METHODS FOR DIAGNOSIS, PROGNOSIS, STRATIFICATION AND TREATING OF MYOCARDITIS
FIELD OF THE INVENTION:
The present invention relates to methods for diagnosis, prognosis, stratification and treatment of myocarditis, and particularly relating to inflammatory myocarditis, and more particularly to myocarditis associated with anti-SARS-CoV-2 immunity.
BACKGROUND OF THE INVENTION:
Coronavirus disease 2019 (COVID-19) is a recently emerged global infectious respiratory disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), a novel betacoronavirus(l, 2). Inter-individual clinical variability over the course of SARS- CoV-2 infection is notorious, and prognosis remains difficult to predict at hospital entry. Respiratory failure is the most prominent feature associated with severe COVID-19, but serious damage to other organs is also observed (1, 3).
COVID-19-related myocarditis has been reported since the beginning of the SARS- CoV-2 outbreak(4-9). Fulminant myocarditis is a rare but life-threatening form of myocarditis leading to significant morbidity and mortality, especially in young patients(lO). Firstly described in children(l l) and subsequently in adults(12), the multisystem inflammatory syndrome (MIS-C or MIS-A, respectively) accounts for a large proportion of CO VID-19 related myocarditis. The United States (US) Centers for Disease Control and Prevention has developed case definition criteria to standardize its diagnosis(13).
Yet, some patients do not meet these criteria suggesting the existence of distinct phenotypes in COVID-19-related fulminant myocarditis. The treatment of the many forms of myocarditis is symptomatic. The definite and etiological diagnosis of myocarditis mainly rely on immunohistochemical and molecular biological analysis of an endomyocardial biopsy (EMB). Given that EMB is infrequently performed there is an important need for biomarkers to reliably identify myocarditis and its underlying causes, in order to identify patients in whom specific therapy will be favourable for the disease outcome.
We conducted a study to compare the clinical, biological and immunological characteristics of patients with fulminant COVID-19-related myocarditis, meeting or not meeting MIS-A criteria. SUMMARY OF THE INVENTION:
A first object of the present invention relates to an in vitro method for diagnosis myocarditis associated with multisystem inflammatory syndrome in a patient comprising the steps of i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with a reference value for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than the reference values for each marker, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' patient) when the level of interferon alpha-2 (IFN-a2) determined at step i) is significantly higher than the reference value.
Another object of the present invention relates to an in vitro method for stratifying and/or classifying patients affected with myocarditis comprising the steps of i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with reference value for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than the reference values for each marker, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' patient) when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value. Another object of the present invention relates to an in vitro method for classifying and/or stratifying patients affected with myocarditis comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected in the group consisting of interleukin-22 and interleukin- 17, and/or interferon alpha-2, ii) comparing the level of interleukin-22 and interleukin- 17), and/or interferon alpha-2 determined at step i) with a reference values for each marker and, iii) - concluding that the patient has a late form of myocarditis when the level of interleukin 22 (IL-22) and interleukin 17 (IL- 17) determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a precocious form of myocarditis when the level of interferon alpha 2 (IFN-a2) determined at step i) is significantly higher than the reference value.
Another object of the invention relates to an in vitro method for assessing a patient’s risk of having or developing severe or critically severe form of myocarditis, comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with reference value for each marker, and iii) - concluding that the patient has a low risk of having or developing severe or critically severe form of myocarditis when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a high risk of having or developing severe or critically severe form of myocarditis when the level of interferon alpha-2 determined at step i) is significantly higher than its reference value.
An additional object of the invention relates to a method for treating a myocarditis patient comprising the steps of: i) determining in a sample obtained from the patient the level of interleukin-22 and interleukin- 17, and/or interferon alpha-2, ii) ii) comparing each level of interleukin-22 and interleukin-17, and/or interferon alpha-2 determined at step i) with reference values for each marker and iii) administrating to said patient at least one drug selected from the group consisting of interleukin-22 inhibitor, interleukin- 17 inhibitor, corticosteroid and immunoglobulin when the level of interleukin-22 and interleukin- 17 determined at step i) is significantly higher than their reference values.
DETAILED DESCRIPTION OF THE INVENTION:
The multisystem inflammatory syndrome accounts for a large proportion of COVID- 19-related myocarditis in adults (MIS-A). Herein, the inventors compared the characteristics of fulminant COVID-19-related myocarditis (n=38) between patients fulfilling MIS-A criteria (MIS-A+) or not (MIS-A-). As compared to MIS-A+ (n=25), MIS-A- patients (n=13) had a shorter delay between first COVID-19 symptoms and the onset of myocarditis, a lower LVEF, higher in-ICU organ failure, need for mechanical circulatory support and in-hospital mortality. Immunological profiles were different between the 2 groups: MIS-A+ had high serum levels of IL-22, IL- 17 and TNF-a, while MIS-A- had high IFN-2a and IL-8 serum levels and an elevated frequency of RNA-polymerase-III autoantibodies (54%).
Stratifying methods of patients affected with COVID-19-related fulminant myocarditis according to the invention
Accordingly, the first object of the present invention relates to an in vitro method for diagnosing myocarditis associated with multisystem inflammatory syndrome in a patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with reference value for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ myocarditis patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than the reference values for each marker, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' myocarditis patient) when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
In some embodiments, the present invention relates to an in vitro method for diagnosing myocarditis in a patient comprising the steps of i) determining in a sample obtained from the patient the level of interleukin-22 and/or interleukin- 17 and the level of interferon alpha-2, ii) comparing the level of interleukin-22 and/or interleukin- 17 and the level of interferon alpha-2 determined at step i) with reference values for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ myocarditis patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than reference values for each marker and the level of interferon alpha-2 is not significantly higher that the reference value, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' myocarditis patient) when the level of interferon alpha-2 determined at step i) is significantly higher than a reference value and when the level of interleukin-22 and/or interleukin- 17 determined at step i) is not significantly higher than the reference values for each marker.
In some embodiment, the level of interleukin-22 and interleukin- 17 are determined in step i).
In some embodiment, the level of interleukin-22 and interferon alpha-2 are determined in step i).
In some embodiment, the level of interleukin- 17 and interferon alpha-2 (are determined in step i).
In some embodiment, the level of interleukin-22, interleukin- 17 and interferon alpha-2 are determined in step i). In some embodiment, the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 and the level of at least one marker selected from the group consisting of tumor necrosis factor-alpha , interleukin-8, and RNA- polymerase III auto-antibodies, are determined in step i), and it is concluded at step iii) that the patient have a COVID-19-related fulminant myocarditis associated with multisystem inflammatory syndrome when the level of at least one marker selected from the group consisting of interleukin-22 and interleukin- 17 and the level of tumor necrosis factor-alpha determined at step i) is significantly higher than the reference values for each marker, or that the patient have not a myocarditis associated with multisystem inflammatory syndrome (such as COVID-19 related fulminant myocarditis associated with multisystem inflammatory) when the level of interferon alpha-2 and at least one marker selected from the group consisting of interleukin-8 and RNA-polymerase III antibodies determined at step i) is significantly higher than the reference value for each marker.
Thus, the invention refers to an in vitro method for diagnosing myocarditis associated with multisystem inflammatory syndrome in a patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22 , interleukin- 17 and interferon alpha-2, and the level of at least one marker selected from the group consisting of tumor necrosis factor-alpha , interleukin-8, and RNA-polymerase III auto-antibodies ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 and the level of at least one marker selected from the group consisting of tumor necrosis factor-alpha, interleukin-8, and RNA-polymerase III auto-antibodies determined at step i) with the reference value for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ myocarditis patient) when the level of interleukin-22 and/or interleukin- 17 and the level of tumor necrosis factor-alpha determined at step i) is significantly higher than the reference values for each marker, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' myocarditis patient) when the level of interferon alpha-2 and at least one marker selected from the group consisting of interleukin-8 and RNA-polymerase III antibodies determined at step i) is significantly higher than the reference value.
In some embodiment, the level of at least one marker selected from the group consisting of interleukin-22 , interleukin- 17 and interferon alpha-2 and the level of tumor necrosis factor alpha (TNFa), and/or interleukin 8 (IL-8) are determined in step i).
In some embodiment, the level of 1, 2 or 3 markers selected from the group consisting of interleukin-22,interleukin-17 and interferon alpha-2 and the level of 1, 2 or 3 markers selected from the group consisting of tumor necrosis factor-alpha, and/or interleukin-8 and RNA-polymerase III auto-antibodies are determined in step i).
In some embodiment, the level of interleukin-22, interleukin- 17, interferon alpha-2, tumor necrosis factor-alpha and RNA-polymerase auto-antibodies are determined in step i).
In some embodiment, the level of interleukin-22, interleukin- 17, interferon alpha-2, tumor necrosis factor-alpha and interleukin-8 are determined in step i).
In some embodiment, the level of interleukin-22, interleukin- 17, interferon alpha-2, tumor necrosis factor-alpha, interleukin-8 and RNA-polymerase auto-antibodies are determined in step i).
As used herein, the term “subject” or “patient” refers to a mammalian being, such as a rodent (e.g., a mouse or a rat), a feline, a canine or a primate. In a preferred embodiment, said patient is a human patient.
In particular embodiments, the patient has been previously afflicted with viral infection or has been previously vaccinated with a vaccine against virus.
In particular, the method of the invention is performed the same day or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days after the vaccination or the detection of the viral infection.
In particular, the viral infection is caused by a virus selected from the group consisting of coronavirus (SARS-coronavirus such as SARS-Covl or SARS-Cov 2), coxsackieviruses A and B, echoviruses, polioviruses, influenza A and B viruses, respiratory syncytial virus, mumps virus, measles virus, rubella virus, hepatitis C virus, dengue virus, yellow fever virus, Chikungunya virus, Junin virus, Lassa fever virus, rabies virus, human immunodeficiency virus- 1, adenoviruses, parvovirus Bl 9, cytomegalovirus, human herpes virus-6, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, variola virus, or vaccinia virus.
As used herein the term “vaccine against virus” has its general meaning in the art and refers to a substance used to stimulate the production of antibodies and cytotoxic T cells and provide humoral and cellular immunity against one or several viruses. Viruses prevented by vaccines include polio, Hepatitis A virus, Hepatitis B virus , smallpox virus, measles virus, mumps virus, Human papillomavirus, rubella virus, influenza virus, SARS-Cov-2 and rotavirus.
In particular embodiments, the patient has been previously afflicted with SARS-CoV-2 infection or has been previously vaccinated with SARS-Cov-2 vaccines.
In particular embodiments, the patient is afflicted with SARS-CoV-2 infection.
As used herein, the term “myocarditis” has its general meaning in the art and refers to an acquired cardiomyopathy due to inflammation of the heart muscle. The inflammation can reduce the heart's ability to pump blood. Myocarditis can cause chest pain, shortness of breath, and rapid or irregular heart rhythms (arrhythmias). The diagnostic gold standard to determine myocarditis is endomyocardial biopsy (EMB) but is infrequently used. Myocarditis can also be diagnosed by established histological, immunological and immunohistochemical criteria, as described in Bonaca et al, Myocarditis in the Setting of Cancer Therapeutics, Circulation, 2016 (11) or in Caforio et al, Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases, European Heart Journal , 2013 (43).
According to the invention, myocarditis includes but are not limited to infectious myocarditis, immune-mediated myocarditis and toxic myocarditis, as described in Caforio et al (43).
According to the invention, a patient exhibiting any of the following is diagnosed as presenting with definite or probable myocarditis patients: a) Tissue pathology diagnostic of myocarditis (e.g., on biopsy or autopsy), b) CMR diagnostic of myocarditis such as Elevated biomarker of cardiac myonecrosis or ECG evidence of myo-pericarditis, c) new wall motion abnormality on echocardiogram not explained by another diagnosis (e.g., acute coronary syndrome, stress induced cardiomyopathy, sepsis) and Clinical syndrome consistent with myocarditis, elevated biomarker of cardiac myonecrosis, ECG evidence of myo-pericarditis, and negative angiography or other testing to exclude obstructive coronary disease, d) CMR with findings diagnostic of myocarditis without any of the following: clinical syndrome consistent with myocarditis, elevated biomarker of cardiac myonecrosis, and ECG evidence of myo-pericarditis, e) nonspecific CMR findings suggestive indicative of myocarditis with either 1 or more of the following: clinical syndrome consistent with myocarditis, elevated biomarker(s) of cardiac myonecrosis and ECG evidence of myo-pericarditis, f) new wall motion abnormality on echocardiogram with a clinical syndrome consistent with myocarditis and either: elevated biomarker(s) of cardiac myonecrosis or ECG evidence of myo-pericarditis, or g) a scenario meeting criteria for Possible Myocarditis, as defined in Bonaca et al (11) with fluorodeoxyglucose positron emission tomography imaging showing patchy cardiac fluorodeoxyglucose uptake without another explanation.
According to the invention, a patient is diagnosed with clinically suspected myocarditis if exhibiting at least one of the following clinical presentations: acute chest pain, pericarditis, or pseudo-ischaemic, new-onset (days up to 3 months) or worsening of: dyspnoea at rest or exercise, and/or fatigue, with or without left and/or right heart failure signs, sub acute/ chronic (>3 months) or worsening of: dyspnoea at rest or exercise, and/or fatigue, with or without left and/or right heart failure signs, or palpitation, and/or unexplained arrhythmia symptoms and/or syncope, and/or aborted sudden cardiac death, or unexplained cardiogenic shock; and at least one of the following diagnostic criteria: newly abnormal lead ECG and/or holter and/or stress testing, any of the following: I to III degree atrioventricular block, or bundle branch block, ST/T wave change (ST elevation or non ST elevation, T wave inversion), sinus arrest, ventricular tachycardia or fibrillation and asystole, atrial fibrillation, reduced R wave height, intraventricular conduction delay (widened QRS complex), abnormal Q waves, low voltage, frequent premature beats, supraventricular tachycardia or new, otherwise unexplained LV and/or RV structure and function abnormality (including incidental finding in apparently asymptomatic subjects): regional wall motion or global systolic or diastolic function abnormality, with or without ventricular dilatation, with or without increased wall thickness, with or without pericardial effusion, with or without endocavitary thrombi or Oedema and/or Late gadolinium enhancement (LGE) of classical myocarditic pattern.
In some embodiment, the myocarditis is fulminant myocarditis.
As used herein, the term “fulminant myocarditis” has its general meaning in the art and refers to a peculiar clinical condition and is an acute form of myocarditis, whose main characteristic is a rapidly progressive clinical course with the need for hemodynamic support.
In some embodiments, the myocarditis is an inflammatory myocarditis.
In some embodiments, the myocarditis is an infectious myocarditis. As used herein the term “infectious myocarditis” refers to the result of an immune response to an infection of the heart. Indeed, most often, myocarditis is caused by an infection including viruses, bacteria, protozoa, and fungi. Many viruses have been implicated as causes of myocarditis. These most commonly include adenoviruses and enteroviruses such as the coxsackieviruses. After infection by a cardiotropic virus, a maladaptive post-viral response ensues, which can cause myocardial cell dysfunction and compromised contractility
In some embodiments, the myocarditis is a viral-related myocarditis.
According to the invention, the term “viral -related myocarditis” has its general meaning in the art and include but is not limited to myocarditis caused by coronavirus (SARS- coronavirus such as SARS-CoV-1 or SARS-CoV-2), coxsackieviruses A and B, echoviruses, polioviruses, influenza A and B viruses, respiratory syncytial virus, mumps virus, measles virus, rubella virus, hepatitis C virus, dengue virus, yellow fever virus, Chikungunya virus, Junin virus, Lassa fever virus, rabies virus, human immunodeficiency virus- 1, adenoviruses, parvovirus Bl 9, cytomegalovirus, human herpes virus-6, Epstein-Barr virus, varicella-zoster virus, herpes simplex virus, variola virus, vaccinia virus.
Thus, the invention refers to an in vitro method for diagnosing viral-related myocarditis associated with multisystem inflammatory syndrome in a patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22,interleukin-17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with a reference value for each marker and, iii) - concluding that the patient has a viral-related myocarditis associated with multisystem inflammatory syndrome (MIS-A+ viral-related myocarditis patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than the reference values for each marker, or
- concluding that the patient has not a viral-related myocarditis associated with multisystem inflammatory syndrome (MIS-A' viral-related myocarditis patient) when the level of interferon alpha-2determined at step i) is significantly higher than the reference value.
In some embodiment, the myocarditis is a COVID-19 related myocarditis. Thus, the invention refers to an in vitro method for diagnosing COVID-19-related myocarditis associated with multisystem inflammatory syndrome comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with a reference value for each marker and, iii) - concluding that the patient has a COVID-19-related myocarditis associated with multisystem inflammatory syndrome (MIS-A+ COVID-19-related myocarditis patient) when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than reference values for each marker, or
- concluding that the patient has not a COVID-19-related myocarditis associated with multisystem inflammatory syndrome (MIS-A' COVID-19-related myocarditis patient) when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
As used herein, the term “COVID-19-related myocarditis” has its general meaning in the art and refers to a definite or probable myocarditis, according to the definition by Bonaca et al (11), or a clinically suspected myocarditis by Caforio et al (43) following a SARS-CoV-2 infection or a dose of SARS-CoV-2 vaccines, such as mRNA SARS-CoV-2 vaccines. Indeed, previous studies indicated that doses of mRNA vaccines were associated with increased risk of myocarditis in patients, especially in young patients (14).
As used herein, the term “COVID-19”, also known as coronavirus disease 2019, refers to an infectious disease caused by the SARS-CoV-2 virus. SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2, known by the provisional name 2019 novel coronavirus (2019-nCoV), and is the cause of the respiratory coronavirus disease 2019 (COVID-19). Taxonomically, it is a strain of the Severe acute respiratory syndrome-related coronavirus (SARSr-CoV), a positive-sense single-stranded RNA virus. It is contagious for humans, and the World Health Organization (WHO) has designated the ongoing pandemic of CO VID-19 a Public Health Emergency of International Concern. The SARS-CoV-2 virion is approximately 50-200 nanometers in diameter. Like other coronaviruses, 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, which has been imaged at the atomic level using cryogenic electron microscopy, is the protein responsible for allowing the virus to attach to the membrane receptor of a host cell.
In some embodiments, the COVID-19-related myocarditis is caused by a dose of SARS- CoV-2 vaccines (i.e the administration of a dose) or SARS-Cov-2 infection.
As used herein, the term “SARS-CoV-2 vaccines” has its general meaning in the art and refers to vaccines to prevent SARS-CoV-2 infection. Several COVID-19 vaccines are available including mRNA vaccines such as Pfizer-BioNTech COVID-19 vaccine (BNT162b2) and Moderna COVID-19 vaccine (mRNA-1273); and adenoviral vector vaccine such as Janssen COVID-19 vaccine (Ad26.COV2.S) and AstraZeneca COVID-19 vaccine (AZD1222); and Novavax COVID-19 vaccine (NVX-CoV2373). The World Health Organization (WHO) maintains an updated list of vaccine candidates under evaluation.
As used herein, “SARS-CoV-2 infection” refers to the transmission of this virus from an animal and/or human to another animal and/or human primarily via respiratory droplets from coughs and sneezes within a range of about 2 meters. Indirect contact via contaminated surfaces is another possible cause of infection.
In some embodiment, the myocarditis is a COVID-19-related fulminant myocarditis.
As used herein, the term “multisystem inflammatory syndrome” or “MIS-A” or “MIS- C” has its general meaning in the art and refers to a rare condition associated with COVID-19 in which different body parts become inflamed, including the heart, lungs, kidneys, brain, skin, eyes, or gastrointestinal organs. A case definition for MIS-A can be found in https://www.cdc.gov/mis/mis-a/hcp.htlm.
In some embodiment, the myocarditis is a COVID-19-related fulminant myocarditis.
According to the invention, a COVID-19-related fulminant myocarditis associated with multisystem inflammatory syndrome, means that the fulminant myocarditis is a post-infectious complication of SARS-CoV-2 infection or of SARS-CoV-2 vaccines
As used herein, the term “sample“ or "biological sample" as used herein refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy. In a particular embodiment regarding the prognostic method of the critical form of the coronavirus according to the invention, the biological sample is a body fluid sample (such as blood) or tissue biopsy of said subject. In a preferred embodiment regarding a method of the invention, the biological sample is a body fluid of said subject. Non-limiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, and aqueous humor.
In preferred embodiment, the fluid sample is a blood sample. The term “blood sample” means a whole blood sample obtained from a subject (e.g., an individual for which it is interesting to determine whether a population of serum biomarkers can be identified).
In particular embodiment, the sample is serum,
In particular embodiment, the sample has been previously obtained from the patient.
As used herein, the term " interleukin 22" or “IL-22”, has its general meaning in the art and refers to an a-helical cytokine. Interleukin-22 binds to a heterodimeric cell surface receptor composed of IL-10R2 and IL-22R1 subunits and that in humans is encoded by the IL-22 gene (gene ID 50616). Interleukin-22 contributes to the inflammatory response in vivo and is produced by several populations of immune cells at a site of inflammation.
As used herein, the term " interleukin 17" or “IL-17” also known as IL-17A or CTLA- 8, has its general meaning in the art and refers to a family of pro-inflammatory cystine knot cytokines. Interleukin- 17 is mainly produced by lymphocytes including CD4+, CD8+, gammadelta T (y5-T), invariant NKT and innate lymphoid cells. A group of T helper cells, known as T helper 17 cells, produce IL-17A in response to their stimulation with IL-23. Interleukin- 17 regulates the activities of NF-kappaB and mitogen-activated protein kinases. This cytokine can stimulate the expression of IL-6 and cyclooxygenase-2 (PTGS2/COX-2), as well as enhance the production of nitric oxide (NO). It is a protein that in humans is encoded by the IL-17A gene (gene ID 3605).
As used herein, the term “interferon alpha-2” or “IFN-a2” has its general meaning in the art and refers to proteins produced mainly by plasmacytoid dendritic cells (pDCs). Interferon alpha-2 is mainly involved in innate immunity against viral infection. The genes found responsible for its synthesis is gene IFNA2, (gene ID for human: 3440) which code for human protein Interferon alpha-2 ((UniProtKB - P01563).
As used herein, the term " tumor necrosis factor-alpha " or “TNF-a” also known as Tumor necrosis factor (TNF), cachexin, or cachectin has its general meaning in the art refers to a cytokine that in humans is encoded by the TNF gene (gene ID 7124). TNF-a is a pro- inflammatory cytokine, used by the immune system for cell signaling. If macrophages (or particular lymphocyte cells) detect an infection, they release TNF in order to alert other immune cells as well as cells of other tissues, leading to inflammation. The primary role of tumor necrosis factor-alpha is in the regulation of immune cells, tumor necrosis factor-alpha, being an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumorigenesis, viral replication, and respond to sepsis via IL-1 and IL-6-producing cells.
As used herein, the term " interleukin-8" (IL-8) also known as IL-8 or chemokine (C- X-C motif) ligand 8, (CXCL8), has its general meaning in the art and refers to a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells (Hedges JC, et al (2000). Am. J. Respir. Cell Mol. Biol. 23 (1): 86-94) and endothelial cells. Endothelial cells store IL-8 in their storage vesicles, the Weibel-Palade bodies (Wolff B, et al (1998). J. Exp. Med. 188 (9): 1757-62). In humans, the interleukin-8 protein is encoded by the CXCL8 gene (gene ID 3576). Interleukin-8 is initially produced as a precursor peptide of 99 amino acids which then undergoes cleavage to create several active Interleukin-8 isoforms. (Brat DJ, et al (2005). Neuro-oncology. 7 (2): 122-133). In culture, a 72 amino acid peptide is the major form secreted by macrophages (Brat DJ, et al (2005)). There are many receptors on the surface membrane capable of binding IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1 and CXCR2. Through a chain of biochemical reactions, Interleukin-8 is secreted and is an important mediator of the immune reaction in the innate immune system response.
As used herein, the term “RNA-polymerase III”, also known as “Pol III”, has its general meaning in the art and refers to a protein that transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose expression is required in all cell types and most environmental conditions. Therefore, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than RNA polymerase II.
As used herein, the term “RNA-polymerase III autoantibodies” has its general meaning in the art and refers to natural antibodies that react with RNA-polymerase III. Natural autoantibodies are mainly IgM, are encoded by unmutated V(D)J genes and display a moderate affinity for self-antigens. In the present application, the “level of marker (i.e., protein)” or the “marker level expression” means the quantity or concentration of said marker. In some embodiments, the “level of protein” means the quantitative measurement of the protein expression relative to a negative control.
Standard methods for detecting the level of specific biomarker such as IL- 17, IL-22, IL- 8, TNF-a, IFN-a2, are well known in the art. Typically, the step consisting of detecting the marker may consist in using at least one differential binding partner directed against the marker.
The level of the markers of the invention may be determined by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectroscopy technique (CE-MS), etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. The level of the markers of the invention may be determined by using other methods well known in the art such as measuring the transcriptomic levels of cytokines by RNA expression arrays or by next generation sequencing (for example RNAseq).
Standard methods for detecting the level of specific biomarker such as IL- 17, IL-22, IL- 8, TNF-a, IFN-a2, are well known in the art. Typically, the step consisting of detecting the marker may consist in using at least one differential binding partner directed against the marker.
For example, the serum concentrations of the biomarker can be determined using the Quanterix SP-X platforms (IL-22, IL-8, and TNF-a) and Quanterix Simoa (IFN-a2, IL-17).
As used herein, the term “binding partner directed against the marker” refers to any molecule (natural or not) that is able to bind the surface marker with high affinity. The binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal antibodies. In another embodiment, the binding partners may be a set of aptamers.
Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.
The binding partners of the invention such as antibodies or aptamers may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.
As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)]) or radioactive molecule or a non-radioactive heavy metals isotopes to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. More particularly, the antibodies are already conjugated to a fluorophore (e.g., FITC-conjugated and/or PE- conjugated).
The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
Such methods comprise contacting a biological sample obtained from the subject to be tested under conditions allowing detection of the markers of the invention (i.e., IL-17, IL-22, IFN-a. Once the sample from the subject is prepared, the level of the markers of the invention may be measured by any known method in the art.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
Particularly, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches [DeSouza and Siu, 2012], Mass spectrometry -based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, a quantification based on extracted ion chromatogram (EIC) and then profile alignment to determine differential level of polypeptides.
Particularly, a mass spectrometry-based quantification method particularly useful can be the use of targeted mass spectrometry methods as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data independent acquisition (DIA) and sequential window acquisition of all theoretical mass spectra (SWATH) [Moving target Zeliadt N 2014 The Scientist;Liebler Zimmerman Biochemistry 2013 targeted quantitation pf proteins by mass spectrometry; Gallien Domon 2015 Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. Methods v81 pl5-23 ; Sajic, Liu, Aebersold, 2015 Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl v9 p 307-21],
Particularly, the mass spectrometry-based quantification method can be the mass cytometry also known as cytometry by time of flight (CYTOF) (Bandura DR, Analytical chemistry, 2009).
Particularly, the mass spectrometry-based quantification is used to do peptide and/or protein profiling can be use with matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), surface-enhanced laser desorption/ionization time of flight (SELDI-TOF; CLINPROT) and MALDI Biotyper apparatus [Solassol, Jacot, Lhermitte, Boulle, Maudelonde, Mange 2006 Clinical proteomics and mass spectrometry profiling for cancer detection. Journal: Expert Review of Proteomics V3, 13, p311-320 ; FDA K130831],
Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins of the invention. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins of the invention.
A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or a "constant factor” are constant numbers that can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the score in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured expression levels of the gene(s) in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the accuracy of the diagnostic result improved as the AUC approaches 1. And the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
Such reference values of expression level may be determined for any marker defined above.
In one embodiment of the present invention, the reference value may also be derived from the level of the markers of the invention determined in a blood sample derived from one or more healthy patients. In some embodiments of the present invention, the reference value is derived from the level of the markers of the invention determined in a blood sample derived from one or more COVID-19-related fulminant myocarditis MIS-A' patients. In one embodiment of the present invention, the reference value may also be derived from the level of the markers of the invention determined in a blood sample derived from one or COVID-19- related fulminant myocarditis MIS-A+ patients. Furthermore, retrospective measurement of the level of the markers of the invention in properly banked historical subject samples may be used in establishing these reference values. Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the classification of patients’ severity by PCA from samples previously collected from the patient under testing.
In another aspect, the invention refers to an in vitro method for classifying or stratifying patients affected with myocarditis the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22,interleukin-17 and interferon alpha-2 determined at step i) with reference value for each marker and, iii) - concluding that the patient has a myocarditis associated with multisystem inflammatory syndrome (MIS-A+ myocarditis patient) when the level of interleukin-22and/or interleukin- 17 determined at step i) is significantly higher than reference values for each marker, or
- concluding that the patient has not a myocarditis associated with multisystem inflammatory syndrome (MIS-A' myocarditis patient) when the level of interferon alpha-2 determined at step i) is significantly higher than a reference value. In some embodiments, the myocarditis associated with multisystem inflammatory syndrome patient (MIS-A+ myocarditis patient) exhibits a late form of myocarditis.
In some embodiments, the myocarditis not associated with multisystem inflammatory syndrome patients (MIS-A' myocarditis patient) exhibits a precocious form of myocarditis.
In particular embodiment, the COVID-19-related fulminant myocarditis associated with multisystem inflammatory syndrome patient (MIS-A+ COVID-19-related fulminant myocarditis patient) exhibits a late form of fulminant COVID-19-related myocarditis.
In particular embodiments, the COVID-19-related fulminant myocarditis not associated with multisystem inflammatory syndrome patients (MIS-A' COVID-19-related fulminant myocarditis patient) exhibits a precocious form of COVID-19-related fulminant myocarditis.
According to the invention, a late form of COVID-19 related fulminant myocarditis refers to a fulminant myocarditis with a high median delay between COVID-19 symptoms onset and occurrence of fulminant myocarditis, i.e., around 25-44 days between first COVID-19 symptoms and myocarditis.
According to the invention, a precocious form of COVID-19 related fulminant myocarditis refers to a fulminant myocarditis with a low median delay between COVID-19 symptoms onset and occurrence of myocarditis, i.e., around 0-8 days between first COVID-19 symptoms and myocarditis, and more particularly 0 to 5 days between first COVID-19 symptoms and myocarditis.
Thus, the method of the invention can be used to classify and/or stratify patients affected with late or precocious form of myocarditis, wherein it is concluded that said patient has a late form of COVID-19-related myocarditis when said patient is diagnosed or classified as MIS-A+ myocarditis patient according to the method of the invention, or that said patient has a precocious form of myocarditis when said patient is diagnosed or classified as MIS-A' myocarditis patient according to the method of the invention.
Thus, the present invention also relates to an in vitro method for diagnosing precocious or late form of myocarditis in a patient comprising the step of concluding that the patient has a late form of myocarditis when said patient has been diagnosed as MIS-A+ myocarditis patient according to the invention, or concluding that the patient has a precocious form of myocarditis when said patient has been diagnosed as MIS-A' myocarditis patients. In other words, the present invention also relates to an in vitro method for diagnosing precocious or late form of myocarditis in a patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected in the group consisting of interleukin-22 and interleukin- 17 , and/or interferon alpha-2 , ii) comparing the level of interleukin-22 and interleukin- 17 , and/or interferon alpha-2 determined at step i) with reference values and, iii) - concluding that the patient has a late form of myocarditis when the level of interleukin-22 and interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a precocious form of myocarditis when the level of interferon alpha2 determined at step i) is significantly higher than a reference value.
In other words, the present invention also relates to an in vitro method for classifying and/or stratifying patients affected with myocarditis comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected in the group consisting of interleukin-22 and interleukin-17, and/or interferon alpha-2, ii) comparing the level of interleukin-22 and interleukin- 17, and/or interferon alpha-2 determined at step i) with a reference values for each marker and, iii) - concluding that the patient has a late form of myocarditis when the level of interleukin-22 and interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a precocious form of myocarditis associated with multisystem inflammatory syndrome (MIS-A' patients) when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
In preferred embodiments, the myocarditis is viral-related myocarditis.
In preferred embodiments, the viral-related myocarditis is COVID-19-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis is COVID-19-related fulminant myocarditis. Prognostic methods of myocarditis severity according to the invention
The inventors shows that a precocious form of myocarditis (i.e., patients classified as MIS-A' myocarditis patients according to the method of the invention) is associated with a severe or critically severe form myocarditis characterised lower left ventricle ejection fraction (LVEF), higher in-ICU organ failure, need for mechanical circulatory support and in-hospital mortality. In some embodiments, the myocarditis associated with multisystem inflammatory syndrome patient (MIS-A+ myocarditis patients) have a low risk of having or developing severe or critically severe form of myocarditis
In some embodiments, the myocarditis not associated with multisystem inflammatory syndrome patients (MIS-A' myocarditis patients) have a high risk of having or developing severe or critically severe form of myocarditis.
Thus, the method of the invention can be used to assess patient’s risk of having or developing severe or critically severe form myocarditis, wherein it is concluded that said patient have a low risk of having or developing severe or critically severe form of myocarditis when said patient is classified as MIS-A+ myocarditis according to the method of the invention, or that said patient have a high risk of having or developing severe or critically severe form of myocarditis when said patient is classified as MIS-A' myocarditis according to the method of the invention.
Thus, the method of the invention can be used to diagnosing severe or critically severe form of myocarditis, wherein it is concluded that said patient has not a severe or critically severe form of myocarditis when said patient is classified as MIS-A+ myocarditis patient according to the method of the invention, or that said patient has a severe or critically severe form of myocarditis when said patient is classified as MIS-A' myocarditis patient according to the method of the invention.
Accordingly, another object of the present invention relates to an in vitro method for assessing a patient’s risk of having or developing severe or critically severe form of myocarditis, comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting in interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with a reference value for each marker, and iii) - concluding that the patient has a low risk of having or developing severe or critically severe form of myocarditis when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a high risk of having or developing severe or critically severe form of myocarditis when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
In other words, the present invention relates to an in vitro method for diagnosing and/or stratifying patients affected with severe or critically severe form of myocarditis comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukinl7 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consistingof interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with a reference value for each marker, and iii) - concluding that the patient has a low risk of having severe or critically severe form of myocarditis when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a high risk of having severe or critically severe form of myocarditis when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
As used herein the term “severe or critical form of myocarditis” refers to a precocious form of myocarditis associated with a lower left ventricle ejection fraction (LVEF), higher in- ICU organ failure, need for mechanical circulatory support. The patient with a severe or critical form of myocarditis had a higher severe sequential organ-failure assessment score (SOFA) as well as a higher simplified acute physiology score-II (SAPS-II). These scores are significant risk factors for in-hospital mortality.
In preferred embodiments, the myocarditis is viral-related myocarditis. In preferred embodiments, the viral-related myocarditis is COVID-19-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis is COVID-19-related fulminant myocarditis.
In some embodiments, the COVID-19-related myocarditis is a COVID-19-related fulminant myocarditis caused by SARS-Cov-2 vaccines (i.e., SARS-CoV-2 vaccine-related myocarditis).
In some embodiment, the patient has been previously vaccinated with SARS-CoV-2 vaccines.
In some embodiment, the patient has been previously or is afflicted with SARS-CoV-2 infection.
Thus, the method of the invention may be used to diagnosis or asses patient’s risk of having or developing severe or critically severe form of myocarditis patient following vaccination against viruses.
Thus, the invention also relates to an in vitro method for assessing a patient’s risk of having or developing severe or critically severe form of myocarditis following vaccination against virus, comprising the steps of: i) determining in a sample obtained from the patient after the vaccination the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2) determined at step i) with the reference value for each marker, and iii) - concluding that the patient has a low risk of having or developing severe or critically severe form of COVID-19-related myocarditis when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a high risk of having or developing severe or critically severe form of myocarditis when the level of interferon alpha-2) determined at step i) is significantly higher than a reference value.
The invention also relates to an in vitro method for diagnosing severe or critically severe form of myocarditis following vaccination against virus, comprising the steps of: i) determining in a sample obtained from the patient after the vaccination the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 , ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 determined at step i) with the reference value for each marker, and iii) - concluding that the patient has not a severe or critically severe form of myocarditis when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient has a severe or critically severe form of myocarditis when the level of interferon alpha-2 determined at step i) is significantly higher than the reference value.
In some embodiment, the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2 is determined between one day and two months after the vaccination.
In some embodiment, the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2is determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days after the vaccination.
Thus, the method of the invention can be used to assess a myocarditis patient’s risk of having a poor prognostic of survival, wherein it is concluded that said patient is at high risk of having a good prognostic of survival when said patient is classified as MIS-A+ myocarditis according to the method of the invention, or that said patient is at high risk of having a poor prognostic of survival when said patient is classified as MIS-A' myocarditis according to the method of the invention.
Accordingly, in other words, the invention relates to an in vitro method for assessing a myocarditis patient’s risk of having a poor prognostic of survival comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of at least one marker selected in the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2determined at step i) with a reference value for each marker, and iii) - concluding that the patient is at high risk of having a good prognostic of survival when the level of of interleukin-22 and interleukin- 17 determined at step i) is significantly higher than their reference values, or
- concluding that the patient is at high risk of having a poor prognostic of survival when the level of interferon alpha-2determined at step i) is significantly higher than the reference value.
In some embodiments, the myocarditis is viral-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis is COVID-19-related fulminant myocarditis.
"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to critical form of COVID-19-related myocarditis, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to critical form of coronavirus disease conversion risk reduction ratios.
"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a late form of of COVID-19-related myocarditis or to one at risk of developing a critical (i.e., precocious) form of COVID-19-related myocarditis. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of critical form of coronavirus disease, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to critical form of COVID-19-related myocarditis, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a critical form of COVID- 19-related myocarditis. In the categorical scenario, the invention can be used to discriminate between late and less severe form of COVID-19-related myocarditis and other subject cohorts at higher risk for critical form of COVID-19-related myocarditis. In other embodiments, the present invention may be used so as to help to discriminate those having precocious and critical form of COVID-19-related myocarditis.
Method for treating myocarditis severity according to the invention
MIS-A- myocarditis patients are more likely to receive mechanical ventilation, extracorporeal membrane oxygenation centralization (ECMO) and renal replacement therapy. The present study shows that marker profiling may have clinical implications for improved personalized treatment.
In another aspect, the invention relates to a method for treating myocarditis in patient in need thereof comprising the steps of: i) diagnosed or classified the patients as MIS-A+ myocarditis patient or MIS-A' patient according to the method of the invention, and ii) administering to said patient a therapeutically effective amount of at least one drug selected from the group consisting of interleukin- 17 inhibitor, interleukin-22 inhibitor, corticosteroid and immunoglobulin when the patient has been diagnosed or classified as MIS-A+ myocarditis patient.
In other words, the invention refers to a method for treating a myocarditis patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 and interferon alpha-2, ii) comparing the level of interleukin-22 and/or interleukin- 17 determined at step i) with a reference values for each marker and iii) administrating to said patient at least one drug selected from the group consisting of interleukin- 17 inhibitor, Interleukin-22 inhibitor, corticosteroid and immunoglobulin when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values
In some embodiment, when the patient has been diagnosed or classified as MIS-A' myocarditis patient, it is concluded that the patient requires transfer to unit able to provide extracorporeal membrane oxygenation (ECMO). The patients diagnosed or classified as MIS- A’ will be clinically monitored and symptomatic treatment could be performed. As used herein, symptomatic treatment include corticosteroid, immunoglobulin as described in Caforio et al (43).
Thus, the invention refers to a method for treating a myocarditis patient comprising the steps of: i) determining in a sample obtained from the patient the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 ii) comparing the level of at least one marker selected from the group consisting of interleukin-22, interleukin- 17 determined at step i) with a reference values for each marker and iii) administrating to said patient at least one drug selected from the group consisting of interleukin- 17 inhibitor, interleukin-22 inhibitor, corticosteroid and immunoglobulin when the level of interleukin-22 and/or interleukin- 17 determined at step i) is significantly higher than their reference values or concluding that said patient requires transfer to unit able to provide extracorporeal membrane oxygenation (ECMO).
In some embodiments, the myocarditis is viral-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis.
In some embodiments, the viral-related myocarditis is COVID-19-related myocarditis is COVID-19-related fulminant myocarditis.
In some embodiments, the COVID-19 related myocarditis is a COVID-19 related myocarditis. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
As used herein, the term “corticoid” or “corticosteroid” means a class of steroid hormones that are produced in the adrenal cortex of vertebrates, as well as the synthetic analogues of these hormones. Two main classes of corticosteroids, glucocorticoids and mineralocorticoids, are involved in a wide range of physiological processes, including stress response, immune response, and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behaviour. Some common naturally occurring steroid hormones are cortisol, corticosterone, cortisone and aldosterone.
The corticoid therapy used synthetic pharmaceutical drugs with corticosteroid-like effects are used in a variety of conditions, ranging from brain tumors to skin diseases. Dexamethasone and its derivatives are almost pure glucocorticoids, while prednisone and its derivatives have some mineralocorticoid action in addition to the glucocorticoid effect. Fludrocortisone (Florinef) is a synthetic mineralocorticoid. Hydrocortisone (cortisol) is typically used for replacement therapy, e.g., for adrenal insufficiency and congenital adrenal hyperplasia.
As used herein, the term “interleukin- 17 inhibitor’ or “IL- 17 inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of interleukin-17. In a particular embodiment, the IL-17 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. In preferred embodiment, the interleukin- 17 inhibitor is an interleukin- 17 antibody. In preferred embodiment, the IL-17 inhibitor is secukinumab, iwekizumab or brodalumab.
As used herein, the term “interleukin-22 inhibitor” or “IL-22 inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of interleukin-22. In a particular embodiment, the interleukin-22 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. In preferred embodiment, the interleukin-22 inhibitor is an interleukin-22 antibody, such as fezakinumab.
As used herein, the term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.
As used herein, the term “aptamers” refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique. In a particular embodiment, the inhibitor is a neutralizing antibody.
In a particular, the inhibitor is an intrabody having specificity for target molecule, i.e., - 22 or IL-17. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
In some embodiments, the inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of target molecule, i.e., IL-22 or IL-17. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Antisense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. As used herein, the term “anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of target mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of target proteins, and thus activity, in a cell.
As used herein, the term “immunoglobulin” also known as “intravenous immunoglobulin” or “IVIG”, has its general meaning in the art and refers to antibodies produced naturally by the body’s. IVIG is a blood product prepared from the serum of different donor.
In some embodiments, the drug is administered intravenously.
As used herein, the term “extracorporeal membrane oxygenation” or “ECMO” has its general meaning in the art and refers to an extracorporeal technique of providing prolonged cardiac and respiratory support to patient in need thereof. In some embodiments, the extracorporeal membrane oxygenation is a venoarterial-extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO is a ECMO where the cannulas are placed in vein (for extraction) and in artery (for infusion) and is required when native cardiac function is minimal to mitigate increased cardiac stroke work associated with pumping against retrograde flow delivered by the aortic cannula.
As used herein, the term “renal replacement therapy” or “RRT” has its general meaning in the art and refers to a therapy that replaces the normal blood-filtering function of the kidneys’ patient.
In some embodiments, the methods of diagnosis/stratify/classify/prognostic of survival/ are performed are performed in vitro or ex vivo.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURE:
Figure 1: Circulating cytokines levels in fulminant COVID-19-related myocarditis. Comparison of 6 circulating serum cytokines levels (IL-8, IL-10, IL-17, IL-22, IFN-a2 and TNF-a) in patients with MIS-A+/MIS-A- and healthy controls. MIS-A+ had higher IL-22, IL- 17 and TNF- a, while MIS-A- had higher IFN-a 2 and IL-8. Methods: serum concentrations of IL-8, IL-10, IL-22 and TNF-a were measured on a Quanterix® SP-X™ imaging and analysis platform using the Human CorPlex Cytokine Panel Array kit (Quanterix, Lexington, MA, USA). Single-plex bead-based ultrasensitive immunodetection of IL-17A and IFN-a was performed by digital ELISA using the SimoaTM (single molecule array) HD-1 analyzer (Quanterix), according to the manufacturer’s instructions. For box and whisker plots: the center line denotes the median value (50th percentile), while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles.
Figure 2: Principal component analyses (PCA): of cytokines measured in fulminant COVID-19 related myocarditis and combining clinical and biological features in fulminant COVID-19 related myocarditis. Unsupervised PCA was performed using R v3.6.2 with the FactoExtra and FactoMineR functions, on z-scaled loglO-transformed cytokine concentrations. Samples with missing data were excluded from the PCA analysis for one MIS- A+ and two MIS-A- patients). Ellipses with 66 % confidence interval are drawn for each group. A. The principal component analysis of circulating serum cytokines. B. The principal component analysis including clinical findings, laboratory findings and immunological profiles highlights the main features of MIS-A- and MIS-A+ fulminant COVID-19 related myocarditis phenotypes.
EXAMPLE:
MATERIAL AND METHODS:
Patients and controls
We retrospectively reviewed the database of our 26-bed ICU between March 2020 and June 2021, and included all patients admitted for clinically suspected myocarditis with proven SARS-CoV-2 infection. Clinically suspected myocarditis was then adjudicated as definite or probable myocarditis according to the definition by Bonaca et al.( 5) following clinical investigations. Proven SARS-CoV-2 infection was confirmed by positive RT-PCR in either nasopharyngeal aspirate, lower airway respiratory samples or serum and/or positive serology showing the presence of circulating anti-N or anti-S-RBD antibodies, in patients not vaccinated against COVID-19. All laboratory analyses are performed as the standard of care in the myocarditis work-up of our institution. In addition, healthy SARS-CoV-2 -negative individuals (n=10) were included as controls for cytokine measurements.
Data collection
The following information was collected on standardized forms: epidemiologic parameters; severity of underlying condition according to the McCabe & Jackson criteria; medical history; COVID-19 infection history, manifestations and complications; MIS-A criteria; day-0 Sequential Organ Failure Assessment (SOFA) score and Simplified Acute Physiology Score (SAPS) II; day-0 and in-ICU clinical and biological parameters; day-0 and in-ICU organ-failure support treatment; day-0, in-ICU and last-follow-up echocardiography parameters; in-ICU cytokine profiling; in-ICU SARS-CoV-2 and myocarditis-specific treatment; in-ICU and follow-up CT-scan and cardiac magnetic resonance imaging (CMR); complications; vital status at ICU and hospital discharge, as well as at last follow-up.
SARS-CoV-2 RT-PCR and serological analyses
Detection of SARS-CoV-2 was carried out by RT-PCR in clinical specimens, using Cobas®6800 SARS-CoV-2 Test-Roche Diagnostics (Roche Diagnostic, Meylan, France) and serological detection of IgG anti-N and IgG anti-S SARS-CoV-2 was performed by ELISA on the Abbott platform (Abbott Diagnostics, Rungis, France) in accordance with the manufacturer’s specifications.
Cytokine measurement
Whole blood was collected in anticoagulant-free tubes, serum was separated by centrifugation and stored at -80°C. Serum concentrations of IL-ip, IL-4, IL-5, IL-6, IL-8, IL- 10, IL-12p70, IL-22, IFN-y, and TNF-a were measured on a Quanterix® SP-X™ imaging and analysis platform using the Human CorPlex Cytokine Panel Array kit (Quanterix, Lexington, MA, USA). Single-plex bead-based ultrasensitive immunodetection of IL-17A and IFN-a was performed by digital ELISA using the SimoaTM (single molecule array) HD-1 analyzer (Quanterix), according to the manufacturer’s instructions. Serum IFN-P levels were quantified using a highly sensitive ELISA kit (PBL Assay Science, Piscataway, NJ, USA), according to the manufacturer’s instructions. Serum cytokine concentrations were interpolated from the correspondent calibration curve taking into account the dilution factor. All cytokine concentrations were expressed in pg/mL. Samples with non-detectable values or those above the detection range were replaced by the limit of detection value (LOD) and the upper limit of quantification (ULOQ), respectively.
Anti-IFN-a and RNA-polymerase-III autoantibodies
Auto-antibodies against IFN-a were quantified using the anti-IFN-a Antibody Human ELISA Kit (Thermo Fisher, Invitrogen), according to the manufacturer's instructions. Calibrators were run in duplicate and fit with a 4-parameter logistic (4PL) regression. The concentration of anti-IFN-a antibodies in samples was interpolated from the calibration curve by multiplying the obtained values with the dilution factor. The positivity threshold was 15 ng/mL. For RNA-polymerase-III autoantibodies screening, an indirect immunofluorescence assay was run on HEp-2000 cells (Immuno Concepts, Sacramento, CA, USA). When positive (>1/80) and when the immunofluorescence labeling pattern was evocative of RNA-polymerase Ill autoantibodies (fine-speckled nuclear-labeling pattern with small dots), a confirmatory immunodot assay (Euroline Systemic Sclerosis Test, Bio Advance, Bussy Saint-Martin, France) was carried out.
Statistical analyses
Continuous variables are expressed as median [interquartile range] and compared with Wilcoxon’s signed-rank tests. Categorical variables are expressed as number (percent) and compared with %2 tests or Fisher’s exact tests. Cumulative probabilities of survival were calculated using Kaplan-Meier method and compared with Log-Rank tests. A two-tailed p- value < 0.05 was considered statistically significant. Analyses were computed with StatView v5.0 (SAS Institute, Cary, NC) and IBM SPSS Statistics v22.0 software (IBM Corp, Armonk, NY). Unsupervised principal component analysis (PCA) was performed using R v3.6.2 with the FactoExtra and FactoMineR functions, on z-scaled loglO-transformed cytokine concentrations. Samples with missing data were excluded from the PCA analysis for one MIS- A+ and two MIS-A- patients).
Ethical considerations
This study was conducted in accordance with the declaration of Helsinki using the database registered at the Commission Nationale de I’lnformatique et des Libertes (CNIL, registration no. 1950673). In agreement with the ethical standards of our hospital’s institutional review board, the Committee for the Protection of Human Subjects and French law, written informed consent was not needed for demographic, physiological and hospital-outcome data analysis, because this observational study does not modify existing diagnostic or therapeutic strategies; however, patients and/or their relatives were informed of their anonymous inclusion in the study.
RESULTS :
General Patients characteristics
Between March 2020 and June 2021, 38 patients requiring ICU admission for clinically suspected fulminant COVID-19-related myocarditis were included in this study. They were mostly men (66%) of young age (median [IQR25-75] age, 27.5 [19-37] years) with few comorbidities. All had positive SARS-CoV-2 RT-PCR (37%) or serology (68%) with a median delay of five days between COVID-19 symptoms onset and the first manifestation of myocarditis. None had previously received any COVID-19 vaccine. Most frequent symptoms were fever (95%), abdominal pain or nausea (60%), chest pain (47%) and dyspnea (42%). At admission, patients had severely impaired left ventricle function (LVEF 20% [14-10 37], LVOT-VTI 11 cm [6-15]), an increased high-sensitivity T-troponin (median 1,300 [IQR 11 = 486-4,750] ng/mL) and 79% presented with cardiogenic shock. When performed (n=10), coronary angiography was normal. COVID-19 pneumonia was noted on CT scan examination in 29% of cases. In the 26 patients who had CMR evaluation, myocardial edema and late gadolinium enhancement were reported in 73% and 54% respectively.
Three patients without recovery of cardiac function underwent myocardial biopsy. None were MRI-guided, as all were taken under mechanical circulatory support. Two surgical biopsies were taken in patients during venoarterial-extracorporeal membrane oxygenation (VA- ECMO) centralization. The first one was an apical surgical biopsy in a patient cannulated under cardiopulmonary resuscitation which was nonconclusive. The second surgical biopsy highlighted myocarditis with an inflammatory infiltrate associated with myocyte dystrophy and oedema. SARS-CoV-2 RT-PCR was negative and electron microscopy analysis failed to identify viral particles in cardiomyocytes despite active myocarditis lesions on the evaluated sample. Last biopsy was endomyocardial and disclosed a mild lympho-histiocytic myocarditis with no oedema but severe necrosis. SARS-CoV-2 RT-PCR was negative. Twenty-nine (76%) patients met the Bonaca classification criteria for definite myocarditis while the others had probable myocarditis.
In-ICU evolution and Outcomes
Median length of stay in ICU was 6 days. Seventy nine percent of the patients received dobutamine, 60% norepinephrine, 50% mechanical ventilation and 29% renal replacement therapy. Four patients had a large pericardial effusion requiring drainage. Sixteen (42%) patients required mechanical circulatory support with VA-ECMO 1 [0-1] day following ICU admission, for a median duration of 7 days. Twenty-eight (74%) were treated with corticosteroids and 27 (74%) with intravenous immunoglobulins. In-hospital mortality was 13%. None of the survivors required cardiac transplantation or long-term ventricular assist device. Among the five deceased patients, all had multiorgan failure before VA-ECMO implantation, including three cannulations during cardiopulmonary resuscitation. None could be weaned from VA-ECMO because of severe cardiac dysfunction. Median LVEF at ICU and hospital discharge was 42% [30-54] and 60% [50-64], respectively. Twenty one survivors (64%) received betablockers at discharge and 25 (76%) were treated with angiotensin- converting-enzyme inhibitors, until distant evaluation with a cardiologist. At the last follow-up (median [IQR] 235 [155-359] days), 32 patients were alive, all but one had normal LVEF. One patient was lost of follow-up. Comparison between MIS-A+ and MIS-A- patients
Twenty -five (66%) patients met the MIS-A criteria. By definition, MIS-A+ patients had more frequent fever, skin rash, enanthema, pharyngitis and conjunctivitis, as compared to MIS- A- patients. In addition, they had, as expected by the MIS-A definition, higher levels of systemic inflammation markers, including circulating leucocytes, procalcitonin, C-reactive protein and fibrinogen.
The median delay between COVID-19 symptoms onset and occurrence of myocarditis was shorter in MIS-A- patients: 3 vs. 8 days. Noteworthy, the delay between first COVID-19 symptoms and myocarditis was 32 [25-44] days among the 12 MIS-A+ patients with prior proven symptomatic SARS-CoV-2 infection. The rate of positive serology was lower in MIS- A- patients (15% vs. 96%) and their titer was also much lower than in MIS-A+ patients (p<0.0001). Conversely, positive nasopharyngeal RT-PCR at the time of myocarditis was infrequent in MIS-A+ (16%), as compared to MIS-A- (85%) patients.
MIS-A- patients had swifter ICU admission after myocarditis onset (1 vs. 4 days) with a more severe presentation (day-0 SOFA score of 11 versus 6 in MIS-A+ patients). They had a lower left ventricular ejection fraction (LVEF 10 vs. 30% and LVOT-VTI 5 vs. 13 cm) and were more likely to receive norepinephrine, mechanical ventilation and renal replacement therapy. Large pericardial effusions were also more frequently observed in MIS-A-patients. The median lactate level was 5.5 versus 2.1 mmol/L in MIS-A- and MIS-A+ patients, respectively. Finally, MIS-A- patients were more likely to require VA-ECMO than MIS-A+ patients (92% vs. 16%), and had a higher in-ICU mortality (31% vs. 4%, p=0.04). The 3-month cumulative probabilities of survival ± standard errors for MIS-A- and MIS-A+ patients were respectively: 68±13% and 96±4%, Log-Rank test p=0.01.
Cytokine profiling highlighted the presence of two distinct cytokine production profiles (Figure 1): MIS-A+ had higher IL-22 (9.93 vs. 1.5 pg/mL, p<0.0001), IL-17 (3.2 vs. 0.15 pg/mL, p<0.0001) and TNF-a (21.1 vs. 8.0 pg/mL, p=0.05) levels, as compared to MIS-A- patients, while the latter had higher IFN-a2 (2.4 vs. 0.013 pg/mL, p=0.001) and IL-8 (158.7 vs. 65.7 pg/mL, p=0.02), respectively. Moreover, RNA-polymerase III autoantibodies were found in seven (54%) MIS-A- patients, five of them being female.
Finally, to elucidate the relative importance of the various bio-clinical parameters listed above with the clinical profile of MIS-A+ or MIS-A- patients, we performed non-supervised PCA using study parameters contributing, in a statistically significant manner, to inter-patient variation. The results from PCA underlined important overall differences between MIS-A+ and MIS-A- patients, (Figure 2). The data also further highlight parameters most contributing to either clinical status i.e.'. fibrinogen (p<0.0001), CRP (p<0.0001), IL-17 (p<0.0001), IL-22 (p<0.0001), IFN-a2 (p=0.001) levels, SARS-CoV-2 serology (p<0.0001) and SARS-CoV-2 RT-PCR (p<0.0001), LVEF (p=0.01) values on admission and the presence of RNA polymerase III autoantibodies (p=0.001) (data not shown).
DISCUSSION:
In this retrospective monocenter cohort of fulminant COVID-19-related myocarditis, we applied the MIS-A criteria case definition and we identified two subsets of patients with very different clinical/biological presentations, outcomes, and immunological profiles. This phenotypic heterogeneity being likely explained by important differences in pathophysiological mechanisms.
The patients in this cohort were mostly young men with severely impaired cardiac function, frequently requiring VA-ECMO, with infrequent concomitant COVID-19-associated pneumonia. All survivors recovered a near normal cardiac function at distant follow-up. To our knowledge, this study is the largest cohort of COVID-19-related fulminant myocarditis and extends prior reports of COVID-19-related myocarditis(9, 16-19) and fulminant non-COVID- 19 myocarditis(10, 20-23).
This analysis underscores the major clinical and immunological differences between patients with fulminant COVID-19-related myocarditis fulfilling or not MIS-A criteria. The original description of MIS-C was reported in May 2020(11) and MIS-A a few months afterwards(12, 24, 25). This somewhat delayed description, together with the rarity of the disease may have participated to an under-recognition of MIS in the adult population. Furthermore, whereas MIS-C is now well defined with classification criteria established by the World Health Organization and the Center for Disease Control and Prevention(26, 27), only the latter has adapted its criteria to the adult population(13).
The main differences between the phenotypes of MIS-A+ and MIS-A- patients are summarized in the Central Illustration. MIS-A+ COVID-19 related myocarditis appears to be a post-infectious complication of SARS-CoV-2 infection, as suggested by the higher delay between COVID-19 symptoms and myocarditis, as well as by frequently positive serology and negative (or slightly positive) RT-PCR. Mucocutaneous manifestations are frequent in addition to laboratory evidence of severe systemic inflammation. Heart failure is more progressive, leading to fewer accounts of refractory cardiogenic shock, and is associated with a lower mortality rate. Conversely, MIS-A- fulminant COVID-19-related myocarditis occurred at the early phase of SARS-CoV-2 infection (negative or slightly positive serology and positive RT- PCR) with an explosive and refractory cardiogenic shock in nearly all patients leading to high morbidity and mortality.
Interestingly, these different clinical phenotypes are supported by immunological findings. The frequency of RNA-polymerase III autoantibodies is high in MIS-A-, while absent in MIS-A+ patients. The presence of these rare autoantibodies, usually associated with severe systemic sclerosis, has been previously reported by Pineton de Chambrun M et al. in patients with severe recurrent myocarditis and/or pericarditis, especially related to influenza virus(28). Their role in the susceptibility to viral myocarditis is not yet elucidated. They might reflect altered immune defenses toward viral infections or alternatively exaggerated antiviral responses leading to organ damage. Another patient with recurrent viral myocarditis, including COVID- 19-related myocarditis, has been recently reported(29).
The cytokine profiles of these patients were also found definitely different in the two clinical phenotypes (Figure 2). In MIS-A- patients, high levels of systemic circulating antiviral IFN-a2 likely arise from the ongoing viral infection, in relation to detectable viral replication and yet undetectable anti-SARS-CoV-2 IgG humoral responses. Levels of IL-8, a proinflammatory cytokine, were also more elevated in MIS-A-, as compared to MIS-A+ patients, further underlining the dominance of an innate type of immune response in the former group. Conversely, elevated IL-17 and IL-22 levels were found particularly associated with the MIS-A+ phenotype, in agreement with the mucocutaneous manifestations observed in these patients. IL- 17 and IL-22 shape innate defenses at mucosal and epithelial surfaces, IL- 17 being pro-inflammatory and involved in the pathogenesis of several autoimmune diseases, while the latter cytokine is playing an important role in tissue regeneration(30). Of note, the extremely high serum IL-10 levels observed both in MIS-A- and MIS-A+ patients have been previously associated with severe myocardial injury(31), and increased risk of death in severe COVID-19 patients(29).
MIS pathogenesis is not fully understood, but the delay between SARS-CoV-2 infection and disease onset, and over-expression of mucosal T cell cytokines (IL-22 and IL-17) suggest a role for the adaptive immune response in MIS-A+ patients. Conversely, in MIS-A- cases, innate anti-viral immunity and/or direct toxicity of the virus are more likely involved in heart tissue injury. In our series, SARS-CoV-2 RT-PCR on 2 MIS-A- endomyocardial biopsies (EMBs) were negative. This is in line with previous cases reports(5, 33), even if positive SARS- CoV-2 RT-PCR in myocardial samples have also been sporadically reported(34, 35). To the best of our knowledge, only one study demonstrated the presence of viral particles in cardiomyocytes by electronical microscopy(36), with only mild interstitial inflammatory infiltrate and no necrosis or microthrombosis, thereby suggesting that the underlying mechanism of myocarditis development was mainly related to a virus-mediated immune response. The EMBs published results from fulminant COVID-19 related myocarditis often reported important myocardial edema with no or mild inflammatory infiltrate or necrosis(2, 37), a finding which is also consistent with cardiac MRI 4 observations^, 8).
The phenotypic clustering of patients with fulminant COVID-19-related myocarditis seems relevant for their management. Indeed, MIS-A- cases, owing to the high risk of evolution towards refractory cardiogenic shock should be urgently referred to a center with VA-ECMO capability and closely monitored to avoid a too late cannulation, especially under cardiopulmonary resuscitation, known to be associated with poor outcome(38). The five patients who died in our series had late VA-ECMO implantation, while having multiple organ failure or under resuscitation. Conversely, the risk of evolution towards refractory cardiogenic shock is lower in MIS-A+ cases. Our results are consistent with those of a large series of 186 MIS-C from the United States, where only eight patients required VA-ECMO and four died(39). MIS-A+ patient identification is all the more important given that numerous data support the efficacy of corticosteroids and/or intravenous immunoglobulins in MIS-C(40). The best treatment regimen is yet to be determined because conflicting results have been reported with standalone or combination treatment(41, 42). However, one should take with caution the results of non-randomized/non-blinded therapeutical intervention in a disease where spontaneous recovery occurs in most patients in a few days.
This study has some limitations that deserve mentioning. First, the external validity is limited by its monocentric and retrospective nature. Notably, as an ECMO center, there might be a selection bias towards the inclusion of the most severe patients. Thus, MIS-A- patients could have been better identified and transferred earlier in our center. Second, while being the largest series of fulminant COVID-19 related myocarditis, the sample size remains small, limiting the power of the study. Lastly, EMBs were performed in only 3 patients, while expert consensus and guidelines recommend to consider EMB in fulminant presentation, for its diagnostic and therapeutic implications(43-45). However, coagulation disorders are frequent in COVID-19-related myocarditis and VA-ECMO patients. The benefit/risk ratio was evaluated against EMB in all but three cases, especially given the known diagnosis of SARS-CoV-2 infection. It is nevertheless unfortunate that we cannot provide a more extensive characterization of COVID-19-related myocarditis histopathological findings in MIS-A+ and MIS-A- patients. REFERENCES:
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