Patent Description:
A new strain of coronavirus, severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>) was recognized in December <NUM> in China. Since then, more than <NUM> million cases have been reported in <NUM> countries, causing more than <NUM>,<NUM>,<NUM> deaths. Incubation time is approximately <NUM> days. Clinical presentation is broad. Most common symptoms include fever, cough and fatigue. However, it can be also presented from a totally asymptomatic infection to an acute respiratory distress syndrome with multiorgan dysfunction.

Hospitalized patients usually present moderate or severe respiratory disease with high fever and pneumonia. Certain biomarkers have been related to this situation as well as radiological alterations. Nevertheless, clinical, radiological and laboratory features cannot be easily distinguished from pneumonia induced by other viral or bacterial infections.

Precise and early diagnosis is the cornerstone therefore for optimal both prevention and treatment, especially in hospitals. Polymerase chain reaction (PCR) is the main test but its sensitivity is variable and, in some reports, reaches <NUM>%. The correct sample collection is necessary, which requires the performance of new PCR tests if the result is negative and a high probability of infection exist. All of this causes a delay in the diagnosis and optimization of treatment, so necessary not only in each patient survival but also in preventive quarantine and hospital organization.

<NPL>) discloses IP-<NUM> to be elevated compared to healthy controls and that IL-<NUM> levels were significantly decreased in patients who succumbed to infection.

<NPL>) discloses that high IL-<NUM> levels are often strongly correlated with respiratory failure in Covid-<NUM> patients.

So, there is an unmet medical need of finding new tools for improving the diagnosis of coronavirus infection. The present invention is thus focused on solving this problem and, consequently, a biomarker fingerprint which efficiently contributes to the proper diagnosis of coronavirus infection is herein presented.

The present invention refers to an in vitro method for the diagnosis of a coronavirus infection which is preferably carried out by using minimally-invasive biological samples obtained from the subject, most preferably plasma samples.

The inventors of the present invention consider that the cytokine profile plays an important role in the pathogenesis of hospitalized patients and, consequently, finding a specific cytokine profile could be of utmost importance for performing an early diagnosis of the coronavirus infection, thus giving rise to the possibility of administering targeted treatments in an early stage of the disease.

Hence, the inventors of the present invention have identified a cytokine fingerprint that allows an early diagnosis of COVID-<NUM>. It is also important to note that the present invention is implemented in by using minimally-invasive biological samples obtained from the subject, preferably plasma samples.

Particularly, the examples and figures provided below show strong statistical results with respect to three specific cytokines, particularly: Interferon gamma-induced protein <NUM> (IP-<NUM>), platelet-derived growth factor (PDGFBB) and interleukin <NUM> (IL4). According to the present invention, any of these cytokines show a relevant statistical result (IP10 shows AUC = <NUM>, PDGFBB shows AUC = <NUM> and IL4 shows AUC = <NUM>) and may independently contribute to the diagnosis of a coronavirus infection. Moreover, when the three cytokines were combined in a model, an AUC of <NUM> was obtained with <NUM>% of sensitivity and <NUM>% of specificity.

So, the present invention describes the molecular fingerprint of cytokines and chemokines that define COVID-<NUM> infection and that therefore could have a clinical impact as novel diagnose biomarkers complementary to the PCR or the antigen tests. Particularly, high levels of IP10 and PDGF-BB coupled with low levels of IL-<NUM> can predict COVID-<NUM> infection. This model was internally validated revealing a <NUM>% accuracy. Moreover, another validation in other cohort of patients, the external one, confirmed our results.

In summary, the inventors of the present invention have developed and validated a simple logistic prediction model for COVID-<NUM> disease based on the determination of just <NUM> blood cytokines: IL4, IP10 and PDGFBB. Their implementation in routine would be easy and quick, associating extraordinary capacities of accuracy, sensitivity and specificity in patients admitted to the hospital. It can be used as a complementary tool to aid clinical decision-making.

So, the first embodiment of the present invention refers to an in vitro method for the diagnosis of a coronavirus infection (hereinafter the method of the invention) in a subject which comprises: a) Measuring the level of IP10 in a minimally-invasive biological sample obtained from the subject, and b) wherein if a deviation or variation of the level of IP10 is identified, as compared with a pre-established reference level measured in healthy control subjects, it is indicative that the subject is suffering from coronavirus infection.

In a preferred embodiment, the method of the invention further comprises: a) Measuring the level of PDGFBB or IL4 in a minimally-invasive biological sample obtained from the subject, and b) wherein if a deviation or variation of the level of PDGFBB or IL4 is identified, as compared with a pre-established reference level measured in healthy control subjects, it is indicative that the subject is suffering from coronavirus infection.

In a preferred embodiment, the method of the invention comprises: a) Measuring the level of [IP10, PDGFBB and IL4] in a minimally-invasive biological sample obtained from the subject, and b) wherein if a deviation or variation of the level of [IP10, PDGFBB and IL4] is identified, as compared with a pre-established reference level measured in healthy control subjects, it is indicative that the subject is suffering from coronavirus infection.

In a preferred embodiment, the method of the invention is carried out in in plasma, serum or blood samples obtained from the subject.

In a preferred embodiment, the method of the invention comprises: a) Measuring the level of [IP10 PDGFBB and IL4] in plasma samples obtained from the subject, and b) wherein if a deviation or variation of the level of [IP10 PDGFBB and IL4] is identified, as compared with a pre-established reference level measured in healthy control subjects, this is indicative that the subject is suffering from coronavirus infection.

In a preferred embodiment, if the level of the biomarkers or the risk score value measured in the subject is statistically higher or lower as compared with the level measured in healthy control subjects, this is indicative that the subject is suffering from coronavirus infection. So, according to the method of the invention, after measuring the level of the cytokines, a score value is obtained for the signature and this score value is compared with a threshold value which defines the diagnostic rule. If this score value is higher/lower than the threshold, then the corresponding sample is classified as a positive sample, which is an indication that the patient might be suffering from a coronavirus infection. The threshold value has been defined in order to optimize sensitivity and specificity values. Consequently, in a preferred embodiment, the method of the invention comprises: a) Measuring the level of the above cited combination of cytokines, in a biological sample obtained from the subject, b) processing, using for instance a processor unit, the concentration values in order to obtain a risk score and c) wherein if a deviation or variation of the risk score value obtained for any of the above cited combinations of biomarkers is identified, as compared with a reference value, this is indicative that the subject is suffering from a coronavirus infection.

In this context, a computer comprising a processing unit can be used, which is configured to:.

In a preferred embodiment, the method of the invention the method of the invention is focused on the diagnosis of a coronavirus infection is caused by SARS-CoV-<NUM>.

In a preferred embodiment, the result obtained with the method of the invention is confirmed by an image technique, for example (non-exhaustive list): Echography, CT scan or computed tomography scan (CAT scan) or Magnetic resonance imaging (MRI); or PCR (Polymerase Chain Reaction).

Also disclosed is the in vitro use of IP10, [IP10 and PDGFBB], or [IP10, PDGFBB and IL4] for the diagnosis of a coronavirus infection in a subject, departing from a minimally-invasive biological sample obtained from the subject.

In a preferred embodiment, the present invention refers to the use of IP10, [IP10 and PDGFBB], or [IP10, PDGFBB and IL4] for the diagnosis of a coronavirus infection caused by SARS-CoV-<NUM> in a subject departing from a minimally-invasive biological sample obtained from the subject.

In a preferred embodiment, the present invention refers to the use of [IP10, PDGFBB and IL4] for the diagnosis of a coronavirus infection departing from plasma, serum or blood samples obtained from the subject.

Also disclosed is a kit configured for implementing the method of the invention which comprises: a) Reagents for obtaining a plasma sample from the subject, and b) reagents for determining the level of [IP10, PDGFBB and IL4].

Also disclosed is the use of the kit for the diagnosis of a coronavirus infection, preferably a coronavirus infection caused by SARS-CoV-<NUM>.

Also disclosed is an antiviral composition for use in the treatment of a coronavirus infection which has been diagnosed by following performing the method of the invention. Alternatively, the present disclosure refers to (not claimed) method for treating patients who may be suffering or are suffering from coronavirus infection and have been diagnosed with the method of the invention, which comprises administering a therapeutically effective amount of an antiviral composition. In a preferred embodiment the antiviral treatment is selected from the treatments disclosed in [<NPL>].

For the purpose of the present invention the following terms are defined:.

Prospective study including <NUM> cases and <NUM> controls. Cases were over <NUM> years old patients who were diagnosed with COVID-<NUM> and admitted at the Hospital Clinico Universitario (Valladolid, Spain) between the <NUM>th of March and the <NUM>th of April <NUM>. Positive result in severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>) infection was confirmed by polymerase chain reaction (PCR) testing of a nasopharyngeal sample. Patients with other infections and/or any chronic terminal illnesses were excluded from the study. Controls were represented by <NUM> age and gender matched healthy volunteers for the normalization of the analytical data of the cytokines. Written consent was obtained by all study participants following ethics approach by the Hospital's Clinical Ethics Committee (PI <NUM>-<NUM>).

Blood samples were collected from the patients following their first night at the hospital and <NUM>:<NUM> using <NUM>% sodium citrate tubes and centrifuging them at <NUM> for <NUM> minutes at room temperature. Plasma was aliquoted and cryopreserved at -<NUM> until used.

Plasma aliquots were used for the quantification of soluble mediators by the kit <NUM>-plex Human XL Cytokine Luminex Performance Panel (R&D). A duplicate analysis was performed on each sample.

Cytokines or chemokines included in the Panel were BDNF, EGF, Eotaxin (also known as CCL11), FGF-<NUM>, GM-CSF, GRO-α (CXCL1), HGF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-<NUM>, IL-12p70, IL-<NUM>, IL-<NUM>, IL-17A (CTLA-<NUM>), IL-<NUM>, II,-1RA, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM> (also known as CXCL8), IL-<NUM>, IP-<NUM> beta (CCL4), IP-<NUM> (CXCL10), LIF, MCP-<NUM> (CCL2), MIP-1α (CCL3), NGF-β, PDGF-BB, PIGF-<NUM>, RANTES (CCL5), SCF, SDF-1α, TNFα, TNFβ, VEGF-A, VEGF-D.

Demographic, clinical and analytical data (Leukocytes, Lymphocytes, Neutrophils, Platelets, C-Reactive Protein, Ferritin and D-Dimer) of each patient were recorded to describe the clinical phenotype.

The hospital protocol for the treatment of COVID-<NUM> pneumonia in March and April <NUM> included: Lopinavir/Ritonavir (Kaletra®. Abbott) <NUM>/<NUM>/ml solution twice a day. Hydroxychloroquine (Dolquine®. Rubió) <NUM> twice a day. According to inflammatory criteria, treatment would also add Interferon 1β (Betaferon®. Bayer) <NUM> every <NUM> hours, corticosteroids <NUM> every day for three days, Tocilizumab (RoActemra ®. Roche), Baricitinib (Olumiant®. Lilly) or Anakinra (Kineret®. In case of suspected bacterial superinfection, antibiotic treatment was required. Oxygen support (nasal cannula, high flow nasal cannula and non-invasive or invasive mechanical ventilation) was administered to patients based on the severity of hypoxemia.

Demographic and clinical characteristics were evaluated using the Chi-square test for categorical variables and the Mann-Whitney U test for continuous variables when appropriate. Mean values and <NUM>% confidence intervals (<NUM>% CI) were used to describe quantitative variables, while median values and interquartile range (IR) were used for ordinal ones.

Individual logistic regression models, in which the exponential of the coefficients can be directly interpreted as odds ratio, was applied. Multivariate model used all those markers that showed a certain degree of association with the disease. A cutoff point < <NUM> in the p-value was required in the individual model. When there are a large number of variables to be included in the multivariate model, especially when the sample size is small, the problem arises of selecting a good subset of variables that intervene in the multivariate model. To overcome that, we used an algorithm that lists all possible sets of biomarkers, leaving us with the best model. The choice of the model was made using the akaike information criterion (AIC).

Internal validation tried to determine the discrimination capacity of the model and its capacity to classify new individuals correctly. As a global measure, we use the area under the ROC curve (AUC), knowing that a model is a perfect classifier when the AUC is <NUM>.

For cytokine analysis, to impute cytokine values below detection limit, robust regression on order statistics was used: this method performs a regression to impute low values assuming log-normal quantiles for samples with a detection rate of at least <NUM>%, after checking that the data follows a log-normal distribution. To accomplish this, the non-detects and data analysis (NADA) R package was used. Molecules detected in less than <NUM>% of the samples were not statistically analyzed any further. Cytokine expression data were transformed using the logarithmic base <NUM> scale. Pro-inflammatory/anti-inflammatory cytokine ratios were also calculated.

Statistical analysis was performed by a PhD-licensed statistician (co- author IF) using the R statistical package version <NUM>. <NUM> (R Core Team; Foundation for Statistical Computing, Vienna, Austria; URL: https:// www. Statistical significance was set at P ≤ <NUM>.

There were no statistical differences in gender and age among cases and controls (Table <NUM>). Median age was <NUM> years in the COVID-<NUM> group, with <NUM>% male, while the control group median age was <NUM> years, with male the <NUM>%. Main comorbidities in the cases were hypertension (<NUM>%), diabetes (<NUM>%), chronic obstructive pulmonary disease (<NUM>%) and coronary disease (<NUM>%). Referred to laboratory variables, there were statistically significant differences between both groups as COVID patients had lower leukocytes, lymphocyte and platelets and higher neutrophils levels. Patients were hospitalized for a median of <NUM> days, which was increased up to <NUM> days in those who required access to the intensive care units. Finally, death rate of the COVID-<NUM> patients was <NUM>% (<NUM> patients in total).

Of the total <NUM> studied cytokines (see Table <NUM> showing the level of cytokine comparing cases and control) , <NUM> out of them had to be excluded (FGF-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, NGFβ and TNFβ) as they could not be detected in at least <NUM>% of the patients. Of the remaining <NUM> cytokines, <NUM> showed statistically significant differences between the patients and the controls (Table <NUM>). Most of these cytokines were increased in the patients (BNDF, HGF, IL-1α IL-1β, IL-<NUM>, IL-17A, IL-<NUM>, IL-1RA, IL-<NUM>, IL-<NUM>, IL-<NUM>, IP1b, IP10, MCP1, PDGF-BB, RANTES, VEGFA and VEGFB). Of note, IL-1RA and PDFGBB were over <NUM> times expanded in the patients referred to the control group. On the other hand, a total of <NUM> cytokines (Eotaxin, IFNγ, IL-<NUM>, IL-<NUM>, IL-<NUM> and SDF1α) were decreased in the COVID-<NUM> patients.

The individual logistic regression models, adjusting the results for age and gender, showed statistically significant overexpression of several cytokines (BDNF, HGF, IL-1β, IL-<NUM>, IL-17A, IL-<NUM>, IL-1RA, IL-<NUM>, IL-<NUM>, IL-<NUM>, IP1b, IP10, MCP1, PDGFBB, VEGFA, VEGFD, RANTES) while IFN γ, IL-<NUM>, IL-<NUM> and SCF were under-expressed (<FIG>).

The best multivariate model, the one with the lower AIC, was chosen. After M3 it was not possible to find valid models. In our case, we chose model <NUM> (AIC =<NUM>) that included <NUM> cytokines: IL-<NUM>, IP10 and PDGF-BB. See Table <NUM> wherein it is identified the best multivariate model following AIC ("Akaike's Information Criterion").

This multivariate model showed a clear association between the levels of these <NUM> cytokines and the high probability of COVID-<NUM> infection (see Table <NUM> showing the multivariate model and the association between cytokine levels and risk of Covid infection. CI, confidence interval; OR, Odds ratio). High IP10 [OR <NUM>, <NUM>% CI (<NUM>-<NUM>), p = <NUM>] and PDGF-BB [OR <NUM>, <NUM>% CI (<NUM>-<NUM>), p = <NUM>] levels increased the risk of infection, reaching <NUM> and <NUM> times more risk if the values were doubled. Low IL-<NUM> [OR <NUM>, <NUM>% CI (<NUM>-<NUM>), p = <NUM>] levels also increased the risk due to their under expression in the patients. According to the level of each cytokine, <FIG> represents the probability of infection.

Claim 1:
In vitro method for the diagnosis of coronavirus infection in hospitalized patients which comprises: a) Measuring the level of IP10, PDGFBB and IL4 in a minimally-invasive biological sample obtained from the hospitalized patient, and b) wherein the identification of higher levels of IP10 and PDGFBB coupled with low levels of IL-<NUM>, as compared with a pre-established reference level measured in healthy control subjects, is indicative that the hospitalized patient is suffering from coronavirus infection.