Patent Application
20230393121
The present invention relates to a method of diagnosing and/or monitoring of virus infection and/or response to vaccination by generating a profile of a virus-specific T cell response that can (i) discriminate between virus infected and uninfected individuals, (ii) determine the effect of vaccination on T cell response, and (iii) determine the effect of viral variants on T cell response. More particularly, the described virus-specific T cell profiling is based on the detection of activated antigen-specific T lymphocytes responding to pools of selected short peptides from virus proteins. These peptide sequences have been selected for their immunogenicity. The profiling is typically performed using ELISPOT, but may also be performed using other techniques such as qPCR, more particularly direct qPCR. The present invention also includes kits for use in the methods of the invention.
The control and the long-term protection against viral infections require the coordinated activation of humoral (antibodies) and cellular (T cells) immunity. Viruses are intracellular pathogens, and CD8 T cells are necessary to recognize and lyse the infected cells. In addition, CD4 helper T cells are necessary to boost the maturation of antibody production.
Despite this important role in antiviral immunity, quantification of virus-specific T cells is technically complex in comparison to methods of antibody detection. As such virus-specific T cells are not routinely measured as a potential correlate of protection.
This is particularly problematic in the present COVID-19 pandemic, since measuring SARS-CoV-2-specific antibodies might not be sufficient to fully gauge the level of antiviral immunity induced by the infection.
The inventors and others have recently shown that SARS-CoV-2-specific T cells are present in 100% of COVID-19 convalescents (Grifoni A., et al., Cell Host Microbe 27(4): 671-680 (2020); Braun J., et al., 2020 medRxiv 1-12. doi:10.1101/2020.04.17.20061440; Dong T., et al., 2020 bioRxiv 1-36. doi:10.1101/2020.06.05.134551; Le Bert N., et al., Nature 2020; 584(7821): 457-62), while observations of SARS-CoV-2 infection with fading antibody titers have also been reported (Long Q.-X., et al., 2020 Nat Med 1-15. doi:10.1038/s41591-020-0897-1). A discrepancy between virus-specific T cell and antibody response was also reported in infections with the Middle East Respiratory Syndrome (MERS), where a quarter of MERS-infected patients showed only MERS-specific T cells (Zhao J., et al., 2017 Science Immunology 2, eaan5393. doi:10.1126/sciimmunol.aan5393).
However, quantification of SARS-CoV-2-specific T cells and, indeed, other virus-specific T cells in the general population is rarely performed since the methods of detection are complex and cumbersome and require very specific technical personnel and assays that require an initial step of separation of peripheral blood mononuclear cells (PBMC) from whole blood and experimental procedures (ELISPOT or Intracellular cytokine staining or qPCR) that necessitate specialized skills and equipment.
There is a need for a test that indicates a patient's level of virus-specific cellular immunity.
The present invention provides methods to quantify virus-specific T cell activation, which have several applications.
SARS-CoV-2 and HBV are exemplified herein.
In a first aspect the invention provides an in vitro method of discriminating past or currently virus-infected subjects from virus un-infected subjects, comprising:
In some embodiments the virus is an enveloped virus. The antigenic structural and non-structural proteins of these viruses are well-known. An example is the membrane (M), nucleoprotein (NP) and/or Spike (S) proteins of an enveloped virus such as a coronavirus. Other enveloped viruses include Hepatitis B virus (HBV), wherein the antigenic proteins are Polymerase (Pol), Envelope (E), Core (C) and X.
In some embodiments of any aspect of the invention, the virus is a coronavirus, such as SARS, MERS or SARS-CoV-2.
In some embodiments, the virus is SARS-CoV-2 or HBV or variant thereof.
In a second aspect the invention provides an in vitro method of determining whether a vaccinee or previously virus-infected subject has T cells whose activation may be reduced by a virus variant, such as a variant of concern (VOC), comprising:
In a third aspect the invention provides a method to quantify the presence of virus-specific T cells in a biological sample comprising or derived from blood, bronchoalveolar lavage (BAL fluid), nasal swabs, or nasopharyngeal aspirate from a subject, comprising;
It would be understood that whether a single peptide pool is sufficient for an assay method may depend on whether a subject has only been exposed to a part of the virus (e.g. spike protein vaccine) or a whole virus (virus-infected). Using a peptide pool specific against a single viral protein only informs the response against that particular protein. Therefore, to know if an individual was infected before using the PCR method, one would still need to assess the response against multiple proteins. This logic does not change with different readouts like PCR, ELISPOT or cytokine release assay.
In a fourth aspect the invention provides a method of treatment comprising administering, to a subject with T cells reactive to a majority of peptide pools derived from virus antigenic proteins, an effective amount of a virus inhibitor.
In some embodiments of the method of treatment, the peptide pools are selected from:
In some embodiments of the method of treatment, the peptide pools are selected from:
In a fifth aspect the invention provides a method of prophylaxis comprising administering, to a subject with T cells reactive to a minority of peptide pools derived from virus antigenic proteins, an effective amount of a virus vaccine.
In a sixth aspect the invention provides a method of monitoring the efficacy of a virus vaccine, comprising testing whether the recipient of said vaccine has T cells reactive to a minority or majority of peptide pools derived from said virus antigenic proteins.
In a seventh aspect the invention provides a kit to discriminate past or currently virus-infected subjects from virus un-infected subjects, the kit comprising a plurality of virus antigenic peptides that stimulate virus-exposed T cells, wherein the virus antigenic peptides are in peptide pools derived from:
In some embodiments,
In some embodiments the kit can quantify virus-specific T cell activation in an isolated patient sample, comprising one or more peptide pools, wherein said peptide pools are separately derived from virus antigenic proteins, such as membrane (M), nucleoprotein (NP) and/or Spike (S) proteins; or Core (C), Polymerase (Pol), X and/or Envelope (E) proteins.
In some embodiments, the kit further comprises:
In an eighth aspect the invention provides a set of 2 to 4 separate pools of peptides suitable to discriminate;
In a ninth aspect the invention provides use of a kit of any one of aspects 7 to 9 in a method according to any one of aspects 1 to 6.
Certain terms employed in the specification, examples and appended claims are collected here for convenience.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
As used herein, the term ‘majority’ refers to a value over 50%. Conversely, the term ‘minority’ refers to a value under 50%. For example, if 3 or 4 of a total of 4 peptide pools positively activate T cells in a sample, compared to a control, a majority of pools are positive and the sample is indicative of the subject having been exposed to SARS-CoV-2 infection. When 4 pools M, NP1, NP2 and S were used, 50% or more (2, 3, or 4 pools out of 4) positive pools was considered to indicate the subject had been infected by SARS-CoV-2.
As used herein, the term ‘a subject uninfected by SARS-CoV-2’ refers to a subject who is considered to not have been exposed to and infected by SARS-CoV-2, for the purpose of the invention.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base, or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.
Bibliographic references mentioned in the present specification are for convenience listed at the end of the examples. The whole content of such bibliographic references are herein incorporated by reference.
The present invention relates to a method of discriminating past or currently virus-infected subjects from virus un-infected subjects, based on the detection of activated antigen-specific T lymphocytes responding to selected peptide sequences from the virus in an isolated sample from said individual. These peptide sequences are selected for their immunogenicity and are represented in peptide pools separately derived from virus antigenic proteins, such as membrane (M), nucleoprotein (NP) and/or Spike (S) proteins; or Polymerase (Pol), Envelope (E), Core (C) and X proteins.
In addition, the present invention provides a method of testing T cell responses in vaccinated subjects. In this way, the efficacy of a vaccine to stimulate a T cell response can be determined.
Further, the invention provides a method of testing the impact of amino acid mutations in a virus strain on T cells that have previously been exposed to a parent or comparator strain. For example, different variants of concern (VOC) of SARS-CoV-2 have replaced world-wide the original SARS-CoV-2 Wuhan isolate. These VOCs are characterized by amino acid substitutions that provide biological advantages such as increased infectivity or escape humoral (Antibodies) but also cellular (T cells) immunity. We have designed a method based on specific combination of peptide pools covering both different SARS-CoV-2 proteins (i.e., Spike) and the corresponding regions affected by the amino acid mutations that are used to stimulate T cells, The results of this multiple stimulation strategy define with accuracy the impact of these amino acid mutations on the SARS-CoV-2-specific T cells induced by infection with Wuhan strain or by vaccination with the present vaccines based on the Wuhan strain. Presented herein as an example is a method to analyze the impact of amino acid mutations present in Spike in different VOCs (using Delta variant as an example) on the SPIKE specific T cells induced by vaccination or previous infection.
Discrimination can be achieved based on the number or proportion of peptide pools that stimulate the isolated T cells above a control value.
Each of the M, NP and S, or Pol, C, E and X, proteins may contribute at least one pool of immunogenic peptides. For example, the NP protein consists of 419 amino acids. It is possible that the NP protein could be divided into more than one pool comprising 15-mer peptides, such as 2 pools where 1 pool comprises 15-mer peptides which overlap adjacent peptides by 10 amino acids covering amino acids 1-215; and a second pool comprising 15-mers which overlap adjacent peptides by 10 amino acids covering amino acids 216-419. The peptide overlap can be seen, for example, in the NP peptides listed sequentially in Table 2. It would be understood that the degree of overlap between 15-mer peptides could be varied from 10 without substantially affecting the ability to activate T cells and obtain a valid result. Likewise, the S protein, which is 1273 amino acids in length, could be divided up into 1, 2, 3 or more pools of 15-mer peptides. The number and size of the pools needs to be balanced with practical considerations, such as the amount of blood sample available, the cost of generating peptide pools, and whether the number of pools improves discrimination.
In a first aspect the invention provides an in vitro method of discriminating past or currently virus-infected subjects from virus un-infected subjects, comprising:
In some embodiments the virus is an enveloped virus or a non-enveloped virus. The antigenic structural and non-structural proteins of these viruses are well-known. An example is the membrane (M), nucleoprotein (NP) and/or Spike (S) proteins of an enveloped viruses such as coronaviruses, whereas and Hepatitis B viruses (HBV) comprise polymerase (Pol), envelope (E), core (C) and X antigenic structural and non-structural proteins.
In some embodiments of any aspect of the invention, the virus is a coronavirus.
In some embodiments, the virus is a coronavirus, selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, HKU1, OC43, NL63 and 229E or variant thereof.
In some embodiments, the virus is SARS-CoV-2 or variant thereof.
In some embodiments, the virus is HBV or variant thereof.
In some embodiments,
In some embodiments;
In some embodiments, the NP peptide pool is divided into 2 pools, NP1 and NP2.
In some embodiments, the S peptide pool comprises or consists of at least one peptide selected from peptides having the amino acid sequences set forth in SEQ ID Nos: 126-180 (Table 4).
In some embodiments NP1 comprises or consists of at least one peptide selected from peptides having the amino acid sequences set forth in SEQ ID Nos: 44-84, and the NP2 peptide pool comprises or consists of at least one peptide selected from peptides having the amino acid sequences set forth in SEQ ID Nos: 85-125.
In some embodiments;
In some embodiments;
In some embodiments, the Pol peptide pool is divided into a plurality of pools, such as 2, 3 or 4 pools of peptides. Preferably, each of the plurality of Pol pools has approximately equal numbers of peptides. In some embodiments, the Pol peptide pool is divided into 4 pools, Pol-1, Pol-2, Pol-3 and Pol-4. An example is shown in Tables 24-27.
In some embodiments, the E peptide pool is divided into 2 pools, E-1 and E-2. An example is shown in Tables 22-23.
In some embodiments,
In some embodiments;
In some embodiments, the method comprises the steps of:
In some embodiments, the method comprises the steps of:
In some embodiments, if the sample T cells are reactive to 50% or more of the peptide pools derived from SARS-CoV-2 M, NP and S, in comparison to unstimulated or DMSO-treated cells, the subject is identified as past or currently infected by virus.
In a second aspect the invention provides an in vitro method of determining whether a vaccinee or previously virus-infected subject has T cells whose activation may be reduced by a virus variant, such as a variant of concern (VOC), comprising:
In some embodiments, the virus is a coronavirus, selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, KHU1, OC43, NL63 and 229E or variants thereof.
In some embodiments, the virus antigenic protein is an M, NP, or S protein.
In some embodiments, the virus is HBV and the virus antigenic protein is an E, Pol, C or X protein.
In some embodiments, peptide pool A and pool B are derived from a wild-type virus.
It would be understood that peptide pool A and pool B could be derived from a variant if it became a reference point due to becoming endemic or if future vaccines employ the variant sequence instead of the original wildtype virus.
In some embodiments of the method;
In some embodiments, the wildtype virus is SARS-CoV-2 wildtype and the variant is selected from the group comprising B.1.617.2 (Delta), B.1.1.7 (Alpha V1), B.1.351 (Beta V2), P.1 (Gamma, V3), B.1.617.1 (Kappa), P.2, B.1.427/9 (Epsilon), B.1.525 (Eta), B.1.526 (Iota), C.37 (Lambda), B.1.621 and B.1.620; or the virus is HBV C.
In some embodiments, the method comprises the steps of:
In some embodiments, the secreted cytokine is selected from the group comprising IFN-gamma (IFN-γ), IL-2, CXCL9, CXCL10, TNF-alpha, IL-6, IL-10 and IL-1. Preferably IFN-gamma, IL-2, or CXCL10 levels are measured. Detection of the cytokine CXCL10 is preferred if qPCR is used to quantify T cell activation.
In some embodiments, the said cytokine level is determined by immunoassay, such as ELISA or ELISPOT, or by qPCR or direct qPCR.
According to the method, a sample is tested by separately mixing aliquots from the sample with a peptide pool representing at least a portion of M, NP or S protein, or with a peptide pool representing at least a portion of E, Pol, C or X protein to determine whether the sample comprises T cells reactive to M, NP and/or S peptides; or E, Pol, C and/or X peptides, respectively.
In some embodiments, the sample comprises whole blood, broncholavage (BAL fluid), nasal swabs, nasopharyngeal aspirate, or isolated peripheral blood mononuclear cells (PBMCs).
In some embodiments, when whole blood, broncholavage (BAL fluid), nasal swabs, or nasopharyngeal aspirate is used, the incubation period in step b) may be for between about 6 to 24 h.
In some embodiments of the method:
In some embodiments, the sample also comprises a concentration of DMSO and/or heparin. Heparin may be required particularly if whole blood is to be assayed, to inhibit coagulation.
In some embodiments, the control sample or threshold control value may be derived from an assay sample comprising a subject sample that is unstimulated or DMSO-treated.
In some embodiments of the method:
In a third aspect the invention provides a method to quantify the presence of virus-specific T cells in a biological sample comprising or derived from blood, broncholavage (BAL fluid), nasal swabs, or nasopharyngeal aspirate from a subject, comprising;
In some embodiments the virus is an enveloped virus. The antigenic structural and non-structural proteins of these viruses are well-known. An example is the membrane (M), nucleoprotein (NP) and/or Spike (S) proteins of a coronavirus. The antigenic proteins of Hepatitis B virus (HBV) are Pol, E, C and X proteins.
In some embodiments, the virus is a coronavirus, selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, KHU1, OC43, NL63 and 229E or variant thereof; or a non-enveloped virus, such as HBV.
In some embodiments, the virus is SARS-CoV-2 or HBV.
In some embodiments:
In some embodiments, the peptide pools comprise one or more M, NP and S peptides listed in Tables 1-4 and 7-19; or one or more Pol, E, C and X peptides listed in Tables 20-27.
In a fourth aspect, the invention provides a method of treatment comprising administering, to a subject with T cells reactive to:
In some embodiments, the virus is a coronavirus such as a coronavirus selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, HKU1, OC43, NL63 and 229E or variants thereof.
In some embodiments, the virus is SARS-CoV-2.
In some embodiments the virus is HBV.
In some embodiments, the invention provides a method of treatment comprising administering, to a subject with T cells reactive to 3 or 4 of the peptide pools M, NP1, NP2 and S listed in Tables 1-4, an effective amount of a SARS-CoV-2 inhibitor.
In some embodiments, the invention provides a method of treatment comprising administering, to a subject with T cells reactive to 3 or 4 of the peptide pools M, NP1, NP2 and S listed in Tables 1-4, an effective amount of a SARS-CoV-2 inhibitor.
In some embodiments, the invention provides a method of treatment comprising administering, to a subject with T cells reactive to 5 to 8 of the pools E1, E2, Pol1, Pol2, Pol3, Pol4, C and X listed in Tables 20-27, an effective amount of a HBV inhibitor.
In a fifth aspect, the invention provides a method of prophylaxis comprising administering, to a subject with T cells reactive to 0, or a minority of peptide pools derived from virus antigenic structural and non-structural proteins, an effective amount of a virus vaccine.
In some embodiments the virus is an enveloped virus.
In some embodiments, the virus is a coronavirus such as a coronavirus selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, HKU1, OC43, NL63 and 229E or variants thereof.
In some embodiments, the virus is SARS-CoV-2. In some embodiments the virus is HBV.
In some embodiments, the invention provides a method of prophylaxis comprising administering, to a subject with T cells reactive to 0, 1 or 2 of the peptide pools M, NP1, NP2 and S listed in Tables 1-4, an effective amount of a SARS-CoV-2 vaccine.
In some embodiments, the invention provides a method of prophylaxis comprising administering, to a subject with T cells reactive to 0, 1, 2, 3 or 4 of the peptide pools E1, E2, Pol1, Po12, Po13, Po14, C and X listed in Tables 20-27, an effective amount of a HBV vaccine.
In a sixth aspect the invention provides a method of monitoring the efficacy of a virus vaccine, comprising testing whether the recipient of said vaccine has T cells reactive to a minority, 50%, or majority of peptide pools derived from virus antigenic structural and non-structural proteins, such as virus M, NP and S proteins; or virus E, Pol, C and X proteins.
In some embodiments, the virus is a coronavirus such as a coronavirus selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, HKU1, OC43, NL63 and 229E or variants thereof. In some embodiments, the virus is SARS-CoV-2.
In some embodiments the virus is HBV.
In some embodiments, the invention provides a method of monitoring the efficacy of a SARS-CoV-2 vaccine, comprising testing whether the recipient of said vaccine has T cells reactive to 0, 1, 2, 3 or 4 of the peptide pools M, NP1, NP2 and S listed in Tables 1-4 and 7-19.
In some embodiments, the invention provides a method of monitoring the efficacy of a HBV vaccine, comprising testing whether the recipient of said vaccine has T cells reactive to 0, 1, 2, 3, 4, 5, 6, 7 or 8 of the peptide pools E1, E2, Pol1, Pol2, Pol3, Pol4, C and X listed in Tables 20-27.
It would be understood that the pools used may depend on which of the virus antigenic proteins is/are used in the vaccine. The pool may be for a particular protein from a wildtype, or variant virus. Table 4 contains selected peptides of the spike protein that were tested and demonstrated to be good enough to estimate the total spike T cell response. This table of peptides do not cover the entire spike protein. This is used in conjunction with peptides from Tables 1-3 to detect if the subject is infected or not infected. Table 7 is the reference peptides that may be used to assess the T cell response against the delta VOC with the wildtype Wuhan as a reference. Tables 8-19 contain peptides derived from the VOC that are different from the Wuhan wildtype SARS-CoV-2 virus.
In a seventh aspect the invention provides a kit to discriminate past or currently virus-infected subjects from virus un-infected subjects, the kit comprising a plurality of virus structural or non-structural peptides that stimulate virus-exposed T cells, wherein the virus peptides are in peptide pools derived from virus antigenic structural or non-structural proteins.
In some embodiments the virus is an enveloped virus or a non-enveloped virus.
An example of the antigenic proteins is the membrane (M), nucleoprotein (NP) and/or Spike (S) proteins of an enveloped virus.
In some embodiments, the virus is a coronavirus such as a coronavirus selected from the group comprising MERS-CoV, SARS-CoV, SARS-CoV-2, HKU1, OC43, NL63 and 229E or variants thereof.
In some embodiments, the virus is SARS-CoV-2. In some embodiments the virus is HBV.
In some embodiments of the kit:
In some embodiments:
In some embodiments the kit further comprises one or more reagents to detect cytokines and/or chemokines secreted from activated T cells.
In some embodiments the kit further comprises:
In some embodiments, suitable primers and probes are as shown in Table 5.
In some embodiments, the kit comprises one or more peptide pools selected from the pools in Tables 7-19 rather than the pools in Tables 1-4. Such pools could be used to analyse the effect of virus variants, including variants of concern (VOC), on T cell activation in vaccinated or previously infected subjects.
In some embodiments the VOC are selected from the group comprising B.1.617.2 (Delta), B.1.1.7 (Alpha V1), B.1.351 (Beta V2), P.1 (Gamma, V3), B.1.617.1 (Kappa), P.2, B.1.427/9 (Epsilon), B.1.525 (Eta), B.1.526 (Iota), C.37 (Lambda), B.1.621 and B.1.620.
Herein is presented a method based on specific combination of peptide pools covering both different SARS-CoV-2 proteins (i.e., Spike) and the corresponding regions affected by the amino acid mutations that are used to stimulate T cells, The results of this multiple stimulation strategy define with accuracy the impact of these amino acid mutations on the SARS-CoV-2-specific T cells induced by infection with Wuhan strain or by vaccination with the present vaccines based on the Wuhan strain. Peptide pools directed to non-conserved regions of the Wuhan strain variants are shown in Tables 8 to 19, while the sequences of the regions of the Wuhan strain that correspond to the regions of the Delta variant (B.1.617.2) are shown in Table 7.
Presented herein is an example of a method to analyze the impact of AA mutations present in the Spike protein in different VOCs (using Delta variant as an example) on the SPIKE specific T cells induced by vaccination. It would be understood that pools derived from non-conserved regions of M or NP may be used to analyse the effect of VOCs, depending on the antigens the subject's T cells have been exposed to.
In an eighth aspect the invention provides a set of at least 2, at least 3, or at least 4 separate pools of peptides suitable to discriminate:
In a ninth aspect the invention provides a use of a kit of aspect 7 in a method according to any one of aspects 1 to 6.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.
Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).
An integral part of our invention is the selection of peptide pools necessary to define a profile of T cell responses that can differentiate individuals that have been primed by SARS-CoV-2 infection or individuals that were infected by other common cold coronaviruses. The SARS-CoV-2 specific T cell responses were profiled in individuals who were in contact with SARS-CoV-2 and tested antibody positive (Abbot test/anti-NP antibody) or were positive in a surrogate virus neutralization assay (sVNT) at the time of the T cell test (symptomatic n=35; asymptomatic n=73), and compared the profile to that obtained from healthy donors (n=51) without any history of SARS-CoV-2 contact and negative for anti-NP SARS-CoV-2 antibodies.
Four SARS-CoV-2 peptide pools of 15-mers (Tables 1-4) covering NP (NP-1, NP-2), membrane (M), and one pool of 55 peptides covering the most immunogenic regions of Spike (S)(
Whole blood samples (8 ml) were collected from individuals who were in contact with SARS-CoV-2 and tested antibody positive (Abbot test/anti-NP antibody), or were positive in a surrogate virus neutralization assay (sVNT) at the time of the T cell test (symptomatic n=35; asymptomatic n=73), or were healthy donors (n=51) without any history of SARS-CoV-2 contact and negative for anti-NP SARS-CoV-2 antibodies.
Schematic representations of suitable assays of the invention are presented in
SARS-CoV-2-specific T cells were tested as described previously [Le Bert N, et al., Nature 584(7821): 457-62 (2020)]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from 8 ml whole blood by density-gradient centrifugation using Ficoll-Paque™ (Sigma-Aldrich). Isolated PBMC were either studied directly or cryopreserved and stored in liquid nitrogen until used in the assays.
ELISpot plates (Millipore) were coated with human IFNγ antibody (1-D1K, Mabtech; 5 μg/ml) overnight at 4° C. Then, 4×105 PBMCs were seeded per well and stimulated for 18 h with the different pools of SARS-CoV-2 peptides (2 μg/ml final concentration per peptide) described in Tables 1-4. For stimulation with peptide matrix pools or single peptides, a concentration of 5 μg/ml was used. Subsequently, the plates were developed with human biotinylated IFNγ detection antibody (7-B6-1, Mabtech; 1:2,000), followed by incubation with streptavidin-AP (Mabtech) and KPL BCIP/NBT Phosphatase Substrate (SeraCare). Spot forming units (SFU) were quantified with ImmunoSpot. To quantify positive peptide-specific responses, the highest number of spots of the unstimulated (or DMSO stimulated) wells was subtracted from the peptide-stimulated wells, and the results expressed as SFU/106 PBMCs. We excluded the results if negative control wells had >30 SFU/106 PBMCs or positive control wells (phorbol 12-myristate 13-acetate/ionomycin) were negative.
The ELISPOT assays showed that patients who have been infected by SARS-CoV-2 and cleared the virus up to 3 months ago have T cells that are reactive to peptide pools covering Membrane, Nucleoprotein and Spike (
Whole blood was isolated from each subject and tested within 24 hours after blood draw. 400 μl aliquots were separately mixed with 100 μl RPMI containing each of the SARS-CoV-2 peptide pools (2 μg/ml final concentration per peptide) or a DMSO control and incubated for a period extending overnight, to allow activation of responsive T cells. A plasma fraction was isolated from each of the incubated samples (about 30 minutes) and the level of cytokines in the sample measured using an EIIa™ multi-analyte ELISA machine (ProteinSimple, CA, USA).
It would be understood that the parameters described in the example may be changed and still achieve a valid assay. For example, the ratio of blood to RMPI media can range from 100% blood to 50% blood; 100 μl aliquots or more than 400 μl might be used, but a larger blood sample would be required to test multiple peptide pools. RPMI may be exchanged with other cell culture media. The final concentration of peptides in an assay mixture may be from 1 to 5 μg/ml. The control sample may contain DMSO or may be an unstimulated blood sample. The incubation period may range between about 6-24 h.
Having showed that the distinct peptide pools are able to define individuals that were recently in contact with SARS-CoV-2 (
SARS-CoV-2 reactive T cells in infected individuals can be detected by quantifying the amount of secreted IFN-γ after the direct addition of the peptide pools into whole blood (
qPCR and dqPCR Testing of PBMCs
The complexity of quantifying the presence of virus-specific T cell has so far prevented large scale studies of the cellular immune response to viral infection or, more recently, the vaccine. To address this problem, we have implemented a qPCR-based rapid T cell Activation (qTACT) assay, based on in vitro stimulation of whole blood samples with a pool of viral peptides covering the spike or other SARS-CoV-2 viral proteins (i.e., nucleoprotein [N]) [Le Bert, N., et al. Nature 584: 457-462 (2020); Kalimuddin S, et al., Med (N Y). 2021 Jun. 11; 2(6):682-688.e4. doi: 10.1016/j.medj.2021.04.003; Le Bert, N. et al., J Exp Med 218(5):e20202617 (2021)], followed by direct amplification of IFN-γ or IL-2 (directly produced by SARS-CoV-2 antigen-specific T cells) or CXCL10, a molecule expressed by monocytes in response to T cell activation. A further technical implementation of the assay allows quantification of T cell immunity directly from blood, bypassing the need for red blood cell (RBC) lysis or RNA purification. We call this latter assay direct qPCR-based rapid T cell Activation (dqTACT) assay
Primers and probes should be resuspended at 100 μM and stored at −20° C. Keep probes away from light when working with them.
dqTACT PCR 1× Mix:
10 μl SCRIPT Direct RT-qPCR ProbesMaster mix; 0.1 μl ACTIN primer 1; 0.1 μl ACTIN primer 2; 0.1 μl CXCL10 primer 1; 0.1 μl CXCL10 primer 2; 0.05 μl ACTIN probe (HEX); 0.05 μl CXCL10 probe (FAM); 2 μl blood/buffer A mixture; 7.5 μl PEC-1
qTACT PCR 1× Mix:
5 μl TaqPath 1 Step Multiplex Master Mix (no ROX); 0.1 μl ACTIN primer 1; 0.1 μl ACTIN primer 2; 0.1 μl CXCL10 primer 1; 0.1 μl CXCL10 primer 2; 0.05 μl ACTIN probe (HEX); 0.05 μl CXCL10 probe (FAM); 5 μl RNA; 9.5 μl PEC-1
dqTACT Run Conditions (Hyris bCUBE ONLY):
Whole Blood Culture with SARS-CoV-2 Peptide Pools
320 μl of whole blood drawn on the same day into sodium heparin tubes (BD) were mixed with 80 μl RPMI and stimulated with pools of SARS-CoV-2 peptides (S or NP; 2 μg/ml) or DMSO control at 37° C. After 15-17 hours of stimulation, the supernatant (plasma) was collected and stored at −80° C. until quantification of cytokines.
qTACT Assay
Samples used for RNA extraction were diluted 1:1 with RNA/DNA shield (Zymo) and incubated at room temperature with proteinase K at a 1:100 dilution (20 mg/ml stock). Samples were then frozen at −80° C. until RNA extraction could be performed. Samples stored in RNA/DNA shield were thawed at room temperature prior to RNA extraction. Samples were vortexed and mixed with Trizol reagent (Life Technologies) at a 1:1 dilution. After vortexing, samples were processed using the Direct-zol 96 well extraction kit (Zymo) as per the manufacturer's instructions. Eluted RNA was diluted in TE buffer, aliquoted, and stored at −80° C. or used immediately for qPCR analysis. Real-time quantification was performed on a BioRad CFX96/CFX384 or Hyris bCUBE 2.0. 5 μl of diluted RNA was used with the TaqPath 1-Step Multiplex MasterMix (Applied Biosystems) and primers/probes targeting ACTIN (internal control) and other target genes, as described.
dqTACT Assay
Samples used for direct amplification from whole blood were diluted 1:3 with Buffer A and stored at −80° C. or used immediately for qPCR analysis. 2 μl of diluted whole blood was mixed with SCRIPT Direct RT-qPCR ProbesMaster (Jena Bioscience) and primers/probes targeting ACTIN (internal control) and other target genes, as described. Quantification was performed using the Hyris bCUBE 2.0.
Reagents:
The present study demonstrates that distinct SARS-CoV-2 peptide pools are able to define the individuals that were recently infected with SARS-CoV-2. This T cell response profile can be evaluated using other laboratory techniques capable of detecting T cell activation after peptide stimulation, including the direct activation of antigen-specific T cells in whole blood. A proposed algorithm to interpret the SARS-CoV-2 T cell response profile is summarized in Table 6.
Table 6 summarizes the interpretation of the SARS-CoV-2 T cell response profile in Example 1. When 50% or more of the pools (thus 2, 3 or 4 out of 4) are positive, the subject is categorized as having SARS-COV2 specific T cells induced by SARS-COV-2 infection (thus previously or currently SARS-COV-2 infected).
Different variants of concern (VOC) of SARS-CoV-2 have replaced world-wide the original SARS-CoV-2 Wuhan isolate. These VOCs are characterized by amino acid substitutions that provide biological advantages like increased infectivity or escape humoral (antibodies) but also cellular (T cells) immunity. We have designed a method based on specific combination of peptide pools covering both different SARS-CoV-2 proteins (i.e., Spike) and the corresponding regions affected by the amino acid mutations that are used to stimulate T cells, The results of this multiple stimulation strategy define with accuracy the impact of these amino acid mutations on the SARS-CoV-2-specific T cells induced by infection with Wuhan strain or by vaccination with the present vaccines based on the Wuhan strain. Peptide pools directed to non-conserved regions of the Wuhan strain variants are shown in Tables 8 to 19, while the sequences of the regions of the Wuhan strain that correspond to the regions of the Delta variant (B.1.617.2) are shown in Table 7.
The inventors present here as an example the method to analyze the impact of AA mutations present in Spike in different VOCs (using Delta variant as an example) on the SPIKE specific T cells induced by vaccination.
An embodiment is shown in schematic diagram
These peptide pools can be, for example, used in a classical ELISPOT assay and thus used to stimulate PBMC of different vaccinated individuals (
Thus, the inventors can obtain a measurement of the alteration that the mutations present in VOCs can exert on total SARS-CoV-2 T cell response.
The inventors show in this Example that qPCR can be used to quantify the presence of virus-specific T cells, based on ex vivo stimulation of whole blood samples with a pool of viral peptides covering the spike or other SARS-CoV-2 viral proteins (i.e. nucleoprotein [NP]), followed by direct amplification of IFN-γ or IL-2 (directly produced by SARS-CoV-2 antigen-specific T cells) or CXCL10, a molecule expressed by monocytes in response to T cell activation.
In order to select genes whose induction would correlate with the presence and activation of antigen specific T cells; we first evaluated the transcriptional profile of whole blood after overnight stimulation with SARS-CoV-2 peptide pools by RNA sequencing (
qPCR
Next, we validated transcriptional induction of shortlisted genes by qPCR (as described in Example 2) in 11 naïve and 8 COVID-19 convalescent subjects. Out of all genes tested (
To assess the reliability of the qTACT test, we compared CXCL10 and IFN-γ expression levels to IFN-γ cytokine secretion as quantified by ELLA in a larger cohort comprised of 89 subjects (43 naïve and 46 COVID-19 convalescent. For this cohort, only samples stimulated with DMSO (control) and the spike peptide pool (Table 4) were considered for downstream analysis. CXCL10 was selected as preferred because our previous data had confirmed its reliability and reproducibility when distinguishing between naïve and COVID-19 convalescent subjects. For this larger cohort, we chose to keep IFN-γ, despite its inferiority relative to CXCL10, to include a gene that is expressed by antigen-specific T cells and to directly correlate mRNA expression (qTACT) with IFN-γ protein secretion (ELLA). The cohort was recruited prior to vaccination and followed at day 10 and 20 after the first and second dose of the BNT162b2 vaccine and the data on IFN-γ an IL-2 cytokine secretion have been described elsewhere (Camara C., et al., bioRxiv 2021.03.22.436441; doi:/10.1101/2021.03.22.436441).
Compared to naïve subjects, COVID-19 recovered individuals had a higher median expression of CXCL10 prior to vaccination (2.53 [N=21] vs 0.0087 [N=19] in naïve subjects) (
The effect of a second dose of the vaccine was next studied. Sampling on day 10 and 20 after a second dose confirmed the beneficial effects of the recall vaccine in naïve individuals who increase their CXCL10 and IFN-γ expression to significant levels. On the contrary, the second dose in COVID-19 recovered individuals did not have a boost effect (no significant increase in CXCL10 and IFN-γ levels) (
qPCR Quantification of CXCL10 as a Proxy for Cellular Immunity
We used the qPCR assay to quantify the levels of CXCL10, as a proxy for cellular immunity, over time. We assessed the level of T cell activation in a small cohort of COVID-19 convalescent subjects at different time points post infection. We observed that, while for some patients CXCL10 levels decreased to background levels after 9 months from SARS-CoV-2 infection, we were able to detect the activation of antigen specific T cells in the majority of subjects even 9-12 months post infection (
dqPCR
Blood samples were prepared as described in Example 2. To this end, following overnight incubation with DMSO control, or SARS-CoV-2 nucleoprotein or spike peptide pools (Table 4), we took 50 μl of blood, diluted it (1:3) to avoid PCR inhibition by anticoagulants (i.e., heparin), and loaded 2 μl directly onto a qPCR instrument (dqTACT)(
IFN-γ and CXCL10 were tested as potential readouts for the dqTACT assay (described in Example 2), being optimized for use on the Hyris bCUBE given its high range of detection compared to other tested instruments, the reduced cost, and the ease of assay set up (
A lack of rapid, accessible, and accurate diagnostic methods to quantify cellular immunity hinders long-term vaccination strategies and public health responses to global pandemics, such as the one being caused by SARS-CoV-2. Considering that diagnostic centers around the world have ramped up the setup of RT-qPCR based facilities, we developed a qPCR-based dqTACT assay, which is amenable to periodic and repeated testing of patient samples, as it requires only 1 ml of blood and a 24-hour turnaround time. Clinical validation of this assay in response to recent draft guidance from the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) is ongoing in a Clinical Laboratory Improvement Amendments (CLIA)-certified microbiology laboratory. First, we implemented a Next Generation Sequencing (NGS) approach. The pros of this approach, which could be further implemented by a targeted amplification panel of 15-20 genes, is the possibility of capturing the variability of the response and measure cytokines produced by both T cells and other myeloid cells in the blood. The cons are a longer turnaround time, a higher cost, and the need for skilled technical personnel.
Second, we developed a qPCR-based method (qTACT assay) on a 96 or 384 well platform (BioRad CFX). The advantages of this approach are the accuracy and sensitivity of qPCR probes, the opportunity to combine more than 2 fluorophores to measure the expression of 2-4 genes, and the scalability and potential automation of the process. The cons include a relatively longer processing time (48 hours per 200 samples), the need to purify RNA (by standard RNA-purification kits/columns), higher associated costs, and a certain level of technical skill (although less than that required for NGS).
Third, we optimized a direct qPCR-based method (dqTACT assay) on the HYRIS bCUBE platform. The advantages of this approach are the accuracy of qPCR probes, the increased accuracy of the bCUBE platform over other tested qPCR machines, and the reduced processing time/cost/skill required. Overall, this is an easy to implement protocol that requires minimal training of the operator, thus reducing technical errors. The cons include a relatively lower scalability (18 samples on the bCUBE as opposed to 48/192 samples on the CFX 96/384) and the limitation to a 2 fluorophore/2 genes detection system.
The derived profile of SARS-CoV-2-specific T cell activation by qTACT/dqTACT assays in different cohorts of naïve, infected or vaccinated individuals, will provide information about their level of SARS-CoV-2-specific cellular immunity.
Eight HBV peptide pools of 15-mers (Tables 20-27) covering the proteome (Core, X, Envelope and Polymerase) of HBV (AB112063 (HBV Gen C)) were generated (
Whole blood was isolated from either a patient with chronic HBV infection or a healthy individual who was vaccinated for HBV (with recombinant HBV envelope vaccine) and tested within 24 hours after blood draw (
It would be understood that the parameters described in the example may be changed and still achieve a valid assay. For example, the ratio of blood to RMPI media can range from 100% blood to 50% blood; 100 μl aliquots or more than 400 μl might be used, but a larger blood sample would be required to test multiple peptide pools. RPMI may be exchanged with other cell culture media. The final concentration of peptides in an assay mixture may be from 1 to 5 μg/ml. The control sample may contain DMSO or may be an unstimulated blood sample. The incubation period may range between about 6-24 h.
HBV reactive T cells in infected individuals can be detected by quantifying the amount of secreted cytokines after the direct addition of the peptide pools into whole blood. At the moment, we have selected the detection of both IFN-γ and IL-2, two cytokines commonly secreted by T cells, as a means of detection.
From the data (
The assays presented here are based on the ability of SARS-CoV-2 T cells to respond to different peptides covering different proteins of the virus. With the possibility to use different peptides pools, our approach represents a flexible strategy that can be easily utilized to detect the presence of T cells responding to emerging mutant strains and, thus, immediately gauge the impact that viral mutation might have on cellular immunity. Moreover, the methods exemplified herein are applicable to viruses other than SARS-CoV-2 and its variants.
Hence, a diagnostic method that can be easily adapted to detect the degree of cellular immunity is an urgently needed complement to the currently available tests measuring viral presence or antibody titers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202010411P | Oct 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050627 | 10/18/2021 | WO |
