Methods for predicting the course of viral diseases

Abstract

The present invention relates to a method for predicting the course of a viral disease in a male subject infected with influenza virus or coronavirus, the method being based on measuring testosterone and / or estradiol levels in the subject. The present invention further relates to a method for monitoring the course of a viral disease in a male subject infected with influenza virus or coronavirus, the method comprising predicting the course of the disease in the subject and allocating the subject to preventive or therapeutic measures if a severe course of the viral disease is predicted. The present invention further relates to an aromatase inhibitor for use in a method for treating or preventing a severe course of a viral disease in a male subject infected with influenza virus or coronavirus, the subject having reduced testosterone and / or elevated estradiol levels compared to reference values. Finally, the present invention also relates to a kit for carrying out one of the methods.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the course of a viral disease in a male subject infected with an influenza virus or coronavirus, the method being based on the measurement of testosterone levels and / or estradiol levels in the subject. The present invention further relates to a method for monitoring the course of a viral disease in a male subject infected with an influenza virus or coronavirus, the method being based on predicting the course of the disease in the subject and assigning the subject to preventive or therapeutic measures if a severe course of the viral disease is expected. The present invention further relates to an aromatase inhibitor for use in a method of treating or preventing a severe course of a viral disease in a male subject infected with an influenza virus or coronavirus, wherein the subject has decreased testosterone levels and / or increased estradiol levels compared to a reference value. Finally, the present invention also relates to a kit for carrying out one of the above methods. [Background technology]

[0002] Influenza can lead to a severe disease progression with a high mortality rate. In severe cases, patients may require treatment in the intensive care unit (ICU). Approximately 30% of all patients receiving intensive care for influenza develop severe respiratory complications, particularly acute respiratory distress syndrome (ARDS) leading to pulmonary failure. Severe respiratory complications can occur very rapidly in influenza patients, sometimes within just a few hours.

[0003] Similarly, ARDS is also regularly observed in subgroups of patients infected with coronaviruses, particularly those infected with severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While approximately 80% of people infected with SARS-CoV-2 recover without special treatment, about 6% of infected individuals experience severe respiratory complications, including ARDS. The risk of a severe course is increased in older adults and those with pre-existing conditions such as asthma, diabetes, or heart disease. Here again, the onset of severe respiratory complications can occur very rapidly.

[0004] To date, it has been impossible to reliably predict whether patients infected with influenza virus or coronavirus will develop severe respiratory complications such as ARDS. Therefore, there is a need for new predictive methods that will enable physicians to identify patients at specific high risk of developing a severe course of the disease. Such methods would allow for the assignment of these patients to specific treatments even before the onset of respiratory complications, thereby significantly improving their chances of survival. [Overview of the Initiative]

[0005] The research underlying this invention has shown that determining testosterone and / or estradiol levels in a subject's bodily fluid sample, preferably a serum sample, makes it possible to predict whether an infection caused by influenza virus or coronavirus will follow a severe or moderate course.

[0006] Specifically, retrospective analysis revealed that testosterone levels detectable in samples from male patients infected with influenza virus or coronavirus were significantly lower in patients who later exhibited a severe course of the disease, including severe respiratory complications such as ARDS. Simultaneously, estradiol levels were higher in patients who later developed complications. Furthermore, animals infected with SARS-CoV-2 showed increased expression of aromatase (also known as CYP19A1), an enzyme that catalyzes the conversion of testosterone to estradiol in the lungs, compared to uninfected animals. In summary, these studies suggest that decreased testosterone levels and / or elevated estradiol levels are prominent features of influenza virus or coronavirus infection.

[0007] Based on this insight, the present invention makes it possible to provide a test that reliably predicts, based on testosterone and / or estradiol levels, whether an influenza virus infection or coronavirus infection will take a severe course that is likely to require intensive care measurements such as mechanical ventilation. In this way, the method of the present invention enables improved risk analysis in hospitals and intensive care units.

[0008] Therefore, in the first aspect, the present invention provides a method for predicting the course of a viral disease in a male subject infected with influenza virus or coronavirus, and the method is (a) Provide a bodily fluid sample from a subject infected with the influenza virus or coronavirus; (b) Determining the concentration of testosterone and / or estradiol in the sample; and (c) Compare the concentration obtained in step (b) to at least one testosterone and / or estradiol reference value; Includes, A method in which the concentration obtained in step (b) is compared with the at least one reference value indicates whether a severe course of the viral disease is expected in the subject.

[0009] Step (a) of the above method provides a bodily fluid sample obtained from an infected male subject. The sample used in the above method may, in principle, be any type of bodily fluid obtained from the subject to be diagnosed. In a preferred embodiment, the sample may be a blood sample such as a whole blood sample, or a plasma or serum sample. In a more preferred embodiment, the sample may be a serum sample such as a human serum sample.

[0010] The samples are derived from male subjects who have already been diagnosed with influenza virus or coronavirus infection. The male subjects may be adults between 18 and 120 years of age, but it is preferable that the subjects are at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 years of age.

[0011] A diagnosis of influenza virus or coronavirus can be obtained by any method suitable for confirming the presence of the virus in a subject, such as PCR-based detection of virus-specific nucleic acids, electron microscopy, detection of antibodies against viral proteins, or immunodetection of viral components using conjugated antibodies, for example, in the form of enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, a diagnosis of influenza virus or coronavirus in a subject is obtained by ELISA.

[0012] In this specification, the term influenza virus refers to a group of RNA viruses that cause infectious influenza. Common symptoms of influenza include fever, headache, and malaise. These symptoms are caused by massive releases of pro-inflammatory cytokines and chemokines, including interferon or tumor necrosis factor (TNF), by influenza-infected cells. It has been suggested that massive releases of cytokines can lead to life-threatening cytokine storms. The methods described herein can be used to predict the severe course of such diseases. These methods can be applied to patients infected with any influenza subtype, including influenza A subtypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and influenza B subtypes of lineages Victoria, Yamagata, Yamaguchi, Yokohama, Yunnan, and Zhuhai.

[0013] The term coronavirus refers to a group of related viruses that cause disease in mammals and birds. In humans, coronaviruses cause respiratory infections that can be associated with symptoms ranging from mild to severe. Mild infections cause symptoms similar to those of a common cold. More severe coronavirus infections can cause life-threatening complications such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus infection 2019 (COVID-19). According to the present invention, subjects may be infected with any type of coronavirus, including viruses of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, but preferably coronaviruses known to cause respiratory infections such as SARS, MERS, and COVID-19. It is particularly preferred that subjects are infected with severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0014] Step (b) of the method described above determines the concentration of testosterone and / or estradiol in a sample from an infected male patient. In one embodiment, the concentration of testosterone in a sample from an infected patient is determined. Testosterone is the major male hormone and plays a crucial role in the development of male reproductive tissues such as the testes and prostate, but also plays a crucial role in regulating the male immune response pathway. Testosterone is an androstane class steroid synthesized from cholesterol in several steps. In men, testosterone is mainly secreted by the testes. In women, who typically have 7 to 8 times lower testosterone levels than men, testosterone is produced in the ovaries.

[0015] Methods for determining testosterone concentration are well-known in the art and are described in scientific literature, e.g., van Nuland et al. (2019), Star-Weinstock & Dey (2019), Wooding et al. (2015), and Ankarberg-Lindgren et al. (2018). Furthermore, kits for quantifying testosterone in samples are commercially available, such as the Testosterone ELISA Assay Kit (Eagle Biosciences, Amherst, USA) or the Testosterone ELISA Kit (Abcam, Berlin, Germany).

[0016] In another embodiment, the concentration of estradiol in a sample from an infected male patient is determined. Estradiol, also known as E2 in the literature, is an estrogen steroid hormone and the major female hormone. Thus, it is involved not only in regulating the estrous and menstrual female reproductive cycle, but also in regulating immune response pathways in women. Estradiol is essential for the development and maintenance of female reproductive tissues such as the mammary glands, uterus, and vagina during puberty, adulthood, and pregnancy. Estradiol is produced from cholesterol through a series of reactions and intermediates. In women, its production occurs particularly in the ovarian follicles. In men, estradiol is mainly produced by the catalytic conversion of testosterone, a reaction catalyzed by the enzyme aromatase (also known as CYP19A1).

[0017] Methods for determining estradiol concentration are well known in the art and are described in scientific literature, for example, Wooding et al. (2015), Siqueira Ferreira et al. (2017), and Keski-Rahkonen et al. (2015), Analytical Chemistry, 87, 14, 7180-7186. Furthermore, kits for the quantification of estradiol in samples are commercially available, such as the Estradiol Parameter Assay Kit (R&D Systems, Inc., Minneapolis, USA), the Estradiol ELISA Kit (Eagle Biosciences, Amherst, USA), or the Human Estradiol E2 ELISA Kit (Abcam, Berlin, Germany).

[0018] It is particularly preferable that step (b) of the above method includes determining both the testosterone concentration and the estradiol concentration in the sample from the infected male patient. The testosterone concentration and the estradiol concentration can be determined in the same or different aliquots of the sample, in either order.

[0019] Once the testosterone and / or estradiol concentrations are determined in step (b) of the method described above, these concentrations are compared to at least one testosterone and / or estradiol reference value. The comparison of the testosterone and / or estradiol concentrations measured in the sample with at least one reference value indicates whether a severe course of the viral disease is expected in the subject.

[0020] In one preferred embodiment, the method of the first aspect of the present invention includes, in step (b), determining the testosterone concentration in a body fluid sample, particularly a blood or serum sample, and in step (c), comparing the testosterone concentration of the sample with a testosterone reference value, wherein if the concentration obtained in step (b) is below the reference value, a severe course of the disease is expected.

[0021] In men between 18 and 50 years of age, testosterone concentrations between 8.69 and 29.00 nMol per liter of serum are considered normal. Conversely, testosterone concentrations below 8.69 nMol / l are considered lower than normal for men of that age and therefore indicate potentially severe complications in patients infected with influenza virus or coronavirus. Thus, in one embodiment, if the reference value for adult men of that age is 8.69 nMol / l and the concentration in the sample is below 8.69 nMol / l, a severe course of the disease is expected. In another embodiment, if the reference value for men of that age is 8.5 nMol / l and the concentration in the sample is below 8.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 7.5 nMol / l and the concentration in the sample is below 7.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 6.5 nMol / l and the concentration in the sample is below 6.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 5.5 nMol / l and the concentration in the sample is below 5.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 4.5 nMol / l and the concentration in the sample is below 4.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 3.5 nMol / l and the concentration in the sample is below 3.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 2.5 nMol / l and the concentration in the sample is below 2.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, if the reference value for men of that age is 1.5 nMol / l, and the concentration in the sample is below 1.5 nMol / l, a severe course of the disease is expected.

[0022] In men over 51 years old, a testosterone concentration between 6.68 and 25.8 nMol per liter of serum is considered normal. Instead, a testosterone concentration value below 6.68 nMol / l is considered lower than normal in men of that age and, therefore, indicates potentially severe complications in patients infected with influenza virus or coronavirus. Thus, in one embodiment, the reference value for adult men of that age is 6.68 nMol / l, and when the concentration in the sample is below 6.68 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 6.5 nMol / l, and when the concentration in the sample is below 6.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 5.5 nMol / l, and when the concentration in the sample is below 5.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 4.5 nMol / l, and when the concentration in the sample is below 4.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 3.5 nMol / l, and when the concentration in the sample is below 3.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 2.5 nMol / l, and when the concentration in the sample is below 2.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 1.5 nMol / l, and when the concentration in the sample is below 1.5 nMol / l, a severe course of the disease is expected. In yet another embodiment, the reference value for men of that age is 1.0 nMol / l, and when the concentration in the sample is below 1.0 nMol / l, a severe course of the disease is expected.

[0023] In the case of estradiol, an estradiol concentration between 27.1 and 52.2 pg per milliliter of serum is considered normal for men, regardless of their age. Instead, an estradiol concentration value exceeding 52.2 pg / ml is considered higher than normal and, therefore, indicates potentially severe complications in men infected with influenza virus or coronavirus. Thus, in one embodiment, the reference value for adult men is 52.2 pg / ml, and when the estradiol concentration in the sample exceeds 52.2 pg / ml, a severe course of the disease is expected. In another embodiment, the reference value for adult men is 55 pg / ml, and when the estradiol concentration in the sample exceeds 55 pg / ml, a severe course of the disease is expected. In another embodiment, the reference value for adult men is 60 pg / ml, and when the estradiol concentration in the sample exceeds 60 pg / ml, a severe course of the disease is expected. In another embodiment, the reference value for adult men is 70 pg / ml, and when the estradiol concentration in the sample exceeds 70 pg / ml, a severe course of the disease is expected. In yet another embodiment, the reference value for adult men is 80 pg / ml, and when the estradiol concentration in the sample exceeds 80 pg / ml, a severe course of the disease is expected. In yet another embodiment, the reference value for adult men is 90 pg / ml, and when the estradiol concentration in the sample exceeds 90 pg / ml, a severe course of the disease is expected. In yet another embodiment, the reference value for adult men is 100 pg / ml, and when the estradiol concentration in the sample exceeds 100 pg / ml, a severe course of the disease is expected.

[0024] The above method can be used as a tool for patient monitoring. Thus, in a second aspect, the present invention provides a method for monitoring the course of a viral disease in male subjects infected with influenza virus or coronavirus, the method comprising (a) repeatedly performing the method according to the first aspect of the present invention as defined above at predetermined time intervals, and (b) If, based on the results obtained in step (a), a severe course of viral disease is anticipated, assign the subject to preventive or therapeutic measures. This method includes [something].

[0025] A predictive method according to a first aspect of the present invention can be used to monitor the course of a viral illness in a male subject infected with influenza virus or coronavirus. Since subjects infected with influenza or coronavirus may experience complications fairly rapidly, it is useful to predict the further course of the disease at predetermined time intervals, such as every 24 hours, every 18 hours, every 12 hours, or every 6 hours. If a decrease in testosterone levels and / or an increase in estradiol levels may be observed, a severe course of the infection with respiratory complications may be expected. In this case, there is a possibility of a severe course of the disease, and the subject may be assigned to preventive or therapeutic measures. Such measures include increased clinical monitoring, initiation of mechanical ventilation, or administration of an antiviral agent such as remdesivir. Measures may also include treatment of the patient with one or more aromatase inhibitors as described elsewhere in this specification, treatment of the patient with one or more testosterone or testosterone derivatives as described elsewhere in this specification, or a combination of such treatments. In a method according to a second aspect of the present invention, a severe course of the viral illness may include the development of ARDS.

[0026] The observation that patients with reduced testosterone levels and / or elevated estradiol levels are at higher risk of experiencing a severe course of the disease after infection with influenza virus or coronavirus suggests that these patients have increased amounts and / or elevated activity levels of the aromatase enzyme that catalyzes the conversion of testosterone to estradiol. This is consistent with the observation, reported in the following examples, that aromatase expression is increased in hamsters infected with SARS-CoV-2 compared to uninfected animals. Therefore, inhibition of aromatase may have a therapeutic effect in these patients.

[0027] Accordingly, in a third aspect, the present invention provides an aromatase inhibitor for use in a method of treating or preventing a severe course of a viral disease in a male subject infected with an influenza virus or coronavirus, wherein the subject has (a) a reduced testosterone level compared to the normal reference level discussed above and / or (b) an elevated estradiol level compared to the normal reference level discussed above.

[0028] As described in the following examples, aromatase inhibitors have been shown to be able to effectively block viral transmission. Therefore, in a fourth aspect, the present invention provides aromatase inhibitors for use in a method for inhibiting viral transmission in subjects infected with influenza virus or coronavirus.

[0029] The aromatase inhibitors mentioned in the third and fourth aspects of the present invention are preferably, compared to a reference value, (a) Decreased testosterone levels, and / or (b) Elevated estradiol levels It is administered to subjects with the following characteristics, more preferably to male subjects.

[0030] Aromatase inhibitors are formulated to suit the intended route of administration. Various routes of administration are feasible to provide aromatase inhibitors to a target. Preferably, aromatase inhibitors are formulated for oral administration in the form of, for example, tablets, capsules, granules, powders, or liquids. Alternatively, aromatase inhibitors may be formulated for parenteral administration, for example, intravenous or subcutaneous administration. Aromatase inhibitors may also be formulated for administration by implantation, for example, by mixing the aromatase inhibitor with a three-dimensional carrier or scaffold such as a hydrogel.

[0031] Suitable aromatase inhibitors for use here include, but are not limited to, aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, volozol, formestan, and fadrozol. Preferably, the aromatase inhibitors are intended for use in methods of treating or preventing severe courses of viral diseases, including the development of ARDS.

[0032] Preferably, the administration of aromatase inhibitors can be combined with testosterone replacement therapy. Therefore, it is particularly preferable to administer testosterone to the patient receiving the aromatase inhibitor. The administration of testosterone and aromatase inhibitors can be carried out simultaneously, sequentially, or in any order.

[0033] In a fifth aspect, the present invention provides testosterone or a testosterone derivative for use in a method of treating or preventing a severe course of a viral disease in a male subject infected with an influenza virus or coronavirus, wherein the male subject has (a) a reduced testosterone level compared to the normal reference level discussed above and / or (b) an elevated estradiol level compared to the normal reference level discussed above. The reference values ​​are those discussed above in relation to the method according to the first aspect of the present invention.

[0034] In this case as well, testosterone or testosterone derivatives are formulated to be compatible with the intended route of administration. Various routes of administration are feasible to provide testosterone or its derivatives to the target. Preferably, testosterone or testosterone derivatives are formulated for oral administration in the form of, for example, tablets, capsules, granules, powders, or liquids. Alternatively, testosterone or testosterone derivatives may be formulated for parenteral administration, for example, intravenous or subcutaneous administration. However, it is preferable that testosterone or testosterone derivatives be formulated for transdermal or transmucosal administration, for example, in the form of a patch that releases testosterone into the skin.

[0035] Preferably, as described above, testosterone administration can be combined with the administration of one or more aromatase inhibitors. Therefore, it is preferable that the recipient of testosterone or a testosterone derivative also receives one of the aromatase inhibitors described above. Testosterone administration and aromatase inhibitor administration can be carried out simultaneously, sequentially, or in any order.

[0036] Finally, in a sixth aspect, the present invention is a kit for carrying out the methods described herein above, (a) Means for determining whether a subject is infected with the influenza virus or coronavirus; (b) means for determining the concentration of testosterone and / or estradiol; and (c) Buffers and diluents, as needed We provide a kit that includes this.

[0037] In one embodiment, the kit contains antibodies useful for detecting influenza virus or coronavirus antigens, for example, by ELISA. The kit may also include appropriate immunoassays for determining testosterone and / or estradiol concentrations. [Brief explanation of the drawing]

[0038] [Figure 1]This figure shows the testosterone and estradiol levels determined in several COVID-19 patients. (A) A table showing the testosterone and estradiol levels measured in male and female COVID-19 patients. (B) A graph display of testosterone (a, b) and estradiol (c, d) levels measured in serum or plasma from COVID-19 patients and age-matched (≥40 years) healthy controls. Male COVID-19 patients (a, c) were subdivided into patients requiring ECMO connection (+ECMO) and patients not placed on ECMO (-ECMO). Bar graphs (a, c) represent males (COVID-19+ECMO, n=5; COVID-19-ECMO, n=34; healthy controls, n=30), and bar graphs (b, d) represent females (COVID-19, n=11; healthy controls, n=20). Statistical significance was assessed using Student's t-test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 2] This figure shows the results of total testosterone expression level measurements in H7N9 men infected with the H7N9 influenza A virus. [Figure 3] This figure shows the results of measuring CYP19A1 expression in Syrian golden hamsters. (a) CYP19A1 mRNA expression levels in the lungs of SARS-CoV-2 infected male and female Syrian golden hamsters 3 days post-infection (n=9~10). The relative CYP19A1 mRNA expression values ​​in PBS-treated hamsters of each sex were normalized to HPRT (hypoxanthine phosphoribosyltransferase 1) and set to 1. Values ​​are shown as mean values, and error bars are shown as SD. Statistical significance was evaluated by Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison test (*p<0.05, ****p<0.0001). (b) CYP19A1 protein expression in the lungs of SARS-CoV-2 infected male (upper panel) and female (lower panel) Syrian golden hamsters 3 days post-infection. Representative photographs of each sex are shown (n=5). Arrowheads indicate positive signals for CYP19A1 expression. [Figure 4] This figure shows the results of measuring viral titers and MIP-1a / MIP-1b expression levels in various organs of hamsters treated with placebo or letrozole. (a-c) Viral titers (n=6 each) in the lungs (a), brain (b), and testes (c) of PBS and SARS-CoV-2 infected male Syrian golden hamsters treated with placebo or letrozole 3 days post-infection. (d-e) Protein expression levels (n=6 each) of MIP-1a (d) and MIP-1b (e) in the lungs of PBS and SARS-CoV-2 infected male Syrian golden hamsters treated with placebo or letrozole 6 days post-infection. (f-h) Viral titers (n=6 each) in the lungs (f), brain (g), and plasma (h) of PBS and SARS-CoV-2 infected female Syrian golden hamsters treated with placebo or letrozole 3 days post-infection. (i-j) Protein expression levels of MIP-1a(i) and MIP-1b(j) in the lungs of PBS and SARS-CoV-2 infected female Syrian golden hamsters treated with placebo or letrozole 6 days post-infection (n=6 for each). Values ​​are shown as mean values, and error bars are shown as SD. Statistical significance was assessed by Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). nd: Undetectable, ns: Not significant. [Figure 5]This figure shows the results of measuring CYP19A1 expression in the lungs of fatal Covid-19 cases. (a) CYP19A1 mRNA expression levels in the lungs of fatal male Covid-19 cases and controls (non-Covid-19) who died for other reasons (non-Covid-19: n=5, Covid-19: n=9). Values ​​are shown as mean values; error bars are shown as SD. Statistical significance was assessed by Kruskal-Wallis one-way ANOVA with Dunn's multiple comparison test (*p<0.05). (b) CYP19A1 protein expression in the lungs of fatal male (upper panel) and female (lower panel) Covid-19 cases and controls (non-Covid-19) who died for other reasons. Detection of SARS-CoV-2 RNA by in situ hybridization. Representative photographs are shown (male: n=8, female: n=3). Squares indicate macrophages. [Examples]

[0039] The present invention is described below based on exemplary examples that do not limit the invention. It will be apparent to those skilled in the art that modifications and variations of the described examples are possible without departing from the concept of the present invention.

[0040] Example 1 Determining the hormonal status of COVID-19 patients Forty-five COVID-19 patients requiring intensive care were examined at the University Hospital Hamburg Eppendorf. Of these patients, 35 were male and 10 were female. The median age within the male and female groups was similar, at 62 and 67.5, respectively. The majority of patients showed elevated body mass index (BMI) (31.4% of males and 30% of females had a BMI ≥ 30). All patients, both male and female, had comorbidities such as hyperlipidemia (69% of males; 50% of females), followed by type 2 diabetes (22.9% of males; 20% of females), hypertension (45.7% of males, 33.3% of females), and cancer (22.9% of males, 33.3% of females). Detected acute respiratory distress (ARDS) was classified as moderate or severe in most male (37% or 26%) and female (33% or 33%) patients. Sequential organ failure assessment (SOFA) scores were evaluated in men and women, with high (4-7) or very high (8-11) scores observed in men (35% or 25%) and women (40% or 60%). Due to the strong male-to-female sex difference of 3.5:1, sex hormones, known to play important roles not only in fertility but also in innate and adaptive immunity, were measured.

[0041] resultThe results are shown in Table 1. Total testosterone levels were decreased in 69% of men. Of these, 26% of men showed very low testosterone levels, and 43% showed extremely low testosterone levels. In 60% of women, testosterone levels were elevated to high levels (50%) or very high levels (10%). Estradiol levels were elevated to either high (30%) or very high (16%) levels in male COVID-19 patients (46%). Similarly, 60% of women also showed elevated estradiol concentrations up to high (40%) or very high (20%) levels. Thus, the majority of male COVID-19 patients have very low testosterone levels and very high estradiol levels. In contrast, female COVID-19 patients tend to have high testosterone and estradiol levels. The sex hormone shift observed in male patients here suggests an increase in aromatase (CYP19A1) activity, i.e., the enzyme that converts testosterone to estradiol.

[0042] Example 2 Determination of hormonal status in H7N9 influenza patients We enrolled 44 avian H7N9 influenza-positive cases in the reproductive age group (18-49 years), with a median age of 42 years. A total of 54 avian H7N9 influenza-positive cases were included in the group with a median age of 61 years, representing those aged 50 years or older. Male H7N9 cases accounted for 75% in the younger age group and 70% in the older age group, consistent with previous epidemiological studies based on larger laboratory-confirmed H7N9 cohorts. Blood samples from H7N9 patients were collected during the acute phase after disease onset.

[0043] To evaluate the role of testosterone in H7N9 infection outcomes, testosterone levels were measured in all cohorts. Testosterone levels were significantly lower in H7N9-infected men in both age groups evaluated compared to virus-negative H7N9 controls. Low testosterone levels were strongly associated with fatal outcomes in H7N9-infected men aged 18–49 years (P<0.001) (Figure 2). These data indicate that low testosterone levels in H7N9-infected men aged 18–49 years are associated with an increased risk of fatal outcomes.

[0044] Example 3 : Virus isolation and animal infection SARS-CoV-2 isolate (SARS-CoV-2 / Germany / Hamburg / 01 / 2020) was isolated by inoculating VeroE6 cells with 200 μl of human nasopharyngeal swab sample from a male COVID-19 patient confirmed in Hamburg, and propagated in VeroE6 cells for three consecutive passages. VeroE6 cells were cultured at 37°C in DMEM (Sigma-Aldrich GmbH) containing 2% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine for viral replication, and tested negative for Mycoplasma sp. by PCR. All SARS-CoV-2 infection experiments were conducted in a biosafety level 3 (BSL-3) laboratory.

[0045] All animal experiments were conducted in strict accordance with the guidelines of the German Animal Protection Act and with the approval of the relevant German authorities (Behoerde fuer Gesundheit und Verbraucherschutz; Protocol N32 / 2020). Male and female Syrian golden hamsters (8-10 weeks old) were purchased from Janvier and housed under standard conditions (21±2℃, 40-50% humidity, free access to food and water) with a 12:12 light-dark cycle. For infection, the hamsters were anesthetized by intraperitoneal injection with 150 mg / kg ketamine and 10 mg / kg xylazine. The animals were given 10 5SARS-CoV-2 plaque-forming units (pfus) were inoculated intranasally, mock-infected with PBS, or administered 1 mg / kg-1 poly(I:C). Three days post-infection, five animals per group were euthanized by intraperitoneal injection of pentobarbital overdose, and blood was collected by cardiac puncture.

[0046] For RNA isolation, the lungs were preserved in RNAprotect Tissue Reagent (QIAGEN). For histopathological examination, the recovered lungs were fixed by immersion in 10% neutral buffered formalin and embedded in paraffin.

[0047] Example 4 Measurement of CYP19A1 mRNA expression levels To determine CYP19A1 mRNA expression levels by real-time quantitative PCR (RT-qPCR), RNAprotect-fixed lungs from hamsters were homogenized in 700 μl of lysis buffer RL containing five sterile stainless steel beads (2 mm diameter, Retsch) in a Mixer Mill MM400 (Retsch) at 30 Hz and 4°C for 10 minutes. Total RNA was isolated from the homogenized lung supernatant using innuPREP RNA Mini Kit 2.0 (Analytik Jena) according to the manufacturer's instructions and subjected to additional on-column DNase I treatment with an RNase-free DNase Set (QIAGEN). RNA was eluted in RNase-free water and mixed with 1 μl-1 RiboLock RNase inhibitor (Thermo Fisher Scientific). For cDNA synthesis, 2 μg of total RNA was used, along with random nonomer primers (Gene Link, pd(N)9, final concentration: 5 μM) and SuperScript III reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0048] cDNA was generated using the GeneAmp PCR System 9700 (Applied Biosystems; cycles: 5 min at 25°C, 60 min at 50°C, 15 min at 70°C, hold at 4°C). The reaction was set up in a LightCycler® 480 multiwell plate 96 reaction plate (Roche Life Science) with PCR-grade water (Roche Life Science). Briefly, 2 μl of cDNA template was added to 10 μl of FastStart Essential DNA Green Master (Roche Life Science) and 300 nM forward and reverse primers. RT-qPCR was performed using the LightCycler® 96 Real-Time PCR System (Roche Life Science) with endpoint fluorescence detection at 95°C for 10 minutes and 45 amplification cycles (15 seconds at 95°C, 10 seconds at 65°C, and 20 seconds at 72°C). CYP19A1 and the reference gene (hamster: HPRT, human: RPL32) were analyzed in a dual sequence for each sample. Negative controls and reverse transcriptase-free samples were included to detect contaminants.

[0049] Corrected E-ΔΔ Ct Relative expression levels were determined using the following method. Rn values ​​were exported from LightCycler® 96Software v1.1.0.1320 (Roche) to Microsoft Office Excel 2016, and the N0 values ​​of the start concentration of the transcript in the original sample were obtained using LinReg PCR Software v2018.0 (Ruijter et al. 2009). Subsequently, the mean N0 values ​​of the CYP19A1 gene were obtained for each sample using HPRT(N 0(HPRT) ) or RPL32(N 0(RPL32) Normalized by the mean N0 value of the biological replicas. 0(CYP19A1) / N 0(HPRT) - or N 0(CYP19A1) / N 0(RPL32) - Indicates expression levels.

[0050] The following primer sequences were used for qRT-PCR of HPRT1 (hypoxanthine-guanine-phosphoribosyltransferase 1) and CYP19A1 in hamster lung: HPRT1 Forward (Sequence ID 1) 5'-TCCCAGCGTCGTGATTAGTG-3' HPRT1 Reverse (Sequence ID 2) 5'-GTGATGGCCTCCCATCTCTT-3' CYP19A1 Forward (Sequence ID 3) 5'-ATGCGGCACATCATGCTGAA-3' CYP19A1 Reverse (SEQ ID NO: 4) 5'-TCTTTCAAGTCCTTGGCGGAT-3'

[0051] result The results are shown in Figure 3(a). Aromatase expression, as determined by RT-qPCR, is significantly higher in animals infected with SARS-CoV-2 compared to uninfected animals.

[0052] Example 5 :Immunohistochemistry For immunohistochemical detection of aromatase, the EnVision+ System (Dako Agilent Pathology Solutions) was used. Endogenous peroxidase was blocked by degreasing and rehydrating serial tissue sections in isopropanol and 96% ethanol, followed by incubation in 85% ethanol containing 0.5% H2O2 at room temperature for 30 minutes. Antigen retrieval was performed by microwaving in citrate buffer (10 mM citrate, 0.05% Tween 20) at 800 W for 20 minutes, followed by incubation at room temperature for 20 minutes. Subsequently, sections were transferred to Shandon Coverplates™ (Thermo Electron GmbH) and stained overnight at 4°C with a polyclonal antibody against aromatase (Abcam, ab18995) diluted 1:500 in PBS containing 1% BSA and 0.3% Triton X-100. Subsequently, the sections were rinsed and applied as a secondary antibody with peroxidase-labeled polymer for 30 minutes. The reaction was visualized by incubation in PBS with 3,3-diaminobenzidine tetrahydrochloride (DAB, 0.05%) and 0.03% H2O2 for 5 minutes, followed by counterstaining with Meyer hematoxylin for 1 minute. For the negative control, the primary antibody was replaced with normal rabbit serum (1:3,000).

[0053] result The results are shown in Figure 3(b). It can be seen that aromatase protein expression can be detected in the lungs of SARS-CoV-2 infected male (upper panel) and female (lower panel) hamsters. Aromatase protein expression cannot be detected in the lungs of control animals.

[0054] Example 6 Letrozole treatment All animal experiments were carried out strictly in accordance with the guidelines of the German Animal Protection Law and were approved by the relevant German authorities (Behoerde fuer Gesundheit und Verbraucherschutz; protocol N103 / 2020). Male and female Syrian golden hamsters (8–12 weeks old) were purchased from Janvier or bred at the Heinrich Pette Institute (Leibniz Institute for Experimental Virology, Hamburg, Germany) and housed under standard conditions (21 ± 2 °C, 40–50% humidity, food and water ad libitum) on a 16:8 light–dark cycle. For infection, hamsters were anesthetized by intraperitoneal injection with 150 mg / kg ketamine and 10 mg / kg xylazine. The animals were 5 intranasally inoculated with 10 plaque forming units (p.f.u.) of SARS-CoV-2 or mock-infected with PBS. Three hours after infection and every other day thereafter, the animals were treated by intraperitoneal injection with 0.18 mg kg−1 letrzole or placebo. On days 3 and 6 after infection, six animals per group were euthanized by intraperitoneal injection of an overdose of pentobarbital and blood was collected by cardiac puncture. For virus titration and cytokine measurements, lungs, brains and testes were harvested, homogenized in 1 ml of 1× PBS and stored at −80 °C.

[0055] Organ homogenization was performed in 1 ml of 1×PBS containing 5 sterile stainless steel beads (φ2 mm, Retsch) in a Mixer Mill MM400 (Retsch) at 30 Hz for 10 minutes. Plaque assays were performed on a monolayer of VeroE6 cells and stained with crystal violet after 72 hours. Tissue homogenates were titrated on VeroE6 cells at 37°C for 30 minutes in 10-fold serial dilutions, and MEM (Sigma-Aldrich) supplemented with 0,2% BSA, 1% L-glutamine, 1% penicillin-streptomycin, 1 μg ml-1 L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich) and 1.25% Avicel was overlaid. 72 hours post-infection, cells were fixed with 4% paraformaldehyde, and plaques were visualized by crystal violet staining.

[0056] The protein expression levels of macrophage inflammatory proteins 1α and 1β (MIP-1α, MIP-1β) were measured in homogenized lungs using a custom Bio-Plex Pro® mouse cytokine multiplex (Bio-Rad) in a Bio-Plex200 system equipped with high-throughput fluidics (HTF; Bio-Rad), according to the manufacturer's instructions.

[0057] All data were analyzed using Prism software (GraphPad, 9.0.1) with Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison test. Statistical significance was defined as p<0.05. * p<0.05, ** p<0.01, *** p<0.001, **** It was defined as p < 0.0001.

[0058] Results: The results are shown in Figure 4. From Figures 4(a)–(c), it can be seen that treatment with the aromatase inhibitor letrozole resulted in lower viral titers in the lungs, brains, and testes of SARS-CoV-2 infected male animals compared to placebo. Similarly, Figures 4(f)–(h) demonstrate that treatment with the aromatase inhibitor letrozole resulted in lower viral titers in the lungs, brains, and plasma of SARS-CoV-2 infected female animals compared to placebo. These data suggest that aromatase inhibitors may inhibit viral transmission. Figure 4 further shows that the expression levels of MIP-1α and MIP-1β were lower in the lungs of animals treated with letrozole in both male (d)–(e) and female (i)–(j) animals.

[0059] Example 7 CYP19A1 expression in lung autopsy of men with fatal Covid-19 We analyzed whether data obtained from preclinical animal models were reflected in humans. Therefore, we analyzed autopsy specimens (n=54) from the lungs of men and women who died from Covid-19. As a control, lung specimens from men and women who died for other reasons (non-Covid-19 control group) were also analyzed. Pathological evaluations were performed at three independent research facilities: Hamburg (n=26 men, n=8 women), Tübingen (n=8 men, n=3 women), and Rotterdam (n=12 men, n=1 woman).

[0060] Total RNA from formalin-fixed, paraffin-embedded human lung tissue sections was purified using the RNeasy@FFPE Kit (Qiagen) according to the manufacturer's instructions. To detect SARS-CoV-2 RNA in the lung tissue, in situ hybridization (ISH) was performed by hybridizing the lung tissue sections with a SARS-CoV-2 specific probe (ACD, Newark, California, USA) followed by the RNAscope 2.5HD Detection Kit Red (ACD, Newark, California, USA) according to the manufacturer's protocol.

[0061] qRT-PCR was performed as described in Example 4 above. Here, the following primer sequences were used for qRT-PCR of RPL32 (ribosomal protein L32) and CYP19A1 in human lung: RPL32 Forward (Sequence ID 5) 5'-GAAGTTCCTGGTCCACAACG-3' RPL32 Reverse (Sequence ID 6) 5'-GCGATCTCGGCACAGTAAG-3' CYP19A1 Forward (Sequence ID 7) 5'-CGGCCTTGTTCGTATGGTCA-3' CYP19A1 Reverse (Sequence ID 8) 5'-CAGAAGGGTCAACACGTCCA-3'

[0062] Results: In all sites, CYP19A1 was abundantly expressed in the lungs of Covid-19 men compared to non-Covid-19 male controls. Generally, CYP19A was expressed in epithelial and endothelial cells, but was most strongly expressed in macrophages independently at all three research facilities. Notably, while SARS-CoV-2 NP protein or RNA was still detectable in the lungs of most deceased women, viral antigen or RNA was expressed at low levels or had already been cleared at the time of death of the men. Quantification of CYP19A1 mRNA levels revealed up to approximately 10-fold transcriptional increases in the lungs of Covid-19 men compared to non-Covid-19 men. These findings indicate that CYP19A1 is abundantly expressed in the lungs of men with Covid-19 even at the time of death. The results are shown in Figure 5.

[0063] literature 1. Ankarberg-Lindgren et al. (2018), J Steroid Biochem Mol Biol, 183:116-124. 2. Siqueira Ferreira et al.(2017),Journal of Chromatography B 1064,109-114. 3. Star-Weinstock&Dey(2019),Clinical Mass Spectrometry,13,27-35. 4. Wooding et al(2015),Steroids,96:89-94, 5. Van Nuland et al.(2019),J Pharm Biomed Anal.,170:161-168.

Claims

1. A method for testing the severity of the course of a viral disease in men infected with influenza virus or coronavirus, (a) Determining the concentration of testosterone and / or estradiol in a bodily fluid sample taken from the subject infected with the influenza virus or coronavirus; and (b) Comparing the concentration obtained in step (a) with at least one testosterone and / or estradiol reference value; Includes, The testosterone reference value indicates that a severe course of the viral disease is expected if the concentration obtained in step (a) falls below the testosterone reference value, and / or A method that indicates that if the estradiol reference value is greater than the concentration obtained in step (a), a severe course of the viral disease is expected.

2. The method according to claim 1, wherein the bodily fluid sample is a blood, plasma, or serum sample.

3. The method according to claim 1 or 2, wherein the testosterone reference value is 8.69 nMol / l serum in men between 18 and 50 years of age or 6.68 nMol / l serum in men over 51 years of age.

4. The method according to any one of claims 1 to 3, wherein the estradiol reference value is 52.2 pg / ml serum or 60 pg / ml serum.

5. (i) Comparison of the testosterone concentration obtained in step (a) with the testosterone reference value, wherein the reference value is 8.69 nMol / l serum in men between 18 and 50 years of age or 6.68 nMol / l serum in men over 51 years of age, and (ii) Comparison of the estradiol concentration obtained in step (a) with the estradiol reference value, wherein the reference value is 52.2 pg / ml serum. Includes, The method according to any one of claims 1 to 4, wherein if the testosterone concentration obtained in step (a) is below the testosterone reference value and the estradiol concentration obtained in step (a) exceeds the estradiol reference value, a severe course of the viral disease is expected.

6. The method according to any one of claims 1 to 5, wherein the influenza virus is H7N9 or the coronavirus is SARS-CoV-2.

7. A method for monitoring the course of a viral disease in a subject infected with influenza virus or coronavirus, (a) Repeatedly performing the method described in any one of claims 1 to 6 at predetermined time intervals, and (b) If, based on the results obtained in step (a), a severe course of the viral disease is expected, assign the subject to preventive or therapeutic measures. A method that includes this.

8. The method according to any one of claims 1 to 7, wherein the severe course of the viral disease includes the onset of acute respiratory distress syndrome (ARDS).

9. An aromatase inhibitor for use in a method of treating or preventing a severe course of viral disease in a subject infected with influenza virus or coronavirus, wherein the subject exhibits a condition compared to a reference value. (a) Decreased testosterone levels, and / or (b) Elevated estradiol levels An aromatase inhibitor having [specific characteristic].

10. An aromatase inhibitor for use in the method according to claim 9, wherein the inhibitor is selected from the group consisting of aminoglutethimide, testactone, anastrozole, letrozole, exemestane, volozol, formestan, and fadrozol.

11. An aromatase inhibitor for use in the method of claim 9 or 10, wherein the severe course of the viral disease includes the development of acute respiratory distress syndrome (ARDS).

12. An aromatase inhibitor for use in the method of any one of claims 9 to 11, wherein the method also includes the administration of testosterone or a testosterone derivative.

13. Testosterone or a testosterone derivative for use in a method of treating or preventing a severe course of a viral disease in a male subject infected with influenza virus or coronavirus, wherein the male subject has (a) a reduced testosterone level compared to a normal reference level and / or (b) an elevated estradiol level compared to a normal reference level.

14. A kit for carrying out the method according to any one of claims 1 to 8, (a) Means for determining whether a subject is infected with the influenza virus or coronavirus; (b) means for determining the concentration of testosterone and / or estradiol; and (c) Buffers and diluents, as needed A kit that includes this.

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