key: cord-0938398-swb9xn4t
authors: Poluektov, Yuri; Daftarian, Pirouz; George, Marybeth; Delcommenne, Marc C.
title: Assessment of SARS-CoV-2 Specific CD4(+) and CD8 (+) T Cell Responses MHC Class I and II Tetramers
date: 2021-03-05
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.03.008
sha: 5e1546d54e74d69f5d3300c9b1b9597974a48b17
doc_id: 938398
cord_uid: swb9xn4t

The success of SARS-CoV-2 (CoV-2) vaccines is measured by their ability to mount immune memory responses that are long-lasting. To achieve this goal, it is important to identify surrogates of immune protection, namely, CoV-2 MHC Class I and II immunodominant pieces/epitopes and methodologies to measure them. Here, we present results of flow cytometry-based MHC Class I and II QuickSwitchTM platforms for assessing SARS-CoV-2 peptide binding affinities to various human alleles as well as the H-2 Kb mouse allele. Multiple SARS-CoV-2 potential MHC binders were screened and validated by QuickSwitch testing. The screen included 31 MHC Class I and 19 MHC Class II peptides predicted to be good binders by the IEDB web resource provided by NIAID. While several predicted peptides with acceptable theoretical Kd showed poor MHC occupancies, fourteen MHC class II and three MHC class I peptides showed promiscuity in that they bind to multiple MHC molecule types. In addition to providing important data towards the study of the SARS-CoV-2 virus and its presented antigenic epitopes, the peptides identified in this study can be used in the QuickSwitch platform to generate MHC tetramers. With those tetramers, scientists can assess CD4+ and CD8+ immune responses to these different MHC/peptide complexes.

While various antiviral drugs or passive antibody therapies are attractive approaches as a bridge to a vaccine, the consensus is that immunization and mounting durable immune memory against the SARS-CoV-2 is an unavoidable task facing today's scientific community [1] . The basic research to uncover the exact mechanisms of how the SARS-CoV-2 virus is perceived and countered by the human immune system have been more difficult. In the effort to fight this outbreak, a greater understanding of how the viral proteins and their processed peptides stimulate the immune responses mediated by CD8+ and CD4+ T lymphocytes is needed. While some tremendous research has been undertaken with an impressive swiftness to determine the specific viral peptides that lead to an effective T cell response, many of the identified SARS-CoV-2-reactive T-cells were also found in healthy donors [2, 3] . This, in turn, raises even more questions as to the nature of the viral peptides needed to mount an effective immune response. In addition, this creates a vital need for a fast and effective way to generate various MHC tetramers needed to detect the numerous specific T cells that will be researched while trying to determine the best possible viral peptides that would stimulate the most effective T cells.

The ability of a peptide to stimulate the immune system stems from such properties as the availability of the right T cell receptor, antigen processability by the antigen processing system that could generate the peptide in question, and the peptide's ability to occupy the cleft of the MHC molecule [4, 5] . It has been observed that more than 80% of MHC class I-bound peptides derived from a virus can be immunogenic [6] . At least in the case of viral peptides, peptides that that have a high binding affinity to MHC Class I molecules tend to also be immunogenic [7, 8] . To this end, we utilized the QuickSwitch antigen presentation platform (MBL International) based on the peptide exchange principle [9] . This platform allows users to quantitate the relative ability of MHC molecules to bind a particular peptide, as well as to generate an MHC tetramer that can be used to stain T cells in flow cytometry experiments. The system relies on an irrelevant and weak binding peptide, called exiting peptide, which comes pre-loaded into the recombinant MHC molecule that you wish to study. When the QuickSwitch MHC molecule is presented with a competing peptide, the exiting peptide will be replaced proportionally to the MHC binding affinity of the competing peptide. In addition, a FITClabeled antibody against the exiting peptide comes included in every kit so that the extent of the peptide exchange can be measured using a simple flow cytometry procedure with magnetic beads specific for the MHC molecule being tested. In this short study we would like to report how the QuickSwitch platform can be used to screen SARS-CoV-2 peptides for their ability to bind various MHC molecules. This short collection of findings helps avoid unnecessary experiments with SARS-CoV-2 peptides that cannot be presented by MHC molecules and allows for the construction of working MHC tetramers that can be used for productive CD8+ and CD4+ cell staining.

The peptide binding tests to determine the peptide's affinity for a specific MHC haplotype were performed on a 50 µg/mL (MHC concentration) solution of tetramers for the MHC Class I molecules or a 100 µg/mL solution of recombinant MHC biotinylated molecules for MHC Class II. While it is possible to get valid peptide exchange results by decreasing the Tetramer or monomer concentration even further, it is not recommended, as some MHC haplotypes are much more sensitive to dilution than others generating different results depending on the MHC concentration. In Silico prediction of peptide binding affinities for MHC molecules. All of the theoretical binding affinities were derived from the Immune Epitope Database (IEDB) web resource funded by NIAID. This website catalogues experimental data on antibody and T cell epitopes resulting from numerous studies in the context of infectious disease, allergy, autoimmunity and transplantation. The accumulated data is then compiled and analyzed to allow the prediction of peptide binding affinities for specific MHC molecules.

The IEDB ANN and SMM-align methods were used respectively for MHC class I and II prediction [10, 11] . 

PBMCs from healthy donors expressing HLA-DRB1*01:01, HLA-DRB1*04:01 and HLA-DRB1*15:01 were cultured for 2 weeks at 1 x 10 6 cells per mL in Aim V medium (Thermofisher) with supplemented IL-2 at 50 U/mL and 5% human AB serum and stimulated with SARS-CoV-2 peptides each at 10 µg/mL.

The ability of a number of SARS-CoV-2 virus derived peptides as potential binders to MHC molecules were tested. Using the SMMAlign and ANN methods of the IEDB web resource the IC50 theoretical binding affinities of peptides derived from the viral proteome to each of the tested MHC molecules were determined [10, 11] . A few specific HLA-A11. These 2 alleles are tightly related and all the tested peptides that were highly exchangeable on HLA-A3 also happened to be good HLA-A11 binders.

To address similar studies in mouse models, we attempted to assess the utility of the platform in a mouse MHC haplotype. A set of predicted strong binding peptides were tested for their ability to load in H-2 Kb MHC tetramers. Mouse H-2 Kb specific SARS-CoV-2 peptides can be used for T cell immunomonitoring in mouse models of COVID-19 studies [12] . Interestingly, two of the identified H-2 Kb peptides were also determined to be good HLA-A*02:01 binders ( Table 2 ).

The exact exchange ratios of the tested HLA-A*02:01 Tetramer-PE molecules with each SARS-CoV-2 peptide can be visualized in Figure 1 . peptides. This may be due to the higher theoretical binding affinity of these five peptides towards HLA-DRB1*01:01 than towards the other two MHC class II alleles, leading to a more vigorous T cell activation and expansion in HLA-DRB1*01:01 individuals.

Presence of SARS-CoV-2 specific T cells from healthy individuals as shown in these staining data could be explained by TcR cross reactivity against peptides of common endemic coronaviruses [3] .

There are concerns that some vaccine researchers focus only on humoral responses to establish the potency and efficacy of their CoV-2 vaccines. This concern stems from the fact that the durability and potency of SARS-CoV-2 vaccines is indeed partially dictated by elicited T cell immune responses. Not examining the CoV-2 T cell immunomonitoring may result in vaccines that induce short lasting humoral responses in the absence of proper T cell memory responses [19] [20] [21] [22] [23] [24] . Other than healthy hosts, it is also important to analyze CoV-2-specific T cell responses in certain vaccinated sub-populations such as the elderly, and hosts who bear underlying conditions that are expected to impact the immune system such as diabetes. To better understand the breath of T cell immune responses, three essential steps need to be interrogated, i) it is necessary to identify protein pieces (peptides) that can be processed in antigen processing pathways, ii) to identify those that are able to interact with the groove of MHC molecules, and iii) to validate those peptides with human PBMCs to ensure that there are T cell clonotypes recognizing these peptides in the context of self MHC. This short communication addresses the second item which is MHC occupancy testing of a selected sets of peptides that are reported in the past few months based on either in silico prediction models and/or after validation in PBMCs of hosts. These peptides should be validated in a system other than the one they are predicted in [25] . DYVYNPFMI  FLAHIQWMV  FLLNKEMYL  FYGGWHNML  ISDEFSSNV  IWLGFIAGL  KLPDDFTGCV  KSAGFPFNK  KTFPPTEPK  LLLDDFVEI  LLYDANYFL  NYLKRRVVF  SAFAMMFVK  SIIAYTMSL  SMWALIISV  STFNVPMEK  SVLLFLAFV  TLMNVLTLV  TTIKPVTYK  TYKPNTWCI  VTNNTFTLK  VVFLHVTYV  YLDAYNMMI  YLNTLTLAV 

Passive antibody therapy in COVID-19

Phenotype of SARS-CoV-2-specific T-cells in COVID-19 patients with acute respiratory distress syndrome

Presence of SARS-CoV-2 reactive T cells in COVID-19 patients and healthy donors

More than one reason to rethink the use of peptides in vaccine design

The determinants of tumour immunogenicity

Most viral peptides displayed by class I MHC on infected cells are immunogenic

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes

Predicting T cell recognition of MHC class I restricted neoepitopes

The QuickSwitch Quant HLA-A*02:01 Tetramer Kit can be used for determining the biological activity of a cancer vaccine

Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

Reliable prediction of T-cell epitopes using neural networks with novel sequence representations

In Silico-Guided Sequence Modification of Epitopes in Cancer Vaccine Development

A Targeted Vaccine against COVID-19: S1-Fc Vaccine Targeting the Antigen-Presenting Cell Compartment Elicits Protection against SARS-CoV-2 Infection

Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Divergent paths for the selection of immunodominant epitopes from distinct antigenic sources

In Vitro Studies of MHC Class I Peptide Loading and Exchange

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans

Long-term maintenance of lung resident memory T cells is mediated by persistent antigen

Immunogenicity of a DNA vaccine candidate for COVID-19

Human Leukocyte Antigen Susceptibility Map for Severe Acute Respiratory Syndrome Coronavirus 2

Systemically comparing host immunity between survived and deceased COVID-19 patients

Total predicted MHC-I epitope load is inversely associated with mortality from SARS-CoV-2

Analysis of SARS-CoV-2 specific T-cell receptors in ImmuneCode reveals cross-reactivity to immunodominant Influenza M1 epitope

Machine-Learning Prediction of Tumor Antigen Immunogenicity in the Selection of Therapeutic Epitopes

MHC class I and II QuickSwitch peptide exchange platforms allow to identify and validate SARS-CoV-2 peptides that actually bind to MHC molecules Assessment of MHC binding affinity of SARS-CoV-2 peptides in a semi quantitative manner with QuickSwitch platforms. Selection of potentially immunodominant SARS-CoV-2 peptides that bind to multiple MHC class I and II alleles. Staining of human T lymphocytes with SARS-CoV-2 peptide/MHC tetramers generated by peptide exchange.

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:This work is covered by 2 patent applications one by MCD and another by MCD and YP