BACKGROUND AND RATIONALE:

The SARS COV-2 infection (COVID 19) has progressed rapidly to become a global pandemic causing untold health and economic misery . At present, there is no vaccine against SARS-CoV-2, and the excessive inflammation associated with severe COVID-19 can lead to respiratory failure, septic shock, and mortality.The overall mortality rate is 0.5–3.5% , but most people seem to be affected less severely, and either remain asymptomatic or develop only mild symptoms during COVID-19, suggesting varying degrees of immune protection against the disease . The body’s immunity has developed a variety of responses to viral infection. Immunity arises by cells that recognise the virus and attack it, and certain chemicals (antibodies) that are produced by the body to neutralise the virus. These two systems of immunity are interdependent and work together.

Studies of immunity in COVID patients have focussed on the detection and measurement of antibodies. However, antibody responses are not detectable in all patients, especially those with less severe forms of COVID-19.  Also, people may not develop antibodies despite having proven infection Recent work has suggested that SARS-CoV-2 elicits a specific robust and long lasting T cell response. It may be used to design an assay that can measure T cell responses to COVID infection as a marker of exposure or previous infection. Interestingly, recent studies suggest that a robust T cell response in patients who are PCR positive for infection may be correlated with a milder clinical course. Hence T cell assays may help in prognosticating infected patients and identifying those who may need hospital and emergent care.

SARS COV-2 specific T cells have been identified in humans (Grifoni et al., 2020; Ni et al., 2020). It has nonetheless remained unclear to what extent various features of the T cell immune response associate with antibody responses and the clinical course of acute and convalescent COVID-19. To address this knowledge gap, two recent studies have characterized SARS-CoV-2-specific CD4+ and CD8+ T cells in outcome-defined patients.

This test could be used :

1. To complement serological surveys- Antibody responses are  not mounted by all patients who develop CoVID, and do not persist beyond 8 weeks in most people. The use of serological tests for screening will underestimate the number of people who have specific viral immunity. This test could complement serological surveys and provide a better picture of the prevalence of the disease and residual immunity. 

2. To triage CoVID positive patients based on prognosis : Strong cytotoxic T cell responses are  linked with a milder clinical course . Patients who test CoVID positive can be assessed for and triaged based on cellular immunity- strong elicited responses are linked to better recovery.

3. As a companion test to a vaccine program: Vaccines need to be prioritised to a vulnerable group of people initially. Patients who are antibody negative and have low cellular immunity could be triaged to receive the vaccine early on.

Steps :

●      Identifying a refined pool of peptides from a known pool of previously studied peptides which cover several SARS CoV-2 proteins in overlapping sequences. This process has been performed in assays for other infections using the Quantiferon platform (TB and CMV).

●      Obtaining these peptides synthesised in experimental quantities to help create the pool.

●       Conducting a direct in vitro comparison of the performance of the pool in blood samples from COVID infected individuals by assessing stimulated cytokine response

The peptide pool can continue to be refined on further iterations to improve response, but it should be possible to move ahead with creating a viable pool with existing knowledge.

Purpose:

The purpose of this study is to assess how well the immune system (cellular immunity) responds to the SARS COV-2 virus, so that in the future it may be possible to predict which patients are at higher risk of developing complications. It may also identify patients who have already developed immunity to SARS COV-2, and can thus be allowed to safely travel and work.

References:

1. A. Chandrashekar, J. Liu, A. J. Martinot, K. McMahan, N. B. Mercado, L. Peter, L. H. Tostanoski, J. Yu, Z. Maliga, M. Nekorchuk, K. Busman-Sahay, M. Terry, L. M. Wrijil, S. Ducat, D. R. Martinez, C. Atyeo, S. Fischinger, J. S. Burke, M. D. Slein, L. Pessaint, A. Van Ry, J. Greenhouse, T. Taylor, K. Blade, A. Cook, B. Finneyfrock, R. Brown, E. Teow, J. Velasco, R. Zahn, F. Wegmann, P. Abbink, E. A. Bondzie, G. Dagotto, M. S. Gebre, X. He, C. Jacob-Dolan, N. Kordana, Z. Li, M. A. Lifton, S. H. Mahrokhian, L. F. Maxfield, R. Nityanandam, J. P. Nkolola, A. G. Schmidt, A. D. Miller, R. S. Baric, G. Alter, P. K. Sorger, J. D. Estes, H. Andersen, M. G. Lewis, D. H. Barouch, SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science 10.1126/science.abc4776 (2020). doi:10.1126/science.abc4776 Medline 
2. A. E. Oja, A. Saris, C. A. Ghandour, N. A. P. M. Kragten, B. M. Hogema, E. J. Nossent, L. M. A. Heunks, S. Cuvalay, E. Slot, F. H. Swaneveld, H. Vrielink, T. Rispens, E. van der Schoot, R. A. W. van Lier, A. Ten Brinke, P. Hombrink, Divergent responses in severe but not mild COVID-19; https://www.biorxiv.org/content/10.1101/2020.06.18.159202v1. 
3. T. Sekine, A. Perez-Potti, O. Rivera-Ballesteros, K. StrÃ¥lin, J.-B. Gorin, A. Olsson, S. Llewellyn-Lacey, H. Kamal, G. Bogdanovic, S. Muschiol, D. J. Wullimann, T. Kammann, J. EmgÃ¥rd, T. Parrot, E. Folkesson, O. Rooyackers, L. I. Eriksson, A. Sönnerborg, T. Allander, J. Albert, M. Nielsen, J. Klingström, S. Gredmark-Russ, N. K. Björkström, J. K. Sandberg, D. A. Price, H.-G. Ljunggren, S. Aleman, M. Buggert, Karolinska COVID-19 Study Group Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19; https://www.biorxiv.org/content/10.1101/2020.06.29.174888v1.full. 
4. F. Gallais, A. Velay, M.-J. Wendling, C. Nazon, M. Partisani, J. Sibilia, S. Candon, S. Fafi-Kremer, Intrafamilial exposure to SARS-CoV-2 induces cellular immune response without seroconversion; https://www.medrxiv.org/content/10.1101/2020.06.21.20132449v1. 
5. R. Channappanavar, C. Fett, J. Zhao, D. K. Meyerholz, S. Perlman, Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection. J. Virol. 88, 11034–11044 (2014). doi:10.1128/JVI.01505-14 Medline 
6. J. Zhao, A. N. Alshukairi, S. A. Baharoon, W. A. Ahmed, A. A. Bokhari, A. M. Nehdi, L. A. Layqah, M. G. Alghamdi, M. M. Al Gethamy, A. M. Dada, I. Khalid, M. Boujelal, S. M. Al Johani, L. Vogel, K. Subbarao, A. Mangalam, C. Wu, P. Ten Eyck, S. Perlman, J. Zhao, Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses. Sci. Immunol. 2, (2017). doi:10.1126/sciimmunol.aan5393 Medline 
7.  C. J. Thieme, M. Anft, K. Paniskaki, A. Blazquez-Navarro, A. Doevelaar, F. Seibert, B. Hoelzer, M. J. Konik, T. Brenner, C. Tempfer, C. Watzl, S. Dolff, U. Dittmer, T. H. Westhoff, O. Witzke, U. Stervbo, T. Roch, N. Babel, The SARS-CoV-2 T-cell immunity is directed against the spike, membrane, and nucleocapsid protein and associated with COVID 19 severity;https://www.medrxiv.org/content/10.1101/2020.05.13.20100636v1. 
8.  Y. Peng, A. J. Mentzer, G. Liu, X. Yao, Z. Yin, D. Dong, W. Dejnirattisai, T. Rostron, P. Supasa, C. Liu, C. Lopez-Camacho, J. Slon-campos, Y. Zhao, D. Stuart, G. Paeson, J. Grimes, F. Antson, O. W. Bayfield, D. E. D. P. Hawkins, D.-S. Ker, L. Turtle, K. Subramaniam, P. Thomson, P. Zhang, C. Dold, J. Ratcliff, P. Simmonds, T. de Silva, P. Sopp, D. Wellington, U. Rajapaksa, Y.-L. Chen, M. Salio, G. Napolitani, W. Paes, P. Borrow, B. Kessler, J. Fry, N. F. Schwabe, M. G. Semple, K. J. Baillie, S. Moore, P. J. M. Openshaw, A. Ansari, S. Dunachie, E. Barnes, J. Frater, G. Kerr, P. Goulder, T. Lockett, R Levin, Oxford Immunology Network Covid-19 Response T cell Consortium, R. J. Cornall, C. Conlon, P. Klenerman, A. McMichael, G. Screaton, J. Mongkolsapaya, J. C. Knight, G. Ogg, T. Dong, Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients. https://www.biorxiv.org/content/10.1101/2020.06.05.134551v1. 
9.  A. Grifoni, D. Weiskopf, S. I. Ramirez, J. Mateus, J. M. Dan, C. R. Moderbacher, S. A.First release:17 July 2020 immunology.sciencemag.org (Page numbers not final at time of first release) 4 Rawlings, A. Sutherland, L.Premkumar, R. S. Jadi, D. Marrama, A. M. de Silva, A. Frazier, A. F. Carlin, J. A. Greenbaum, B. Peters, F. Krammer, D. M. Smith, S.Crotty, A. Sette, TargetsofT CellResponses to SARS-CoV-2 Coronavirus inHumans with COVID-19 Disease and Unexposed Individuals.Cell181, 1489–1501.e15 (2020).doi:10.1016/j.cell.2020.05.015MedlinB.J. Meckiff,C. Ramirez-Suategui,V. Fajardo,S. J. Chhe, A. Kusnadi, H. Simon, A.Grifoni, E. Peloisi, D. Weiskopf, A. Sette, F. Ay,G. Seumois, C. H. Ottensmeier, P.Vijayanand, Single cell transcriptomic analysis of SARS=CoV-2 reactiveCD4 T Cells; https://www.biorxiv.org/content/10.1101/2020.06.12.148916v1.
10.  D. Mathew, J. R. Giles, A. E. Baxter,A. R.Greenplate, J. Wu, C. Alanio, D. Oldridge,L. Kuri-Cervantes,M. Betina Pampena, K. D’Andrea,S. Manne,Z.Chen, Y.J.Huang, J. P. Reilly, A. R. Weisman, C. A. Ittner, O. Kuthuru, J. Dougherty, K. Nzingha, N. Han, J.Kim, A. Pattekar,E.Goodwin, E. M. Anderson, M. E.Weirick, S.Gouma, C. P. Arevalo, M. J. Bolton, F. Chen, S. F. Lacey, S. E. Hensley, S. Apostolidis, A. Huang,L. A. Vella,TheUPenn COVID Processing Unit, BettsMR,MeyerNJ,Wherry J. Deep immune profiling of COVID-19patients reveals distinct immunotype with implications for therapeutic interventions.Science,10.1126/science.abc8511(2020).
11. .L. Rodriguez, P.Pekkarinen, L.Kanth Tadepally,Z. Tan, C.R. Consiglio,C.Pou, Y.Chen, C. Habimana Mugabo, A. Nguyen Quoc, K. Nowlan, T. Strandin, L. Levanov,J.Mikes, J.Wang, A.Kantele, J. Hepojoki, O. Vapalahti,S.Heinonen, E. Kekalainen,P. Brodin, Systems-level immunomonitoring from acute torecoveryphase of severeCOVID-19;https://www.medrxiv.org/content/10.1101/2020.06.03.20121582v2.
12. D. Weiskopf, K. S. Schmitz, M.P.Raadsen,A. Grifoni, N.M.A.Okba,H. Endeman,J. P. C. van den Akker, R. Molenkamp, M. P. G. Koopmans, E. C. M. van Gorp, B. L.Haagmans, R. L. de Swart, A. Sette,R. D. deVries, Phenotype and kinetics ofSARS-CoV-2-specific T cells in COVID-19patients with acute respiratory distress syndrome. Sci. Immunol.5,eabd2071 (2020).doi:10.1126/sciimmunol.abd2071Medline
13.  M. Liao, Y. Liu, J. Yuan, Y. Wen, G. Xu, J. Zhao, L. Cheng, J. Li, X. Wang, F. Wang, L.Liu, I. Amit, S. Zhang, Z. Zhang, Single-cell landscape of bronchoalveolar immune cells in patients withCOVID-19.Nat. Med.26, 842–844 (2020).doi:10.1038/s41591-020-0901-9Medline
14.  J. Braun, L. Loyal, M. Frentsch, D. Wendisch, P. Georg, F. Kurth, S. Hippenstiel, M.Dingeldey, B. Kruse,F. Fauchere, E. Baysal, M. Mangold,L. Henze, R.Lauster,M.Mall,K.Beyer, J. Roehmel, J. Schmitz,S. Miltenyi, M. A.Mueller,M.Witzenrath, N.Suttorp, F. Kern, U.Reimer, H. Wenschuh, C.Drosten, V.Corman, C. Giesecke-Thiel,L.E. Sander, A.Thiel, Presence of SARS-CoV-2reactiveT cells in COVID-19patients and healthy donors;https://www.medrxiv.org/content/10.1101/2020.04.17.20061440v1.
15. N. LeBert, A. T. Tan, K. Kunasegaran, C. Y. L. Tham, M. Hafezi, A. Chia,M. H. Y.Chng, M. Lin,N. Tan,M. Linster, W.N.Chia,M. I.-C. Chen,L.-F. Wang, E. E. Ooi,S. Kalimuddin,P. A. Tambyah, J.G.-H. Low, Y.-J. Tan, A. Bertoletti, SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.Nature10.1038/s41586-020-2550-z(2020).doi:10.1038/s41586-020-2550-z
16.  K. Ng, N. Faulkner, G. Cornish, A. Rosa, C. Earl, W. Wrobel, D. Benton, C. Roustan,W. Bolland, R. Thompson,A.Agua-Doce,P.Hobson,J.Heaney,H.Rickman,S.Paraskevopoulou, C.F. Houlihan,K.Thomson,E. Sanchez, G. Yen Shin,M. J.Spyer, P. A. Walker, S.Kjaer, A.Riddell, R. Beale, C. Swanton, S. Gandhi,B.Stockinger, S. Gamblin, L. E. McCoy, P. Cherepanov, E. Nastouli, G.Kassiotis,Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans;https://www.biorxiv.org/content/10.1101/2020.05.14.095414v1.
17. L. Ni, F. Ye, M. L. Cheng, Y. Feng, Y. Q. Deng, H. Zhao, P. Wei, J. Ge, M. Gou, X. Li, L.Sun, T. Cao, P. Wang, C. Zhou, R. Zhang, P. Liang, H. Guo, X. Wang, C. F. Qin, F.Chen, C. Dong,Detection of SARS-CoV-2-Specific Humoral and Cellular Immunityin COVID-19 Convalescent Individuals.Immunity52, 971–977.e3 (2020).  doi:10.1016/j.immuni.2020.04.023Medline
18. A. Carfì,R. Bernabei, F. Landi; Gemelli Against COVID-19 Post-AcuteCare StudyGroup, Persistent Symptoms in Patients After Acute COVID-19.JAMA10.1001/jama.2020.12603(2020).doi:10.1001/jama.2020.12603Medline

 Study Design: This open label phase 1 trial in 300 patients will assess the stimulated cytokine response of T cells in whole blood of patients of COVID-19, recovered and exposed individuals. 

The following will be assessed in all subjects:

●      Age, sex, comorbidities, date of symptoms, source of infection, type of admission, CBC with neutrophil counts, lymphocyte count

●      Data as per form attached below

●      Serum or plasma antibody titer to SARS-CoV-2:

This study will be conducted in two parts-

1)    An initial development of the ELISA assay for T cell responses against SARS- CoV2 peptides  with comparison against a gold standard (flow cytometry), and

2)    A prospective validation using assessment of T-cell specific assays in predicting outcome of SARS COV-2 infection.

Clinical cohorts to be studied

a)    Recovered from infection: (n=50)

Healthy individuals who have a documented history of RT PCR positive CoVID infection. The samples will be collected at least 2 weeks after the last symptom.[1] [2] 

b)    Active infection; no organ failure (n=50):

Patients who are admitted with active CoVID infection (RT PCR positive) with no organ failure. These patients will be on the Ward (non ICU) with oxygen requirements <10l/min.

c)     Active infection; organ failure( n=-50):

Patients who are admitted with active CoVID infection (RT PCR positive) with mild or moderate pneumonia (Patient must have at least one of the following features:

                                      i.       Bilateral pneumonia present on chest radiograph or computed tomography

                                    ii.       Partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) on arterial blood gas showing: >100mmHg and ≤ 300mmHg regardless of oxygen dose at time of testing.

                                   iii.       Pulse oxygen saturation (SpO2) at rest ≤ 93% or any degree of hypoxia requiring supplemental oxygen. Blood samples will be collected from 50 people who have been exposed to SARS COV-2 infection.

This cohort of patients (admitted in hospital with active infection; n=100) will be followed up for two weeks to assess the change in clinical condition which can then be correlated with T cell response.  The following endpoints will be studied :

Safety Endpoints: 

1. Cumulative incidence of serious adverse events during the study period
2. Transfer to ICU[5] 
3. Type and duration of respiratory support (and other ICU support) in ICU
4. ICU mortality and LOS
5. Hospital mortality and LOS
6. Ventilator-free days
7. 14 day mortality

3)    Healthy individuals who have been exposed to COVID-19 positive patients ( n=100): who have been primary contacts of a SARS COV-2 infected patient , or health care workers working in patient contact areas.

4)    Individuals admitted for elective surgery with no evidence of CoVID infection (n=50): Adults admitted for non-malignant elective surgery who are CoVID pCR negative, antibody negative and have had no history of previous CoVID on CT scan.

Peripheral blood samples will be collected into collection tubes. These blood samples will be used for assessment of stimulated cytokine response by in tube peptide assay and direct stimulation, which will then be studied by flow cytometric analysis. These responses will be correlated with serology (IgG and IgM antibody) analysis of the study subjects.

Ethics.

Prior ethical approval from the local ethics committee will be sought and obtained for the study. Written informed consent will be obtained from study subjects. Participant privacy and data confidentiality will be maintained at all times.

Inclusion criteria:

1. Above 18 years of age
2. Recent SARS COV-2 infection (recovered or active) detected by rapid antigen test or real time reverse transcription-polymerase chain reaction (RT-PCR) test for COVID-19
3. Primary contact of a SARS COV-2 patient diagnosed as above
4. Patient or their surrogate is willing and able to provide written informed consent and comply with all protocol requirements

Exclusion Criteria

1.     Below 18 years of age

2.     Pneumonia caused by bacteria, mycoplasma, chlamydia, legionella, fungi or other viruses

3.     Obstructive pneumonia induced by lung cancer or other known causes

4.     Significant comorbid illness likely to impact the outcome of COVID-19 including but not limited to active malignancy other than skin cancer.

5.     History of long-term use of immunosuppressive agents including prednisone dose >5mg daily over the 30 days prior to enrollment.

6.     History of severe chronic respiratory disease and requirement for long-term oxygen therapy

7.     Undergoing hemodialysis or peritoneal dialysis

8.     Estimated or actual rate of creatinine clearance < 15 ml/min

9.     History of moderate and severe liver disease (Child-Pugh score >12)

10.  A history of substance abuse sufficient that the patient is unlikely to comply with testing requirements.

11.  History of deep venous thrombosis, pulmonary embolism, cerebral vascular disease within the last 3 years

12.  Known HIV, hepatitis virus, or syphilis infection

13.  Co-Infection of tuberculosis, influenza virus, adenovirus and other respiratory infection virus

14.  Moribund patient not expected to survive > 24hours

15.  Any condition unsuitable for the study as determined by the investigators

16.  Female subjects with a positive pregnancy test, breastfeeding, or planning to become pregnant/breastfeed during the study period.

17.  Receipt of experimental therapy for COVID-19 with the exception of convalescent plasma, dexamethasone or another corticosteroid, or remdesivir in an open label study.

18.  Previous or ongoing immune deficient states – active malignancy , undergoing chemotherapy or radiotherapy, active secondary sepsis, post-transplant , patients with immunological/rheumatologic disorders, active HIV infection

Risks to patient â€“ Minimal. The study requires one single blood draw of approximately 10 ml. Every effort will be made to collect the blood along with a previously scheduled test to avoid extra needle pricks.

This study is for a diagnostic assay, so will not impact treatment plans for infected patients.

Where will this study be carried out?

This study will be performed in Yenepoya Medical College Hospital, Mangalore.

Laboratory plan:

A total of 15 millilitres of blood will be collected by venupuncture – 10 ml in a lithium-heparin (green top) tube and 5 ml in a serum collection tube. Whole blood will be aliquoted within 2 hours of collection into the peptide test tubes. This study will be conducted on fresh whole blood samples.

Whole blood peptide stimulation

 1 ml aliquots of (lithium) heparinized whole blood are collected in three blood collection tubes. The tubes are shaken vigorously for 5 sec. Tubes will contain pooled peptide antigens representing CD4 or CD8 specific peptides (2 tubes) (ii) no antigens (negative control), or (iii) phytohemagglutinin (PHA; positive mitogen control). Pooled peptide antigens will be added in 10μl DMSO to maintain a working concentration of 10 μg/ml of each peptide in the mix. One of the peptide test tubes will contain Golgi blockers to allow for intracellular FACE for Th1/2 cytokine analysis. The amount of DMSO will be limited to 0.1% v/v of the total sample.

The tubes are incubated for 16–24 hr at 37 degrees C.

Following incubation; Whole blood from test peptide tube sample is taken for intracellular cytokine assay by FACS . 100 microliters of whole blood is taken from the second peptide tube (non- Golgi plug) for cytokine studies using FACE. The remaining supernatant is centrifuged and taken for IFN gamma and IL-2 ELISA. 

Serology

Serum samples from all donors will be subjected to  for serology assessment. SARS-CoV-

2-specific antibodies will be detected using both the Anti-SARS-CoV-2 IgG and IgM antibodies.

Cytokine bead assays

Supernatants are collected from 24-hour stimulation cultures of the AIM assays and stored in 96 well plates at 20C. Cytokines in cell culture supernatants of the same samples used for AIM are quantified using a human Th cytokine panel (13-plex) kit (LEGENDplex, Biolegend) according to the manufacturer’s instruction. Supernatants are mixed with beads coated with capture antibodies specific for IL-2, IL-4, IL-6, IL-10, IFNg, TNFa,  and incubated on a 96 well filter plate for 2 hours. Beads are washed and incubated with biotin-labeled detection antibodies for 1 hour, followed by a final incubation with streptavidin-PE. Beads are analyzed by flow cytometry using a FACS Canto cytometer. Analysis was performed using the LEGENDplex analysis software v8.0, which distinguishes between the 13 different analytes on basis of bead size and internal dye.