the assay, since there are cells that produce one, two or all three of the cytokines. All these events may be evaluated as one population. The percentage of detected cells is significantly higher and the fluorescent intensity is significantly increased and this makes it possible for cells producing low levels of cytokine to be distinguished over the background.
  • the fluorochrome used to label the cytokine antibodies is phycoerythrin (PE).
  • PE is widely available and is useful for all commonly available flow cytometers. PE also advantageously has a high signal intensity.
  • the anti-cytokine antibodies may be the following antibodies conjugated with R-PE from Becton Dickinson (bdbiosciences.com):
  • the anti-CD3 antibody is labelled with a first fluorochrome; and the antibody or antibodies against the at least one cytokine is labelled with a different fluorochrome or fluorochromes. It is most preferred that all anti-cytokine antibodies are labelled with the same fluorochrome (e.g. PE)
  • the assay used according to step (d) of the first aspect of the invention may be any assay that determines antigen specific (i.e. peptide induced) cytokine production by cells (e.g peripheral blood cells).
  • assays include ELISA and ELISPOT.
  • cells that are CD3 + and which express the at least one cytokine are detected by flow cytometry.
  • Flow cytometry may be conducted according to standard protocols.
  • the anti-CD3 antibody is labelled with a first fluorochrome and detected in a first channel; and the antibody or antibodies against the at least one cytokine is labelled with a different fluorochrome or fluorochromes and detected in a second channel.
  • a skilled person will appreciate that data obtained when cells are stimulated with peptides according to step (a) of the method of the invention may be best interpreted in view of data generated using positive and/or negative controls instead of the peptides.
  • a negative control may involve a stimulation step which excludes the peptides (and may be useful to evaluate if any artificial T cell activation occurs e.g. because of the blood draw process).
  • a positive control may involve stimulating the sample with a polyclonal stimulus instead of the peptides according to the invention (this may be used to demonstrate the ability of T cells to produce cytokines).
  • PMA and/or lonomycin may be used as a positive control as described in the Example 2. Therefore, according to a preferred embodiment of the invention two or three samples may be taken from a subject and T cells reactivity assessed in view of positive and/or negative controls.
  • the inventor analyzed the genome of Sars-Cov-2 and realized that conserved regions of ORF1 and ORF9 may be useful regions of the genome from which peptides may be derived that will elicit a T cell response. Furthermore, peptides may be selected that may be advantageously used as described above.
  • ORF1 and ORF9 conserved regions of ORF1 and ORF9 and also the Spike protein (ORF2) by the following assessment methods:
  • SAS A Solvent Accessible Surface Areas
  • EDTSurf Euclidean Distance Transform Surface
  • SVMTriP score predicts antigenic epitopes, surface regions of protein that are preferentially recognised by antibodies or T-cells for receptor binding.
  • Support Vector Machine SVM was used by combining the Tri-peptide similarity and Propensity scores (SVMTriP). SVMTriP scores of >0.90 were preferred for peptide selection.
  • Peptides were also selected on the basis of Kolaskar and Tongaonkar Antigenicity Analysis. These analyses provides a useful tool to predict antigenic determinants on proteins by analysing the occurrence of hydrophobic residues Cys, Leu and Vai. Peptides from ORF 1 or ORF9 comprising hydrophobic residues Cys, Leu and Vai are preferred peptides for use according to the invention.
  • peptides were identified by analytical methods (1) - (3) as being antigenic coronavirus peptides which were from conserved regions of ORF 1, ORF 2 or ORF 9 and which would activate T-cells primed to such antigens.
  • the inventor chose the most antigenic for use as peptides according to the invention. It will be appreciated that the total number of peptides and the precise selection of peptides may be varied within the spirit of the invention. Table 1 provides details of a most preferred, but exemplary, mixture or pool of peptides that may be employed according to the invention.
  • Example 1 The peptides selected in Example 1 were employed in a method according to the invention as described below.
  • the 29 peptides identified in Table 1 were used as a peptide mixture in the test.
  • Peptides of SEQ ID No. 1- 29 were synthesized using standard peptide protocols; mixed in equal mass ratio; and lyophilized. After storage the peptides were reconstituted in dimethyl sulfoxide (DMSO) to get a concentrated stock. The stock solution was diluted with HBSS to a 22x concentrated work solution. Peptides are used in an end concentration of 1 pg/mL for each peptide.
  • DMSO dimethyl sulfoxide
  • Reagent A 1 :20 Dilute Reagent A 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent A with (57 x N) pL lx Reagent K thoroughly in a sterile tube.
  • Reagent B 1 :20 Dilute Reagent B 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent A with (57 x N) pL lx Reagent K thoroughly in a sterile tube
  • Reagent C 1 :20 Dilute Reagent C 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent C with (57 x N) pL lx Reagent K thoroughly in a sterile tube 2.3.4 Reagent E Secretion inhibitor (Two vials)
  • Reagent E 1 :20 Dilute Reagent E 1 :20 with lx Dilution buffer (Reagent K) by mixing (10 x N) pL Reagent E with (190 x N) pL lx Reagent K thoroughly in a sterile tube
  • Three Tubes are used per donor (negative control, positive control and peptide mix).
  • i. Label three 5 mL tubes per donor and add 1 mL Li-Heparin treated whole blood to each tube;
  • ii. Add 50pL diluted Reagent A to Tube 1 (negative control), add 50 pl diluted Reagent B to Tube 2 (positive control) and 50 pl diluted Reagent C to Tube 3 (peptide mix).
  • iii. Incubate for 1 hour at 37°C, 5% CO2.
  • iv. Add, after the initial hour of incubation, 50 pl diluted Reagent E to all tubes and gently mix total volume of each tube well v. Incubate for 5 hours at 37°C, 5% CO2.
  • FSC Forward Scatter
  • SSC Sideward Scatter
  • FSC and SSC are plotted linear and the fluorescence channels FL-1 and Fl-2 are plotted logarithmic.
  • v. Create gate 1 by selecting all lymphocytes based on FSC/SSC profile and exclude debris, background noise and other cell types.
  • vi. Make gate 1 active on the second dot plot and only show the events representing the lymphocytes.
  • Create gate 2 by selecting the T-cell marker (FL-1) positive events.
  • vii. Make gate 2 active on the third dot plot and only show the T-cell marker positive events. Only select the cytokine positive events with a quadrant to determine the percentage CD3+/cytokine+ T cells. The correct evaluation is done with the use of a quadrant and use the negative and positive control to set appropriate gating.
  • viii. The percentage of activated T-cells is expressed as percentage of the complete CD3 pos T-cell population.
  • Example 2 The protocols of Example 2 were employed to evaluate the status of T Cells in blood samples taken from healthy volunteers as described below.

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Abstract

The present invention concerns methods of determining T cell activation in response to exposure to viral peptides. The methods involve stimulating a sample containing T cells with peptides derivable from a virus; fixing and permeabilizing cells from the sample; staining the fixed and permeabilized cells with a labelled anti-CD3 antibody and a labelled anti-cytokine antibody against at least one cytokine; and conducting an assay to identify cells that are CD3 + and which express the at least one cytokine. The peptides should include peptides corresponding to peptides encoded by Open Reading Frame (ORF) 1 and Open Reading Frame (ORF) 9 of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome.

Description

METHODS FOR ASSESSING THE STATUS OF THE IMMUNE SYSTEM
Provided herein are methods for testing samples to assess the status of the immune system in a subject from which the sample has been taken. The methods are useful for testing samples from subjects who may have been exposed to a virus; are concerned about being exposed to a virus; and may be used to determine if a subject has inherent protection from a virus.
There is a need to develop tests that will give insight into the status of the immune system and how that status may impact upon the ability of a subject to be resistant to viral infection. This need is clear when the impact of the CO VID 19 pandemic is taken into account. Some individuals are asymptomatic when exposed to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov- 2) whereas, as of January 2022, over 5.5 million people have died from CO VID 19. A better understanding of a subject’s ability to “fight off’ viruses such as SARS-Cov-2 will enable individuals to make life-style choices and will be very useful in the clinical management of such an individual should they subsequently become infected or reinfected.
Viral infection elicits a response in the immune system to fight the infection. This consists of a short term and longer term immune response. After infection, T-cells reactive to a virus can be present in the blood long after recovery, whereas antibodies to the virus may only be present for months after disease recovery. For example, T-cells to the SARS virus have been found in survivors 17 years after the original infection and this kind of long-term protection may be expected for other viruses and including other coronaviruses such as SARS-CoV-2.
The inventor recognized that the development of tests that determine T cell responsiveness may be useful for determining inherent protection from viruses in a subject who has avoided, or at least believes they have avoided, viral infection. Tests that determine T cell responsiveness are also useful for determining the degree of long-term protection a subject may have after recovering from a viral infection. Protection from re-infection or serious disease may be conferred by T cells which may be present long after antibodies have disappeared from the blood. Thus examining T-cell responses can be particularly useful for assessing the degree of long term protection from a viral threat. Tests examining T-cell responsiveness to viruses are known in the art. For instance, MyriadRBM market a trueculture ® test (for research use only) which examines T-Cell activation in response to peptides from the SARS-CoV-2 Spike protein. MyriadRBM employs ELISA to measure cytokines produced by the T Cells. Furthermore, Miltenyi market a SARS-CoV-2 T cell Analysis Kit that examines T-Cell activation in response to a mixture of peptides generated from across the SARS- CoV-2 genome (from their Peptivator® Peptide Pools) and employ flow cyometry to detect cytokine production from the activated T Cells.
According to a first aspect of the present invention there is provided a method of determining T cell activation in response to exposure to viral peptides, comprising:
(a) Stimulating a sample containing T cells that has been obtained from a subject with peptides derivable from a virus;
(b) Fixing and permeabilizing cells from the sample;
(c) Staining the fixed and permeabilized cells with a labelled anti-CD3 antibody and a labelled anti-cytokine antibody or antibodies against at least one cytokine;
(d) Conducting an assay to identify cells that are CD3 + and which express the at least one cytokine; and wherein the peptides include peptides corresponding to peptides encoded by Open Reading Frame (ORF) 1 and Open Reading Frame (ORF) 9 of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome.
The methods according to the invention are useful for determining inherent protection from viruses in a subject who has avoided, or at least believes they have avoided, viral infection. The methods are also useful for determining the degree of long-term protection a subject may have after recovering from a viral infection. The methods are particularly useful for determining the degree of long-term protection present in a subject after recovery from COVID-19 or for assessing whether or not a subject has inherent protection from COVID-19 (e.g. following vaccination).
The assay used according to step (d) of the first aspect of the invention may be any assay that determines antigen specific (i.e. peptide induced) cytokine production by cells (e.g. peripheral blood cells). Example of assays that may be used include ELISA and ELISPOT. It is most preferred that cells that are CD3 + and which express the at least one cytokine are detected by employing flow cytometry. The use of ELISA, ELISPOT and particularly flow cytometry has the advantage that the methods of the invention may employ laboratory equipment that is easy to use and standard in both the developed and developing world. This increases the scope of the test beyond wealthy nations.
Peptides encoded by ORF 1 and ORF 9 of the SARS-CoV-2 genome are advantageously antigenic viral proteins and provide a way of stimulating T-cells to respond as if they have come in contact with the virus in question, but without having to use viral particles within a test. The peptides also enable the methods of the invention to establish whether or not innate immunity to a specific viral threat or antigen is built up through: a) adaptive immunity after exposure to the virus or antigen in the past; b) adaptive immunity after exposure to a similar virus or antigen (providing a cross- reactive immune response to the virus or antigen in question); and c) adaptive immunity through vaccination to the virus in question or a similar virus. The methods provide an indication as to whether or not the immune system of the subject can generate a T-cell response if infected with the specific virus or antigen in question, and also provides an indication as to how well the body would respond if infected in the future.
The methods of the invention may also give an indication as to what disease protection strategies are required for an individual who is concerned about catching a specific virus (vaccination, social distancing, mask wearing, isolation/shielding, and additional pharmaceutical or nutraceutical strategies to boost the immune function where required). This provides additional information about immune function for, in particular, the vaccine hesitant to influence their vaccination decisions.
Data generated by the methods of the invention may be used to help governments and healthcare groups prioritize vaccination more effectively by targeting those who are identified by the methods of the invention of having lower levels of adaptive immunity.
Data generated by the methods of the invention may be used to establish a database of T-cell responses in individuals which allows for the prediction of future immune responses based on individuals with a similar immune profile. For instance, if an individual has been infected with, and recovered from Sars-Cov-1 in the past, it may be possible to predict that they will effectively mount a T-cell response to Sars-Cov-2. Peptide Selection
The peptides may be derived from any virus (e.g. a torovirus) although it is preferred that the peptides are derived from Beta-Coronaviruses. It is more preferred that the peptides are derived from SARS-CoV, HCoV-OC43, HCov-HKUl, MERS-CoV or SARS-CoV-2. It is most preferred that the peptides are derived from SARS-CoV-2.
Coronaviruses, including SARS-CoV-2, has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The Spike protein is encoded by Open Reading Frame (ORF) 1 of the viral genome whereas the E, M and N proteins are encoded by ORF4, ORF5 and ORF9 respectively. Peptide mixes used according to the invention should include peptides derived from ORF1 and ORF 9.
It is more preferred that peptides are derived from regions of ORF1 and ORF 9 which the inventor has associated with (a) being conserved between Sars-Cov-2 variants and also other viruses (particularly coronaviruses); and (b) elicit T-cell and B-cell mediated immune responses after prior infection with Sars-Cov-2 and other related coronaviruses or with no prior Sars-Cov-2 infection.
The inventor has recognized that the ORF 1 region shows conservation between both coronaviruses and toroviruses. Peptides derived from ORF1 are therefore useful because they show cross reactivity to T-cells after infection with different viruses.
It is advantageous that peptides used according to the invention are encoded by these conserved regions because infection with many different viruses, particularly different coronaviruses, can produce a T-cell response to the peptides contained within these regions. Subsequent infection with a different virus that also contains peptide sequences that are the same or similar, can elicit a rapid immune response that is mediated by memory T-cells that were produced to the initial infection.
Methods according to the invention may be used to identify T cells that are: (i) reactive due to previous exposure to Sars-Cov-2; (ii) which are reactive due to exposure to another virus (e.g. another coronavirus) which have the same or similar conserved regions; (iii) which are reactive following vaccination; or (iv) are primed/reactive for an unknown reason which may represent natural immunity. It will be appreciated that knowledge of T cell activation in subjects for each of (i) - (iv) is extremely valuable for an individual, a clinician treating such an individual; and for making decisions for disease management for a population of subjects. For instance, the methods may be used to evaluate the length of time immunity will persist following vaccination and inform authorities when a booster vaccine would be appropriate.
In preferred embodiments the peptides include peptides encoded by ORF 1 or ORF 9 of the SARS- CoV-2 genome and also peptides encoded by ORF2 (encoding the Spike Protein).
Peptides used according to the invention may be of any size that is suitable for activating a T cell. Typically a peptide may be 5 - 50 amino acids in length, is preferably 5- 25 amino acids in length and more preferably 6 -18 amino acids in length.
It is most preferred that a mixture of peptides or a peptide pool is used according to the invention. This mixture may comprise between 10 and 100 different peptides (with at least one peptide from ORF 1 and ORF 9 and preferably at least one peptide from ORF2); more preferably the mixture comprises between 15 and 50 peptides (with at least one peptide from ORF 1 and ORF 9 and preferably at least one peptide from ORF2); and more preferably the mixture comprises between 20 and 40 peptides (with all of the peptides derived from ORF 1, ORF 9 and ORF2). It is most preferred that the mixture comprises about 30 peptides (e.g. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 peptides) and with all of the peptides derived from ORF 1, ORF 9 and ORF2. A most preferred pool of peptides is listed in Table 1.
Peptide according to the invention activate T cells such that they start producing cytokines. It is preferred that the peptides induce T cells to produce at least one of Tumour Necrosis Factor Alpha (TNFa), Interleukin 2 (IL-2) and Interferon Gamma (IFN-y). It is more preferred that the T cells are induced to produce at least two of these cytokines and most preferred that the T cell produce all three. In a preferred embodiment peptides according to the invention are chosen with one or more positive antigenicity characteristics, as determined by the three assessment methods outlined below:
(1) Solvent Accessible Surface Areas (SAS A (A2)) were assessed using the Euclidean Distance Transform Surface (EDTSurf) algorithm. This was used to predict the accessible area for potential protein docking. Peptides that had a SASA >900 A2 were favoured for selection. For a 15 aa peptide a SASA of > 900 A2 transforms into a solvent accessible surface area of > 20%. An amino acid with SASA < 20% is considered not solvent accessible according to the EDTSurf algorithm used in the peptide analysis. > 800 A2 was accepted for peptides where they scored highly on the SVMTriP and KT metrics.
(2) The SVMTriP score - predicts antigenic epitopes, surface regions of protein that are preferentially recognised by antibodies or T-cells for receptor binding. In this method, Support Vector Machine (SVM) was used by combining the Tri-peptide similarity and Propensity scores (SVMTriP). SVMTriP scores of >0.90 were preferred for peptide selection.
(3) Peptides may also be selected on the basis of Kolaskar and Tongaonkar Antigenicity Analysis. These analyses provides a useful tool to predict antigenic determinants on proteins by analysing the occurrence of hydrophobic residues Cys, Leu and Vai. Peptides from ORF 1 or ORF9 comprising hydrophobic residues Cys, Leu and Vai are preferred peptides fro use according to the invention.
It is preferred that peptides according to the invention include peptides derived from ORF 1 and ORF 9 of the SARS-CoV-2 genome and have the characteristics identified in at least one of (1) - (3) above; preferably two out of three of (1) - (3) above; and most preferably each of (1) - (3) above.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Sample Selection
Samples used according to the invention should be derived from a mammal and preferably from a Human.
The sample may be any tissue sample that contains T cells. Preferably the sample is blood or a fraction thereof (e.g. a buffy coat). Preferably the sample is whole blood. A suitable amount of blood should be used. Given that T cell numbers are relatively low in blood it is preferred that samples comprise at least one milliliter of blood. Whole blood is preferably treated with heparin, according to conventional techniques, to prevent coagulation.
Stimulation of Samples
T cells in samples are preferably stimulated by adding a suitable amount of the peptides according to the first aspect of the invention and incubating for a suitable length of time.
Sufficient peptides should be added to the sample to stimulate cytokine production from activated T cells. It will be appreciated that individual laboratories may need to optimize the amount used and this will at least in part depend upon the specific peptides that are used according to the invention. By way of example, the inventor has found, when using the peptide mix specified in Table 1, that an end concentration of about 1 pg/mL of total peptides in a whole blood sample is suitable.
Stimulated samples are preferably incubated at 37°C and for 1-24 hours, preferably 1-12 hours, more preferably 2-8 hour and in one embodiment for about 6 hours.
The secretion of cytokines from a cell can be disadvantageous when employing certain assays according to the invention and particularly when flow cytometry is used. When this is the case, it is preferred that a cytokine secretion inhibitor is included that retains a higher concentration of the cytokine in the T cells. It is therefore preferred that a secretion inhibitor is used during stimulation of the samples in order that the amount of detectable cytokine is maximized. A preferred secretion inhibitor for use according to the invention is Brefaldin A. It is preferred that Brefaldin A is used at a concentration of about 1 pg/mL in a whole blood sample.
In a preferred embodiment a sample is first incubated with the peptides for about an hour. A sufficient amount of a secretion inhibitor is then added to the sample and the sample is incubated at 37°C and for a further 2-7 hour and in one embodiment for about a further 5 hours.
A preferred procedure for stimulating the cells is described in 2.4 of Example 2. Fixing and permeabilization
After the stimulation of a sample the T cells should be isolated from the sample. When the sample is whole blood, red blood cells should be lysed and the white cells fixed and permeabilized.
Conventional fixatives may be used. In a preferred embodiment the fixative is formalin. A preferred procedure for fixing cells is described in 2.5 of Example 2.
Cells may be permeabilized prior to staining using conventional methods. In one embodiment cells may be permeabilized with Phosphate buffered saline containing 10% saponin (which may be added to a sample to 0.1% end concentration). A preferred procedure for permeabilizing cells is described in 2.6 of Example 2.
Staining
Step (c) of the first aspect of the invention requires the fixed and permeabilized cells to be stained with a labelled anti-CD3 antibody and a labelled anti-cytokine antibody against at least one cytokine.
Labelled anti-CD3 antibodies
Cluster of Differentiation 3 (CD3), is a protein complex and T cell co-receptor that is involved in activating both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) and the CD3(^ chain to generate an activation signal in T lymphocytes. The TCR, CD3(^, and the other CD3 molecules together constitute the TCR complex.
The inventor has found that the labelled anti- CD3 antibodies represent an ideal way of identifying T cells in a sample.
The anti-CD3 antibody may be raised against antigens derived from the CD3y chain, CD36 chain, CD3s chain or TCR. A preferred anti-CD3 antibody is raised against CD3s. Antibodies are preferably monoclonal antibodies and a most preferred antibody is clone UCHT1 from ThermoFisher. It is preferred that a label is selected that is suitable for cell sorting by flow cytometry. It is preferred that the label for anti-CD3 antibodies is Fluorescein isothiocyanate (FITC). FITC may be conjugated to the antibody via a succinimidyl ester.
Labelled anti-cytokine antibodies
Anti-cytokine antibodies used according to the invention, should be raised against at least one cytokine selected from TNFa, IL-2 and IFN-y.
It has been reported (e.g. Le Bert et al. (2020) Nature 584 (7821) p457-462) that flow cytometry may be used to detect (specific) antigen induced cytokine production by peripheral blood cells (PBMC). However, such reports concern the detection of single cytokines and extremely low percentages (<1%) of cytokine positive lymphocytes were reported. The signals also strongly vary in the amount of cytokine per cell (signal strength), which makes it difficult to evaluate the data. Furthermore, signals cannot always clearly be discriminated from unstimulated samples (background signal). It is therefore preferred that the detection of cytokine positive T Cells is optimized by evaluation of at least two cytokines by using anti-cytokine antibodies raised against at least two cytokines selected from TNFa, IL-2 and IFN-y. It is most preferred that anti-cytokine antibodies used according to the invention are raised against each of TNFa, IL-2 and IFN-y.
When employing flow cytometry, the detection of multiple cytokines may be achieved by evaluation of all cytokines in the same fluorescence channel. This may be achieved by all cytokine antibodies being conjugated with the same fluorochrome. This strongly increases the sensitivity of the assay, since there are cells that produce one, two or all three of the cytokines. All these events may be evaluated as one population. The percentage of detected cells is significantly higher and the fluorescent intensity is significantly increased and this makes it possible for cells producing low levels of cytokine to be distinguished over the background.
It is preferred that the fluorochrome used to label the cytokine antibodies is phycoerythrin (PE). PE is widely available and is useful for all commonly available flow cytometers. PE also advantageously has a high signal intensity.
It is preferred that monoclonal antibodies are used that are specific to individual cytokines. According to one embodiment of the invention the anti-cytokine antibodies may be the following antibodies conjugated with R-PE from Becton Dickinson (bdbiosciences.com):
PE Rat Anti-Human IL-2 (554566)
PE Mouse Anti-Human TNF alpha (554513)
PE Mouse Anti-Human IFN-y (554552)
According to another embodiment of the invention the anti-cytokine antibodies may be the following antibodies:
Mouse Anti-Human IL-2 (clone MQ1-17H12)
Mouse Anti-Human TNF alpha (clone B-C7)
Mouse Anti-Human IFN-y (clone B-Bl)
These antibodies are conjugated with R-PE by the applicant after delivery using standard conjugation protocols. After conjugation a size exclusion column (Sephadex) is used to separate free antibody and fluorochrome form the conjugate.
In a preferred embodiment the anti-CD3 antibody is labelled with a first fluorochrome; and the antibody or antibodies against the at least one cytokine is labelled with a different fluorochrome or fluorochromes. It is most preferred that all anti-cytokine antibodies are labelled with the same fluorochrome (e.g. PE)
Standard staining techniques are used to introduce the label into the fixed permeabilized cells. A preferred procedure for staining cells is described in 2.7 of Example 2.
The Assay
The assay used according to step (d) of the first aspect of the invention may be any assay that determines antigen specific (i.e. peptide induced) cytokine production by cells (e.g peripheral blood cells). Examples of assays that may be used include ELISA and ELISPOT.
It is most preferred that cells that are CD3 + and which express the at least one cytokine are detected by flow cytometry. Flow cytometry may be conducted according to standard protocols. In a preferred embodiment the anti-CD3 antibody is labelled with a first fluorochrome and detected in a first channel; and the antibody or antibodies against the at least one cytokine is labelled with a different fluorochrome or fluorochromes and detected in a second channel.
Selecting the cells on the basis of their size and scatter profile, followed by the selection of the CD3 positive cells, makes it possible to determine the percentage of COVID-19 reactive cells by looking at the cytokine positive cells.
The use of 3 antibodies within one channel and all attached to the same fluorophore provides a higher signal for the detection of activated cells. This increases the sensitivity of the test and allows detection of activated T cells at lower thresholds than known to the art.
Use of Controls
A skilled person will appreciate that data obtained when cells are stimulated with peptides according to step (a) of the method of the invention may be best interpreted in view of data generated using positive and/or negative controls instead of the peptides. A negative control may involve a stimulation step which excludes the peptides (and may be useful to evaluate if any artificial T cell activation occurs e.g. because of the blood draw process). A positive control may involve stimulating the sample with a polyclonal stimulus instead of the peptides according to the invention (this may be used to demonstrate the ability of T cells to produce cytokines). PMA and/or lonomycin may be used as a positive control as described in the Example 2. Therefore, according to a preferred embodiment of the invention two or three samples may be taken from a subject and T cells reactivity assessed in view of positive and/or negative controls.
EXAMPLE 1
The inventor analyzed the genome of Sars-Cov-2 and realized that conserved regions of ORF1 and ORF9 may be useful regions of the genome from which peptides may be derived that will elicit a T cell response. Furthermore, peptides may be selected that may be advantageously used as described above.
The inventor then further analyzed conserved regions of ORF1 and ORF9 and also the Spike protein (ORF2) by the following assessment methods:
(1) Solvent Accessible Surface Areas (SAS A (A2)) were assessed using the Euclidean Distance Transform Surface (EDTSurf) algorithm. This was used to predict the accessible area for potential protein docking. Peptides that had a SASA >900 A2 were favoured for selection. For a 15 aa peptide a SASA of > 900 A2 transforms into a solvent accessible surface area of > 20%. An amino acid with SASA < 20% is considered not solvent accessible according to the EDTSurf algorithm used in our peptide analysis. > 800 A2 was accepted for peptides where they scored highly on the SVMTriP and KT metrics.
(2) The SVMTriP score - predicts antigenic epitopes, surface regions of protein that are preferentially recognised by antibodies or T-cells for receptor binding. In this method, Support Vector Machine (SVM) was used by combining the Tri-peptide similarity and Propensity scores (SVMTriP). SVMTriP scores of >0.90 were preferred for peptide selection.
(3) Peptides were also selected on the basis of Kolaskar and Tongaonkar Antigenicity Analysis. These analyses provides a useful tool to predict antigenic determinants on proteins by analysing the occurrence of hydrophobic residues Cys, Leu and Vai. Peptides from ORF 1 or ORF9 comprising hydrophobic residues Cys, Leu and Vai are preferred peptides for use according to the invention.
197 peptides were identified by analytical methods (1) - (3) as being antigenic coronavirus peptides which were from conserved regions of ORF 1, ORF 2 or ORF 9 and which would activate T-cells primed to such antigens. The inventor chose the most antigenic for use as peptides according to the invention. It will be appreciated that the total number of peptides and the precise selection of peptides may be varied within the spirit of the invention. Table 1 provides details of a most preferred, but exemplary, mixture or pool of peptides that may be employed according to the invention.
Table 1:
Figure imgf000014_0001
Figure imgf000015_0001
EXAMPLE 2
The peptides selected in Example 1 were employed in a method according to the invention as described below.
2.1 Reagents Used
Table 2:
Figure imgf000015_0002
2.2 Peptide Mix
The 29 peptides identified in Table 1 were used as a peptide mixture in the test.
Peptides of SEQ ID No. 1- 29 were synthesized using standard peptide protocols; mixed in equal mass ratio; and lyophilized. After storage the peptides were reconstituted in dimethyl sulfoxide (DMSO) to get a concentrated stock. The stock solution was diluted with HBSS to a 22x concentrated work solution. Peptides are used in an end concentration of 1 pg/mL for each peptide.
2.3 Preparation of Reagents
2.3.1 Reagent A Negative control
Calculate the volume necessary for the number of donors (N) to be tested.
N x 60 pL = Z pL total volume
Dilute Reagent A 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent A with (57 x N) pL lx Reagent K thoroughly in a sterile tube.
2.3.2 Reagent B Positive control
Calculate the volume necessary for the number of donors (N) to be tested.
N x 60 pL = Z pL total volume
Dilute Reagent B 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent A with (57 x N) pL lx Reagent K thoroughly in a sterile tube
2.3.3 Reagent C Peptide mix
Add 100 pl Reagent D (DMSO) to the Reagent C vial, close vial and vortex thoroughly.
Calculate the volume necessary for the number of donors (N) to be tested.
N x 60 pL = Z pL total volume
Dilute Reagent C 1 :20 with lx Dilution buffer (Reagent K) by mixing (3 x N) pL Reagent C with (57 x N) pL lx Reagent K thoroughly in a sterile tube 2.3.4 Reagent E Secretion inhibitor (Two vials)
Calculate the volume necessary for the number of donors (N) to be tested.
N x 200 pL = Z pL total volume
Dilute Reagent E 1 :20 with lx Dilution buffer (Reagent K) by mixing (10 x N) pL Reagent E with (190 x N) pL lx Reagent K thoroughly in a sterile tube
2.3.5 Reagent F Lysing solution (lOx)
Dilute the lOx Reagent F 1 : 10 by mixing 1 part of the concentrated Reagent F with 9 parts sterile demineralized water. The final pH of this solution should be 7.40 +/- 0.05.
2.3.6 Reagent G Fixative
No sample preparation is needed
2.3.7 Reagent H & I Permeabilization solution
Dilute 1.5 mL Reagent H concentrate ten times with 13.5 mL sterile demineralized water for each donor tested (3 samples) and add 150 pl Reagent I (1 TOO).
2.3.8 Reagent J Antibody cocktail (Two vials)
No sample preparation is needed.
2.3.9 Reagent K Dilution buffer
Dilute 3.5 mL the lOx Dilution buffer (Reagent K) concentrate with 31.5 mL sterile demineralized water for each donor tested (3 samples) in a sterile tube.
2.4 Stimulation of Cells
Three Tubes are used per donor (negative control, positive control and peptide mix). i. Label three 5 mL tubes per donor and add 1 mL Li-Heparin treated whole blood to each tube; ii. Add 50pL diluted Reagent A to Tube 1 (negative control), add 50 pl diluted Reagent B to Tube 2 (positive control) and 50 pl diluted Reagent C to Tube 3 (peptide mix). Gently mix total volume of each tube well. iii. Incubate for 1 hour at 37°C, 5% CO2. iv. Add, after the initial hour of incubation, 50 pl diluted Reagent E to all tubes and gently mix total volume of each tube well v. Incubate for 5 hours at 37°C, 5% CO2. vi. Add 3 mL of lx lysing solution (Reagent F) to all samples and mix immediately. Make sure buffer is at room temperature. vii. Incubate 15 minutes at room temperature. Vortex every 2 minutes. viii. Centrifuge the cell suspension for 5 minutes at 300x g. ix. Remove supernatant, add 3 mL of lx Dilution buffer (Reagent K) and centrifuge for 5 minutes at 300x g. x. Remove supernatant and loosen pellet. ation of Cells i. Resuspend pellet in 500 pl cold fixative (2 to 8 °C, Reagent G), vortex and incubate for 10 minutes at room temperature. ii. Add 4 ml of lx Dilution buffer (Reagent K) and centrifuge at 300x g for 5 minutes. iii. Remove supernatant and loosen pellet. meabilization of cells i. Add 1.5 ml of the prepared permeabilization solution (Reagent H/I), mix well and centrifuge at 300x g for 5 minutes. ii. Remove the supernatant and resuspend the cells in 250 pl of prepared permeabilization solution (Reagent H/I). ining of intracellular antigens i. Add 50 pl of conjugated monoclonal antibody mix (Reagent J) to the tube and mix well by vortexing. ii. Incubate for 20 minutes at room temperature in the dark. iii. Add 1.5 ml of prepared permeabilization solution (Reagent H/I) and centrifuge at 300x g for 5 minutes. iv. Remove the supernatant and resuspend the cells in 1 mL of lx Dilution buffer (Reagent K). lysis by flow cytometry i. Calibrate flow cytometer. ii. Collect all cells from each tube with the flow cytometer. iii. Analyze all events of each measurement, but for convenience reasons only show 5000 events per dot plot. iv. Make three dot plots,
1. a Forward Scatter (FSC) vs. Sideward Scatter (SSC) dot plot; to select the lymphocyte population
2. a FITC (FL-1) vs. SSC dot plot; to select T-cells
3. a R-PE (FL-2) vs. SSC dot plot; to determine the cytokine positive T- cells.
FSC and SSC are plotted linear and the fluorescence channels FL-1 and Fl-2 are plotted logarithmic. v. Create gate 1 by selecting all lymphocytes based on FSC/SSC profile and exclude debris, background noise and other cell types. vi. Make gate 1 active on the second dot plot and only show the events representing the lymphocytes. Create gate 2 by selecting the T-cell marker (FL-1) positive events. vii. Make gate 2 active on the third dot plot and only show the T-cell marker positive events. Only select the cytokine positive events with a quadrant to determine the percentage CD3+/cytokine+ T cells. The correct evaluation is done with the use of a quadrant and use the negative and positive control to set appropriate gating. viii. The percentage of activated T-cells is expressed as percentage of the complete CD3pos T-cell population.
EXAMPLE 3
The protocols of Example 2 were employed to evaluate the status of T Cells in blood samples taken from healthy volunteers as described below.
3.1 Volunteer Selection
Inclusion Criteria:
• Healthy volunteers men or women
• 21 to 65 years old (inclusive) on the day of consent
• Able and willing to provide written informed consent and comply with study procedures
• Fluent in the language of the study site and able to read in this language
• Using a form of contraception with at least a 98% success rate when used correctly
Exclusion Criteria:
• Individuals younger than 21 years or older than 65 years of age
• pregnant women
• Have not had a COVID-19 vaccination or contracted COVID-19 in the last three months
• Individuals taking immune suppressive medication or receiving chemotherapy (cancer treatment)
• Individuals that took non-steroid anti-inflammatory drugs, like ibuprofen, diclofenac, etc. during the last 48 hours.
• Individuals with underlying blood disorders, like leukaemia
• Individuals known to have human anti mouse antibodies (HAMA response)
• Individuals receiving cytokine or anti-cytokine therapy
• Individuals receiving anti -thrombotic medication.
• Subjects who, in the opinion of the Investigator, are not suitable for enrolment for another reason
• No participation in a clinical trial in the last 2 months prior giving consent
The following information was collected:
• Gender
• Age
• Background (race)
• Date & volume of the blood draw
3.2 Sample Collection
From each subject one to three (maximum) 9 ml heparinised tubes of blood were collected. The blood was processed immediately or stored for a defined period as specified in Example 2.
This samples were anonymised using a unique identification number and only the clinical site was able to link this code to a volunteer’s name.

Claims

CLAIMS:
1. A method of determining T cell activation in response to exposure to viral peptides, comprising:
(a) Stimulating a sample containing T cells that has been obtained from a subject with peptides derivable from a virus;
(b) Fixing and permeabilizing cells from the sample;
(c) Staining the fixed and permeabilized cells with a labelled anti-CD3 antibody and a labelled anti-cytokine antibody against at least one cytokine;
(d) Conducting an assay to identify cells that are CD3 + and which express the at least one cytokine; and wherein the peptides include peptides corresponding to peptides encoded by Open Reading Frame (ORF) 1 and Open Reading Frame (ORF) 9 of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genome.
2. The method according to claim 1 wherein the peptides include peptides corresponding to peptides encoded by Open Reading Frame (ORF) 2.
3. The method according to claim 1 or 2 wherein the sample is a whole blood sample.
4. The method according to any one of claims 1-3 wherein the anti-cytokine antibody is against at least one cytokine selected from Tumour Necrosis Factor Alpha (TNFa), Interleukin 2 (IL-2) or Interferon Gamma (IFN-y).
5. The method according to claim 4 wherein anti-cytokine antibodies are used that are raised against each of TNFa, IL-2 and IFN-y.
6. The method according to any preceding claim wherein the anti-CD3 antibody is labelled with a first fluorochrome and the antibody or antibodies against the at least one cytokine is labelled with a second fluorochrome.
7. The method according to any preceding claim wherein the anti-CD3 antibody is labelled with Fluorescein isothiocyanate (FITC).
8. The method according to any preceding claim wherein the anti-cytokine antibody or antibodies is/are labelled with Phycoerythrin (PE).
9. The method according to any preceding claim wherein a cytokine secretion inhibitor is used during step (a).
10. The method according to claim 9 wherein the secretion inhibitor is Brefaldin A.
11. The method according to any preceding claim wherein step (b) the cells are fixed with formalin.
12. The method according to any preceding claim wherein step (d) is conducted by flow cytometry.
13. The method according to any preceding claim wherein T cell activation is determined by comparing data for the sample with data from a positive and/or negative control.
14. The method according to claim 13 wherein data for the positive control and/or negative control is obtained from processing fractions of the sample being tested.
15. The method according to claims 13 or 14 wherein a positive control sample is processed according to claim 1 except Phorbol Myristate Acetate (PMA) is added in step (a) instead of the peptides.
16. The method according to any one of claims 13 - 15 wherein a negative control sample is processed according to claim 1 except Dimethylsulphoxide (DMSO) is added in step (a) instead of the peptides.
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