WO2023139009A1

WO2023139009A1 - A simple assay for identifying defective signaling pathways upstream of nfkb

Info

Publication number: WO2023139009A1
Application number: PCT/EP2023/050808
Authority: WO
Inventors: Ludwig ENGLMEIER
Original assignee: Englmeier Ludwig
Priority date: 2022-01-20
Filing date: 2023-01-16
Publication date: 2023-07-27

Abstract

The present invention relates to a simple non-invasive assay which allows the identification of individuals who have a defect in a signaling pathway upstream of NFkB. The assay is based on administering an agonist of a component of a signaling pathway upstream of NFkB, such as a specific TLR agonist, to the skin, for example in the context o a skin prick assay, and then observing the absence of the development of an inflammatory response, such as missing reddening of the skin at the site of administration in the context of for example a skin prick assay. Individuals who have a functional signaling pathway will, conversely, be identifiable by reddening of the skin at the site of administration.

Description

A SIMPLE ASSAY FOR IDENTIFYING DEFECTIVE SIGNALING PATHWAYS UPSTREAM OF NFKB

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 disease, has caused an international pandemic with numerous casualties. It has been noted that the severity of the COVID- 19 disease course is highly variable and that the obese and elderly are particularly vulnerable, with men being consistently more vulnerable than women of the same age group. Predicting disease severity would be beneficial, as medical treatment could be targeted to vulnerable individuals earlier during the disease and expensive new antiviral treatments, like paxlovid, could be targeted to those that are indeed in need of such a treatment, as their immune system is not able to effectively fight the viral infection without pharmacological support.

Several attempts have been made to predict disease severity after the onset of symptoms. WO2021/257888A1 uses type2 cytokines as predictors of disease severity after the onset of symptoms. WO2021/217505A1 uses specific IgAs for the evaluation of the risk of contracting COVID-19 and developing severe disease. US2021330269A1 uses a computer algorithm for predicting disease severity.

The literature on predicting vulnerable individuals prior to infection is less extensive. Velavan TP et al. “Host genetic factors determining COVID-19 susceptibility and severity.” EBioMedicine. 2021 PMID: 34655949 provide a review of genetic risk factors for the development of severe COVID-19 disease and mention more than 30 genes that, when mutated, can make an individual vulnerable towards the development of severe disease. Solanich X et al. “Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19.” Front Immunol. 2021 PMID: 34367187 suggest genetic screening for variants of one of the genes mentioned in Velavan et al., TLR7 variants, in young men in the absence of other relevant risk factors and suggest that a diagnosis of TLR7 deficiency could not only inform on treatment options for the patient, but also enable pre-symptomatic testing of at-risk male relatives with the possibility of instituting early preventive and therapeutic interventions. However, population-wide genetic screens for rare loss-of-function variants are not feasible, even if limited to the group of young men. There remains a need for a simple screening method which would allow the identification of individuals vulnerable to developing severe disease upon infection with an RNA-virus such as the coronavirus SARS-CoV-2.

SUMMARY OF THE INVENTION

The present inventor has found that activators of DAMP and PAMP receptors, like TLR4-agonists or TLR7 -agonists, produce an observable inflammatory response when administered non-invasively, such as in the context of a skin prick assay, presumably by leading to the activation of NFkB, the master transcription factor for the induction of pro-inflammatory cytokines, and has realized that this readout allows for simple diagnostic assays. Thus, selective activators of signaling pathways upstream of NFkB can be used diagnostically, even in a non-invasive setting, for determining, whether the components of the respective signaling pathway are functional - resulting in an observable inflammatory response - or defective - resulting in a missing or reduced inflammatory response. This has important medical implications, because individuals with, for example, mutant PAMP-receptors, such as mutant or defective TLR7, have an increased risk of developing severe disease from a pathogen which is typically detected by said PAMP receptor. An example of such a situation are the above-described young men with defects in TLR7, who develop severe COVID-19 as a consequence of an infection with SARS- CoV-2. The assay is safe and inexpensive, and its simplicity allows for screening large numbers of individuals al low costs due to material and/or work effort by medical personell. Moreover, by using a collection of different selective PAMP-receptor agonists in separate entities, the functionality of several different pathways of interest can be assayed in one go, for example by applying different samples with different individual selective PAMP-receptor agonists as separate drops to, for example, a forearm as collection of individual prick tests. This is a similar setting to the widely used allergy tests, where several different allergens are tested in one go, simply by applying separate drops containing individual allergens.

Moreover, given the integrative nature of signal transduction at the level of transcription factors, a collection of PAMP -agonists can be used to identify defects in downstream components closer to NFkB, such as MyD88 or IRAK4. A patient with IRAK4 deficiency - which predisposes patients to recurrent life-threatening bacterial diseases - can be expected to be unresponsive or less responsive to more than one PAMP-receptor agonist in the assay of the invention, for example can be expected to fail to produce a response to a TLR4-agonist AND a further TLR-agonist (other than a TLR3 -agonist). Thus, with the appropriate choice of PAMP-receptor agonists, it is possible to differentiate between defects at the receptor level and defects at the level of shared downstream signaling components.

One application, where the diagnostic assay of the invention can be used, is the diagnosis of individuals of being at an increased risk of developing severe viral diseases.

The present invention provides a simple method of diagnosing an individual as having an increased risk of developing severe symptoms from an infection with an RNA-virus, comprising the steps of a) administering a TLR3 and/or a TLR7 agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an at-risk individual.

A preferred example of this method is the topical administration of an imiquimod solution to the skin of an individual to be tested in the context of a skin prick assay. In individuals with intact TLR-signaling the engagement of TLR7 by imiquimod will lead to an inflammatory response, which manifests itself, for example, as an erythema at the site of administration in response to the imiquimod administration, for example a small red dot developing at the site where the epidermis was punctured. If the individual does not develop a symptom of an inflammatory response upon imiquimod stimulation of the skin, for example does not develop erythema at the site of administration, this can be an indication that TLR7 or downstream components of the TLR-signaling pathway towards NFkB are functionally defective or downregulated, which would explain a missing or very weak inflammatory response to the imiquimod challenge. Individuals with a potentially higher risk of developing severe disease from a coronavirus infection can thus be identified simply by the fact that they don’t develop a small red dot at the site of administration upon a challenge with a TLR3 and/or a TLR7 agonist, such as an imiquimod challenge, for example at 2 hours to 4 hours after administration. The skilled person will understand that this is also a method - depending on the type of TLR-agonist used - of diagnosing an individual as having defective or downregulated TLR7/TLR3 or defective or downregulated downstream components of the TLR-signaling pathway between TLR7/TLR3 and IL6 and/or TNFa.

The invention also relates to a TLR3 and/or a TLR7 agonist for use as a diagnostic agent, in particular for use in the diagnosis of susceptibility towards developing severe disease upon infection with an RNA virus, such as a coronavirus like SARS-CoV-2. In particular the diagnostic use is a non-invasive use, such as in the context of a skin prick test where the epidermis, but not the hypodermis is breached.

The invention also relates to a formulation comprising a TLR3 and/or a TLR7 agonist in an amount appropriate for a one-time or two-time diagnostic administration. The invention also relates to a kit of parts comprising a first formulation comprising a TLR3 and/or a TLR7 agonist and optionally a second formulation not comprising the TLR3 and/or TLR7 agonist, but being otherwise identical or at least similar. The second formulation serves as a placebo control without active ingredients, and helps to increase specificity of the diagnosis by identifying individuals which show an inflammatory reaction only because they are allergic towards an excipient of the formulation or particular excipients.

The identification of potentially vulnerable individuals also helps to target antiviral therapies to those in need. The invention thus also relates to a method of preventing severe (course of) disease from an infection with an RNA-virus, wherein an individual having a potentially increased risk of developing severe disease from an infection with an RNA-virus is diagnosed by a method of the invention and an antiviral drug is then administered to said identified at-risk individual after viral infection or prescribed or determined to be administered to said individual. The invention also relates to an antiviral drug for use in the treatment of a viral infection, wherein the patients to be treated with the antiviral drug are potentially vulnerable individuals who are or were identified as being potentially vulnerable by a diagnostic method of the invention.

More broadly the invention relates to a non-invasive method of diagnosing an individual as having defective TLR signaling, comprising the steps of a) non-invasively administering a TLR agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an individual having a defective TLR signaling pathway.

Based on the diagnosis of an individual as having a defect in a particular TLR signaling pathway, important treatment decisions can be guided.

Even more broadly the invention relates to a non-invasive method of diagnosing an individual as having a defective signaling pathway upstream of NFKB, comprising the steps of a) non-invasively administering an agonist of a DAMP- and/or PAMP -receptor, which DAMP- and/or PAMP -receptor activates NFKB when itself being activated by said agonist, at an agonist concentration sufficient to cause an inflammatory response, and b) detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an individual having a defective signaling pathway upstream of NFKB.

Based on the diagnosis of an individual as having a defect in a signaling pathway upstream of NFKB, important treatment decisions can be guided.

The invention also relates to the use of an agonist of a DAMP- and/or PAMP-receptor, which DAMP- and/or PAMP-receptor activates NFKB when itself being activated by said agonist, in a non-invasive diagnostic method, such as a skin-prick assay. The invention also relates to the use of a kit of parts, wherein the kit of parts is a collection of agonists of a DAMP- and/or PAMP-receptor, which DAMP- and/or PAMP-receptor activates NFKB when itself being activated by said agonist, in a non-invasive diagnostic method, such as a skin-prick assay, wherein at least two agonists of a DAMP- and/or PAMP- receptor are an selective agonist and their respective receptor selectivity is different from one another.

REFERENCES

WO2021/257888A1

WO2021/217505A1

US2021330269A1 Velavan TP et al. “Host genetic factors determining COVID-19 susceptibility and severity.” EBioMedicine. 2021 PMID: 34655949

Solanich X et al. “Genetic Screening for TLR7 Variants in Young and Previously Healthy Men With Severe COVID-19.” Front Immunol. 2021 PMID: 34367187

Stockfleth E et al. “A randomized, double-blind, vehicle-controlled study to assess 5% imiquimod cream for the treatment of multiple actinic keratoses. Arch Dermatol. 2002 Nov;138(l l): 1498-502. PMID: 12437457.

DESCRIPTION OF THE INVENTION

The present invention relates to a method of diagnosing an individual as having an increased risk of developing severe disease from an infection with an RNA-virus, comprising the steps of a) administering a TLR-agonist, such as a TLR3 and/or a TLR7 agonist, at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of a symptom of the expected inflammatory response to identify a potential at-risk individual.

Preferably the administration in step a) is topical administration, such as administration to the skin, for example a part of the body where a topical drug can easily be applied to, such as the skin of the forearm, the upper arm, the leg or the back of the hand. Examples of administration to the skin are simply contacting the skin with the TLR-agonist, such as a TLR3 and/or a TLR7 agonist, but also applying the TLR-agonist, such as the TLR3 and/or the TLR7 agonist, in the form of a skin-prick assay similar to current allergy tests.

A TLR-agonist, such as a TLR3 and/or a TLR7 agonist, and in particular a small molecule TLR3- agonist and/or a small molecule TLR7 -agonist like imiquimod, when applied topically in this manner, will lead to an inflammatory response in most healthy individuals, since the TLR-engagement in the skin cells triggers signaling by NFkB and/or IFRs, resulting in the secretion of inflammatory cytokines and/or type I and/or type III interferons. This inflammatory response is visible and/or detectable in individuals who are responsive towards signaling by the respective TLR, such as responsive to TLR3 and/or TLR7 signaling. For example, it has been described that sensory neurons can respond to TLR7 signaling by causing a sensation at the site of administration, such as a tingle, a prickle, an itch and/or a sensation of pressure at the site of administration. A preferred example of a symptom of an inflammatory response, however, is the development of erythema at the site of administration of a TLR- agonist, for example the development of a small red dot at the site where the epidermis was punctured in the context of a skin prick assay. Potential at-risk individuals for developing severe COVID 19 will have a missing inflammatory response after administration of a TLR7 agonist, which allows their diagnosis as potential at-risk individuals.

The present invention therefore relates to the above method of diagnosing an individual as having an increased risk of developing severe disease from an infection with an RNA-virus, wherein the potential at-risk individual is identified by not having a sensation at the site of exposure upon exposure to the TLR3 and/or TLR7 agonist, for example not sensing a tingle, a prickle, an itch and/or a sensation of pressure at the site of administration within 24 hours after administration, in particular not within the first 3 hours after administration.

The present invention also relates to the above method of diagnosing an individual as having an increased risk of developing severe disease from an infection with an RNA-virus, wherein the potential at-risk individual is identified by not having an inflammatory response at the site of exposure upon exposure to the TLR3 and/or TLR7 agonist, for example by lacking all skin-related symptoms of the expected inflammatory response selected from skin erythema, blisters of the skin, burning of the skin, flaking of the skin, pain of the skin, swelling at the place of application, scabs on the skin, scaling and skin rash, with the lacking development of a small red dot at the site where the epidermis was punctured in the context of a skin prick assay being a preferred skin-related symptom.

A male subject, for example a young male from 12 to 50 years of age, such as from 18 to 40 years of age, who is diagnosed as being a potential at-risk patient can then be confirmed as an at-risk patient by sequencing of his TLR7 locus or mRNA and identifying a mutation therein, such as a loss-of-function mutation. Thus, the method of diagnosis of the present invention is beneficial because it can be used as a simple functional screen prior to genetic analysis which narrows down the number of individuals which need genetic analysis to a manageable number.

In one alternative of the method the TLR agonist is a TLR3 agonist, for example a TLR3 agonist selected from a poly(I:C)-based drug, like rintatolimod, PGV-001 or MCT465. Small molecule TLR3 agonists are preferred, as in general they penetrate more easily the skin or enter more easily into the skin. The skilled person can identify a concentration and amount sufficient to cause a symptom of an inflammatory response, for example by testing increasing concentrations of the TLR3 agonist on the site of administration, such topical administration on the skin, for examples in a skin-prick format, and testing for the appearance of a symptom of an inflammatory response, such as the development of an sensation at the site of administration or preferably the development of an erythema at the site of administration.

In another alternative the TLR agonist is a TLR7 agonist, such as imiquimod, R848, CV-8102, BNT- 411, vesatolimod, guretolimod, AL-034, NKTR-262 or RG-7854. Again, it is preferred that the TLR7 agonist is a small molecule, and the TLR7 agonists imiquimod and resiquimod (r848) are particularly preferred, with imiquimod being most preferred, if only for the reason that it is the only FDA approved small molecule TLR7 agonist. The skilled person can identify a concentration and amount sufficient to cause a symptom of an inflammatory response, for example by testing increasing concentrations of the TLR7 agonist on the site of administration, such topical administration on the skin, for examples in a skin-prick format, and testing for the appearance of a symptom of an inflammatory response, such as the development of an sensation at the site of administration or preferably the development of an erythema at the site of administration. An analogous logic applies for identifying suitable concentrations of agonists which are specific for other toll like receptors, such as TLR4.

Imiquimod is approved for the treatment of actinic keratosis, some forms of basal cell carcinoma and genital warts. Imiquimods side effects, which are due to its activity as a TLR7 agonist, are, for example described in Stockfleth E et al. “A randomized, double-blind, vehicle-controlled study to assess 5% imiquimod cream for the treatment of multiple actinic keratoses. Arch Dermatol. 2002 Nov;138(l 1): 1498-502. PMID: 12437457. Stockfleth E explains that “All patients treated with imiquimod experienced mild, moderate, or severe adverse effects. They included erythema, edema, induration, vesicles, erosion, ulceration, excoriation or flaking, and scabbing.”, with erythema and scabbing being the most frequent side effect. The present inventor has further found that a sensation develops at the site of exposure upon administration of the TLR3 and/or TLR7 agonist, for example a tingle, a prickle, an itch and/or a sensation of pressure at the site of administration within 24 hours, such as within 3 hours or even within 90 minutes. Furthermore, the present inventor has found that erythema, for example a visible small red dot, develops at the site of exposure upon administration of a TLR- agonist, such as a TLR4 and/or a TLR7 agonist, at the site of administration of the TLR agonist in a sin prick assay after one hour but within 24 hours, such as within 3 hours or even within 90 minutes. It is the absence of these effects that is indicative of a missing TLR signaling, such as missing TLR7 signaling, upon engagement by an artificial or natural agonist that the present invention suggests can be monitored in order to identify vulnerable individuals. The skilled person will understand that this is also a method of diagnosing an individual as having defective or downregulated TLR signaling, such as defective or downregulated TLR7 signaling when a TLR7 agonist is being used in the assay, or defective or downregulated downstream components of the TLR-signaling pathway, for example between TLR7 and IL6 and/or TNFa. In other words, a simple and inexpensive skin assay can be used for gathering diagnostic information on genetic defects of components of highly relevant innate immune pathways, which - if defect - render the affected patients vulnerable to infectious disease, such as C0VID19.

In a preferred method of diagnosis of the present invention imiquimod is therefore used, preferably as a 5% creme, such as Aldara or its generic equivalents, and administered topically to the skin. The expected response of an individual with an intact innate immune response is skin irritation, for example manifesting itself as the development of a sensation at the site of administration, for example a tingle, a prickle, an itch and/or a sensation of pressure.

The lack of skin irritation, for example the lack of a sensation at the site of administration, such as the lack of a tingle, a prickle, an itch and/or a sensation pressure, or the lack of erythema, is then indicative for a potential at-risk individual.

Skin irritation can also manifest itself as skin erythema. In a further preferred method of diagnosis of the present invention imiquimod is therefore used as a solution and administered intracutaneously. The lack of erythema, such as the lack of a small red dot, is then indicative for a potential at-risk individual. The skilled person will understand that this is also a method of diagnosing an individual as having defective or downregulated TLR7 or defective or downregulated downstream components of the TLR- signaling pathway between TLR7 and IL6 and/or TNFa.

The present invention also relates to a TLR3 agonist and/or a TLR7 agonist for use as a diagnostic agent, such as for a non-invasive diagnostic use, in particular for use in the diagnosis of susceptibility towards developing severe symptoms upon infection with an RNA virus, and in particular a single stranded RNA virus. RNA viruses can be characterized as being enveloped or naked, and SARS-CoV- 2 is an enveloped RNA virus, Single stranded RNA viruses can be positive or negative stranded, and SARS-CoV-2 is a positive strand RNA virus, belonging to the virus order nidovirales, the family coronaviridae and to the group of betacoronaviruses. Viruses can also be characterized by how they enter a host cell, and in the case of SARS-CoV-2 it can enter a human cell via ACE2 and/or Neuropilin- 1.

It might seem surprising that young men with a functional defect in TLR7 would develop severe disease from an infection with SARS-CoV-2, as there are further PAMPs which can sense viral RNA, such as the cytoplasmic proteins RIG-1 and MDA-5. Without wishing to be bound by any theory, the inventor believes that SARS-CoV-2 inhibits the interferon response triggered by the cytoplasmic RLR-family members in the epithelial cells which it infects by way of viral proteins. The hosts interferon response then becomes dependent on TLR3 and/or TLR7 mediated sensing of the viral RNA in the endosomal compartment of macrophages and/or plasmacytoid dendritic cells underlying the epithelium and patrolling it.

Thus, the method of diagnosis is particularly useful for determining vulnerability to severe virus infection of such viruses that, like SARS-CoV-2, can inhibit cytoplasmic RLR-family signaling in infected epithelial cells. Preferred viruses, for which vulnerability can be diagnosed with a TLR3 and/or TLR7 agonist are, therefore, RNA viruses encoding viral proteins which are capable of interfering with RIG- 1- and/or MDA-5 -mediated interferon signaling, for example an RNA virus which encodes a viral protein which shares at least 30%, such as at least 50% or even at least 80% sequence homology at the protein level with any one of the SARS-CoV-2 proteins selected from the group consisting of S, M, N, NsplO, Nspl4, Nspl5, Nspl6, ORF9b, ORF3b, ORF6 and PLP, and in particular the RNA virus is a coronavirus such as SARS-CoV-2. More generally the present invention also relates to a specific TLR-agonist for use as a diagnostic agent, and in particular for a non-invasive diagnostic use. In particular wherein the TLR-agonist is applied intracutaneously, for example in the context of skin prick assay, and wherein development of erythema, such as the development of a red dot, at the site of administration is indicative of a functional TLR signaling pathway, wherein the TLR signaling pathway corresponds to the specific TLR agonist.

The TLR-agonist, such as the TLR3 agonist and/or TLR7 agonist for diagnostic use is preferably used in the context of a diagnostic method of the invention, as described above. A preferred diagnostic use is a non-invasive diagnostic use, for example a diagnostic method that does not rely on taking a blood sample.

As already mentioned, imiquimod is approved as a 5% w/w creme and provided as a collection of 12 or more sachets for repeated administration as required for the treatment of actinic keratosis, genital warts or basal cell carcinoma. Each sachet contains 12.5mg imiquimod in 250mg cream, which is an amount of creme sufficient to cover an area of 25cm². While these sachets are suitable in the context of the present invention, the method of the present invention does not need to cover 25cm², but rather just the one to three cm² for producing a stripe of imiquimod on the skin. Besides, the method of the present invention also does not require the large number of repeated administrations that therapeutic applications with imiquimod need, but rather administration only once or twice, such as only once.

For the sake of convenience and to avoid waste, the present invention therefore also provides a TLR- agonist, such as a TLR3 agonist and/or a TLR7 agonist, for use as a diagnostic agent, in particular for use in the diagnosis of susceptibility towards developing severe symptoms upon infection with an RNA virus, wherein the TLR agonist is comprised in a formulation in an amount and at a concentration appropriate for a one-time or two-time diagnostic administration. It is sufficient that the amount of the TLR-agonist-containing formulation can cover only at most 5cm², such as at most 2cm² or at most 1cm² of skin. As an example, in the case of a 5% imiquimod cream the amount of imiquimod sufficient for a unit dose formulation to cover 2.5cm² is 1.25mg imiquimod in 25mg cream.

For a skin prick assay it is sufficient that the volume of the TLR-agonist-containing solution is enough for a few drops on the sin, such as from 50pl to 5ml.

The present invention also relates to a unit dose formulation comprising a TLR-agonist, such as a TLR3 agonist and/or TLR7 agonist, in an amount and at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR-agonist, for example a TLR3 and/or a TLR7 agonist, wherein the unit dose formulation is for use as a diagnostic agent, in particular for non-invasive diagnostic use. An example is a TLR7 agonist, such as imiquimod, for example wherein the imiquimod concentration is at least 2% w/w and at most 10% w/w, such as the commercial 5% w/w cream of imiquimod. The invention also relates to a unit dose formulation comprising imiquimod in an amount of at least 0. Img and at most 4 mg. A further example is an aqueous solution of a TLR4 agonist, such as LPS, or a TLR7 agonist, such as imiquimod. The invention also relates to a unit dose formulation for diagnostic use comprising a TLR agonist in an amount of at most 1 mg, such as at most lOOpg, for example at most lOpg.

Few vulnerable individuals might show a symptom of an inflammatory response, such as the development of an erythema or the development of a sensation at the site of administration, for example a tingle, a prickle, an itch and/or a sensation of pressure, upon administration of the composition comprising a TLR-agonist, such as a TLR3 agonist and/or a TLR7 agonist, only because they are allergic against a component of the formulation rather than reacting to the TLR agonist. The specificity of the assay can thus be increased by inclusion of a placebo control formulation not containing any active ingredient. The invention therefore also relates to a kit of parts comprising a first formulation comprising a TLR agonist, such as a TLR3 and/or a TLR7 agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist and a second formulation not comprising the TLR3 and/or TLR7 agonist, but being otherwise identical. Preferably the first formulation of the kit of parts is a unit dose formulation, for example of the 5% w/w imiquimod cream, or a unit dose formulation for diagnostic use comprising a TLR agonist in an amount of at most 1 mg, such as at most lOOpg, for example at most lOpg. The invention also relates to the kit of parts of the invention for diagnostic use.

In particular if the absence of the development of a sensation at the site of administration, for example the absence of a tingle, a prickle, an itch and/or a sensation of pressure, is used as the readout of the assay, it is advantageous to blind and code the formulation containing the active ingredient and the placebo, such that the individual to be tested does not know, which cream contains the active ingredient, and to then administer test formulation and placebo to different sites, such as the left and the right inner forearm. The individual to be tested can then record the site where a sensation develops upon administration. By unblinding and decoding after the assay it can then be controlled, whether the site of a sensation corresponded to the site, where the formulation containing the active ingredient was administered.

The fastest way to implement the diagnostic method of the present invention might be to use the approved pharmaceutical Aldara as such, and to supply placebo controls separately so that a medical practitioner can perform an assay on an individual to-be-tested in a way that the individual does not know, where the imiquimod-containing formulation was applied and where the placebo control. The individual to-be-tested is then blind and does not know, where a local effect of the administered formulation is to be expected.

The invention therefore also relates to a placebo-control formulation NOT comprising an active pharmaceutical ingredient, such as a TLR-agonist, for example imiquimod, for use as a diagnostic agent, such as for a non-invasive diagnostic use, in particular for use in the diagnosis of susceptibility towards developing severe symptoms upon infection with an RNA virus, and in particular a single stranded RNA virus. The placebo-control formulation can be provided alone, but with the intention and/or instruction that it should be used in combination with a corresponding formulation comprising a TLR- agonist, such as a TLR3 and/or TLR7 inhibitor. A placebo-control formulation for Aldara 5% cream is a cream that has the same composition as Aldara 5% cream, but does not comprise imiquimod. In the case of Aldara 5% cream (as marketed in Europe) the placebo control formulation comprises isostearic acid, benzyl alcohol, cetyl alcohol, stearyl alcohol, white soft paraffin, polysorbate 60, sorbitan stearate, glycerol, methyl hydroxybenzoate (E 218), propyl hydroxybenzoate (E 216), xanthan gum and purified water, preferably also supplied as 250mg cream in sachets, if only to match the appearance of Aldara 5% cream.

For the diagnostic method relying on the format of a skin-prick test, the present invention provides an aqueous solution of a TLR agonist for use as a diagnostic agent wherein the TLR agonist is comprised in the aqueous solution in an amount appropriate for a one-time or two-time diagnostic administration and at a concentration sufficient to cause an erythema.

As already mentioned, the identification of potentially vulnerable individuals enables the targeting of antiviral therapy, such as the expensive new therapy with the combination of nirmatrelvir and ritonavir marketed as Paxlovid by Pfizer, to those patients where this therapy is really needed because the vulnerable individuals might progress towards severe disease without pharmacological intervention. Therefore the present invention also relates to a method of preventing severe disease from an infection with an RNA-virus, wherein an individual having an increased risk of developing severe disease from an infection with an RNA-virus is identified by a diagnostic method of the invention as described above and wherein an antiviral drug is then administered to said identified at-risk individual.

The present invention also relates to an antiviral drug for use in the treatment of a viral infection in an individual diagnosed as having an increased risk of developing severe disease from the viral infection, wherein the diagnosed individual having an increased risk of developing severe disease from an infection with a virus was identified by a method of the invention, as described above, in particular wherein the antiviral drug is Paxlovid and the viral infection is infection with SARS-CoV-2. Preferably imiquimod was used in the identification of the individuals which are selected for the treatment with paxlovid.

As touched upon above, a particular advantage of the present invention is that the simple skin-assay with imiquimod cream can be used widely, for example among young men in order to narrow down the number of individuals who one might want to evaluate by a more expensive test, like genetic analysis. This allows for a faster and less expensive screen for those young men, who are at risk from a SARS- CoV-2 infection since they have a loss-of-function allele of TLR7. For other viruses, where the interferon response depends on TLR3 rather than on TLR7, similar test can be developed based on TLR3 agonists.

More broadly the invention relates to a non-invasive method of diagnosing an individual as having defective TLR signaling, for example due to having a defective TLR, comprising the steps of a) non-invasively administering a TLR agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an individual having a defective TLR signaling pathway, for example due to having a defective TLR.

Preferably the administration in step a) is topical administration, such as administration to the skin, for example a part of the body where a topical drug can easily be applied to, such as the skin of the forearm, the upper arm, the leg or the back of the hand. Examples of administration to the skin are simply contacting the skin with the TLR agonist, such as a TLR3-, TLR4-, TLR6-, TLR7- or a TLR9-agonist, but also applying the TLR agonist, such as a TLR3-, TLR4-, TLR6-, TLR7- or a TLR9-agonist, in the form of a skin-prick assay similar to current allergy tests.

Preferred TLR-agonists are specific to one particular TLR (toll-like receptor), and non-invasive administration of such a TLR-agonist, and in particular a small molecule TLR-agonist like imiquimod, when applied in this manner, leads to an inflammatory response in most healthy individuals, since the TLR-engagement in the skin cells, such as epithelial cells and/or underlying plasmacytoid dendritic cells and/or macrophages, triggers - depending on the exact receptor - signaling by NFkB and/or IFRs, resulting in the secretion of inflammatory cytokines and/or type I and/or type III interferons. This inflammatory response is visible and/or detectable in individuals who are responsive towards signaling by the particular TLR that is engaged by the specific TLR agonist. For example, for some TLRs, like TLR7, it has been described that sensory neurons can respond by causing a sensation at the site of administration, such as a tingle, a prickle, an itch and/or a sensation of pressure at the site of administration. A preferred example of a symptom of an inflammatory response is the development of erythema at the site of administration. Individuals who have a missing inflammatory response after administration of a specific TLR agonist, can be diagnosed as individuals having a defective TLR signaling pathway, for example due to having a defective TLR, such as due to a genomic loss-of- function mutation.

The present invention therefore relates to a method of diagnosing an individual as having a defective TLR signaling pathway, wherein the individual having a defective TLR signaling pathway is identified by not having a sensation at the site of exposure upon exposure to the TLR agonist, for example not sensing a tingle, a prickle, an itch and/or a sensation of pressure at the site of administration within 24 hours after administration, in particular not within the first 3 hours after administration. The present invention relates to a method of diagnosing an individual as having a defective TLR signaling pathway, wherein the individual having a defective TLR signaling pathway is identified by by lacking skin erythema, blisters of the skin, burning of the skin, flaking of the skin, pain of the skin, swelling at the place of application, scabs on the skin, scaling and skin rash, and in particular by lacking skin erythema at the site of administration of a TLR agonist in the context of a skin prick assay. If skin erythema at the site of administration of a TLR agonist is missing upon administration, then a tested individual can be diagnosed as potentially having defective TLR signaling.

The present invention relates to a method of diagnosing an individual as having a defective signaling pathway upstream of NFkB, such as a defective RIG signaling pathway, wherein the individual having a defective signaling pathway upstream of NFkB, such as a defective RIG signaling pathway, is identified by lacking skin erythema, blisters of the skin, burning of the skin, flaking of the skin, pain of the skin, swelling at the place of application, scabs on the skin, scaling and skin rash, and in particular by lacking skin erythema at the site of administration of an agonist of said signaling pathway upstream of NFkB, such as an agonist of RIG-1 and/or an agonist of MDA5, in the context of a skin prick assay. If skin erythema at the site of administration of an agonist of said signaling pathway upstream of NFkB, such as an agonist of RIG- 1 and/or an agonist of MDA5, is missing upon administration, then a tested individual can be diagnosed as potentially having a defective signaling pathway upstream of NFkB, such as defective RIG-1 signaling if a RIG-1 agonist does not result in erythema or defective MDA5 signaling if a MDA5 agonist does not result in erythema.

The present invention therefore also provides a TLR agonist for use as a diagnostic agent, in particular for use in the diagnosis of an individual as having a defective TLR signaling pathway. Preferably the TLR agonist is comprised in a formulation in an amount and at a concentration appropriate for a onetime or two-time diagnostic administration. It is sufficient that the amount of the TLR-agonist- containing formulation can cover only at most 5cm², such as at most 2cm² or at most 1cm² of skin. As an example, in the case of a 5% imiquimod cream the amount of imiquimod sufficient for a unit dose formulation to cover 2.5cm² is 1.25mg imiquimod in 25mg cream. As a further example a unit dose formulation for diagnostic use comprising a TLR agonist in an amount of at most 1 mg, such as at most lOOpg, for example at most lOpg, can be used, for example for skin prick assays.

The present invention also relates to a unit dose formulation comprising a TLR agonist in an amount and at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR agonist, wherein the unit dose formulation is for use as a diagnostic agent, in particular for non-invasive use in the diagnosis of an individual as having a defective TLR signaling pathway.

The present invention also relates to a unit dose formulation comprising an agonist of a signaling pathway upstream of NFkB, such as a RIG-1 agonist or a MDA5 agonist, in an amount and at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said agonist, wherein the unit dose formulation is for use as a diagnostic agent, in particular for non-invasive use in the diagnosis of an individual as having a defective signaling pathway upstream of NFkB, such as defective RIG-1 signaling or defective MDA5 signaling.

Few individuals having a defective TLR signaling pathway, for example due to having a defective TLR, might show a symptom of an inflammatory response, such as the development of a sensation at the site of administration or an erythema, upon administration of the composition comprising a TLR agonist only because they are allergic against a component of the formulation rather than reacting to the TLR agonist. The specificity of the assay can thus be increased by inclusion of a placebo control formulation not containing any active ingredient. The invention therefore also relates to a kit of parts comprising a first formulation comprising a TLR agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR agonist and a second formulation not comprising said TLR agonist, but being otherwise identical. The invention also relates to the kit of parts of the invention for the use of diagnosing an individual as having a defective TLR signaling pathway, for example due to having a defective TLR.

As already mentioned, the diagnosis of individuals as having defective TLR-signaling, for example due to having a particular defective TLR, enables to guide important therapeutic decisions. For example, TLR agonists are in use as adjuvants for vaccines. Clearly, a vaccine having, for example, a TLR4 agonist such as 3-O-desacyl-4'-monophosphoryl lipid A (MPL; a detoxified form of lipopolysaccharide extracted from Salmonella Minnesota) as an adjuvans, would not work as well in individuals who have a defective TLR4 signaling pathway, for example due to two copies of TLR4 loss-of-fimction alleles. The diagnosis of individuals as having a defective specific TLR-signaling pathway, for example due to having a particular defective TLR, would help in the selection of individuals, where a therapy, such as a vaccination, which uses TLR agonists as part of the therapy, should be avoided, because the individual having a defect in the TLR signaling pathway would not benefit in the same manner from said therapy as somebody who responds properly to said TLR agonist. The diagnosis of an individual as having a defective specific TLR-signaling pathway, for example due to having a particular defective TLR, is of particular importance for TLRs encoded on the X-chromosome, such as TLR7, where only one gene is present in men. Several therapies based on TLR7 agonists are in development, such as cancer treatments based on, among others, TLR7 agonists or vaccine developments which include TLR7 agonists as an adjuvans. Such treatments would not work as well in individuals who are diagnosed as having a defective TLR-7 signaling pathway, for example due to having a defective TLR7.

The present invention therefore also relates to a TLR agonist, which TLR agonist is specific for one TLR, for use in therapy, such as cancer treatment or a vaccination, wherein the patients to be treated exclude individuals who have been diagnosed as having a defective TLR-signaling pathway corresponding to said specific TLR agonist, for example due to having a defective TLR corresponding to said specific TLR agonist, by a non-invasive diagnostic method, for example by the non-invasive diagnostic method of the present invention. In particular, the invention also relates to a specific TLR7 agonist for use in therapy, such as cancer - and in particular melanoma- treatment or a vaccination, wherein the patients to be treated exclude individuals who have been diagnosed as having a defective TLR7-signaling pathway, for example due to having a defective TLR7, by a non-invasive diagnostic method, for example by the non-invasive diagnostic method of the present invention. In simple words, the invention uses the inflammatory response upon non-invasive administration of a TLR agonist which is specific for one particular TLR agonist, for example the development of a local erythema or a sensation at the site of administration, to identify individuals for which a therapy with said specific TLR agonist or another TLR agonist having the same TLR specificity would be counterindicated. A simple prior skin assay would thus be able to guide decisions on more complex treatment regimens, such as vaccinations or cancer treatments.

More generally, the present invention also relates to an agonist of a signaling pathway upstream of NFkB, such as a RIG-1 agonist or a MDA5 agonist, which agonist is specific for one component of a signaling pathway upstream of NFkB, such as specific for RIG-1 or specific for MDA5, for use in therapy, such as cancer treatment or a vaccination, wherein the patients to be treated exclude individuals who have been diagnosed as having a defective signaling pathway corresponding to said specific agonist, for example due to having defective RIG-1 corresponding to said specific RIG-1 agonist, by a non-invasive diagnostic method, for example by the non-invasive diagnostic method of the present invention.

Using the TLR7 agonist imiquimod as an example, the prospective patients should first be diagnosed as having functional TLR7 signaling before a lengthy treatment - that would have little prospect of success if the pharmacological target is not functional - is initiated. The non invasive diagnostic method of the invention would serve to select a patient group for treatment, which will show a better response as a group. Thus, the present invention also relates to the medical use of imiquimod, for example for the treatment of actinic keratosis, the treatment of genital warts and the treatment of superficial basal cell carcinoma, wherein individuals who have been diagnosed as having a defective TLR7-signaling pathway, for example due to having a defective TLR7, by a non-invasive diagnostic method as described herein are counterindicated.

Definitions

The term "gene" means a DNA sequence that codes for an RNA or a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

Within the context of the present invention the terms "mutant" and "mutation" mean a detectable change in genetic material, i.e. genomic DNA. Mutations include deletion, insertion or substitution of one or more nucleotides. The mutation may occur in the coding region of a gene (i.e. in exons), in introns, or in the regulatory regions (e.g. enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, promoters) of the gene. Generally, a mutation is identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population. Where the mutation is within the gene coding sequence, the mutation may be a "missense" mutation, where it replaces one amino acid with another in the gene product, or a "nonsense" mutation, where it replaces an amino acid codon with a stop codon. A mutation may also occur in a splicing site where it creates or destroys signals for exon-intron splicing and thereby lead to a gene product of altered structure. Within the context of the present invention a mutation is not silent, i.e. it results at least in an alteration of the nucleotide sequence (where the gene product is a functional RNA) or an amino acid sequence (where the gene product is a protein) that renders the gene product non-functional or that reduces expression of the gene product at the RNA-level by at least 80%.

As used herein the term “deletion” means that a part of a DNA-sequence is missing compared to a reference sequence.

As used herein the term "expression" may refer to gene expression of a polypeptide or protein, or to gene expression of a polynucleotide, such as miRNAs or IncRNAs, depending on the context. Expression of a polynucleotide may be determined, for example, by measuring the production of RNA transcript levels using methods well known to those skilled in the art. Expression of a protein or polypeptide may be determined, for example, by immunoassay using (an) antibody(ies) that binds the polypeptide specifically, using methods well known to those skilled in the art.

As used herein the “expression of an mRNA” relates to the transcriptional level of gene expression.

As used herein an “exon” is any part of a gene that will encode a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature messenger RNA.

As used herein an "agonist" is a molecule, such as a chemical, that binds to a receptor and activates the receptor to produce a biological response. A specific TLR agonist is a molecule, such as a chemical, that binds to and activates one specific TLR to produce a biological response, but does not bind to and activate other TLRs. A specific TLR7 agonist, for example, would bind to and activate only TLR7, but not other TLRs. R848 binds to and activates TLR7 and TLR8 and is thus not an example of a specific TLR agonist.

As used herein the term “having an increased risk of developing severe disease” means that an individual has a higher individual risk of hospitalization, ventilation or even death upon viral infection than has been determined for a randomly selected cohort of healthy individuals representative for the worldwide age distribution at the priority date. In the case of young men “having an increased risk of developing severe disease” means that an individual has a higher individual risk of hospitalization, ventilation or even death upon viral infection than has been determined for a randomly selected cohort of healthy individuals representative for the population of young men of similar age.

The skilled person will understand that wherever the invention relates to “a method of diagnosing an individual as having an increased risk of developing severe disease from an infection with an RNA- virus”, by “detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an at-risk individual” it conversely relates to “ a method of diagnosing an individual as not having an increased risk of developing severe disease from an infection with an RNA- virus” by “detecting the presence of the expected inflammatory response in an individual who is not an at-risk individual”.

"Population" means a group of individual organisms of the same species that live in the same geographic area. For example all human beings living in the United States of America form the "population" of the USA. Or all cows of a farm form the population of cows on the farm. "Subpopulation" means a proper subset of the population, wherein the individuals from the subset share at least one characteristic with one another that is not shared by all individuals of the population. For example, old people above the age of 60 in the USA would form a subpopulation within the larger population of the USA.

TLR7 as used herein relates, depending on the context, to the TLR7 gene or to the TLR7 protein, an endosomal receptor that plays a key role in innate and adaptive immunity (PubMed: 14976261, PubMed:32433612). It is envolved in host immune response against pathogens through recognition of uridine-containing single strand RNAs (ssRNAs) of viral origin or guanosine analogs (PubMed:31608988, PubMed:27742543, PubMed: 12738885, PubMed:32706371). Upon binding to agonists, the TLR7 receptor undergoes dimerization that brings TIR domains from the two molecules into direct contact, leading to the recruitment of the TIR-containing downstream adapter MYD88 through homotypic interaction (PubMed:27742543). In turn, the Myddosome signaling complex is formed involving IRAK4, IRAKI, TRAF6, TRAF3 leading to activation of downstream transcription factors NF-kappa-B and IRF7 to induce proinflammatory cytokines and interferons, respectively (PubMed:27742543, PubMed:32706371). TLR7 is described in detail as gene ID 51284 in the NCBI Gene database and is located on the X chromosome. TLR3 as used herein relates, depending on the context, to the TLR3 gene or to the TLR3 protein, an endosomal receptor that plays a key role in innate and adaptive immunity. TLR3 is a nucleotide -sensing TLR which is activated by double -stranded RNA, a sign of certain viral infections. TLR3 acts via the adapter TRIF/TICAM1, leading to NF-kappa-B activation, IRF3 nuclear translocation, cytokine secretion and the inflammatory response. TLR3 is described in detail as gene ID 7098 in the NCBI Gene database. TLR3 is located on chromosome 4.

ACE2 as used herein relates, depending on the context, to the ACE2 gene or to the ACE2 protein. ACE2 is a single-pass, transmembrane protease enzyme that has high homology with angiotensin-converting enzyme (ACE). It is the host cell receptor for several coronaviruses but also plays a role in the reninangiotensin system where it inactivates angiotensin II by converting it to angiotensin 1-7. ACE2 is described in detail as gene ID 59272 in the NCBI Gene database.

Neuropilin- 1 as used herein relates, depending on the context, to the NRP1 gene or to the Neuropilin 1 protein. Neuropilin 1 is a cell-surface receptor involved in the development of the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits and in organogenesis outside the nervous system. It mediates the chemorepulsant activity of semaphorins and acts as a host factor for human coronavirus SARS-CoV-2 infection. NRP1 is described in detail as gene ID 8829 in the NCBI Gene database.

RIG-1 as used herein relates, depending on the context, to the DDX58 gene or its expression product, the RIG-1 protein, an innate immune receptor that senses cytoplasmic viral nucleic acids and activates a downstream signaling cascade leading to the production of type I interferons and proinflammatory cytokines. Forms a ribonucleoprotein complex with viral RNAs on which it homooligomerizes to form filaments. The homooligomerization allows the recruitment of RNF135 an E3 ubiquitin-protein ligase that activates and amplifies the RIG-I-mediated antiviral signaling in an RNA length-dependent manner through ubiquitination-dependent and -independent mechanisms (PubMed:28469175, PubMed:31006531). Upon activation, associates with mitochondria antiviral signaling protein (MAVS/IPS1) that activates the IKK-related kinases TBK1 and IKBKE which in turn phosphorylate the interferon regulatory factors IRF3 and IRF7, activating transcription of antiviral immunological genes including the IFN-alpha and IFN-beta interferons (PubMed:28469175, PubMed:31006531). DDX58 is described in detail as gene ID 23586 in the NCBI Gene database.

MDA5 as used herein relates, depending on the context, to the IFIH1 gene or its expression product, the MDA5 protein, an intracellular sensor of viral RNA that triggers the innate immune response. Sensing RNA length and secondary structure, MDA5 binds dsRNA oligonucleotides with a modified DExD/H-box helicase core and a C-terminal domain, thus leading to a proinflammatory response that includes interferons. IFIH1 is described in detail as gene ID 64135 in the NCBI Gene database.

The term RLRs as used herein relates to RIG-like receptors (retinoic acid-inducible gene-I-like receptors.) They are a type of intracellular pattern recognition receptor involved in the recognition of viruses by the innate immune system. Examples are RIG-I, MDA5 and LGP2. RLRs are cytoplasmic pattern recognition receptors (PRRs) with sentinel function for intracellular viral RNA that is a product of viral infection.

NFkB or NFkappaB as used herein relates, depending on the context, to the NFKB1 gene or its expression product, the transcription factor NFkappaB, a rapidly acting primary transcription factor found in all cell types. It is involved in the cellular responses to stimuli such as cytokines and stress and plays a key role in regulating the inflammatory response to infections. NFKB1 is described in detail as gene ID 4790 in the NCBI Gene database.

The term IRF as used herein relates to interferon regulatory factors, proteins which regulate transcription of interferons. Interferon regulatory factors contain a conserved N-terminal region of about 120 amino acids, which folds into a structure that binds specifically to the IRF -element (IRF-E) motifs, which is located upstream of the interferon genes. An important IRF involved in TLR7-signaling is IRF7. IRF7 is described in detail as gene ID 3665 in the NCBI Gene database.

SARS-CoV-2 as used herein is the strain of coronavirus that causes COVID-19. It is a positive-sense single -stranded RNA virus. Its genome contains several essential genes that encode the viral proteins necessary for replication, transcription and infectious virus assembly. The essential genes comprise the open reading frames la and lb (ORF lab) that are translated to produce 16 mature nonstructural proteins (nspl-nspl6, numbered according to their order from the N-terminus to the C-terminus of the ORF 1 polyproteins) that are involved in viral RNA replication and transcription. In addition to these genes, SARS-CoV-2 contains genes that encode four structural proteins which are involved in infectious virus assembly: (S)pike, (E)nvelope), (M)embrane, and (N)ucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike protein is responsible for allowing the virus to attach to and fuse with the membrane of a host cell. Interspersed between these genes in the coronavirus genome are several other genes called "group-specific or accessory genes" and their gene products are called "accessory proteins" that are dispensable for virus growth in vitro, but may play an important role in modulating the host response to virus infection and thereby, contribute to pathogenesis. The reference sequence for SARS-CoV-2 is the NCBI Reference Sequence: NC_045512.2, the complete genome of the Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu- 1. SARS-CoV-2 variants typically show at least 98.0% sequence similarity at the whole genome level with this reference sequence, preferably at least 99.0%.

A “unit dose” as used herein is the amount of a medication administered to a patient in a single dose.

Medical or physiological terms, like “sensation” or “erythema”, as used herein have the meaning as known to the experienced general practitioner or medical doctor; unless specified otherwise. In cases of doubt, the English language version of the book “Pschyrembel” can be used to define medical or physiological terms.

“A concentration sufficient to cause an inflammatory response” as used herein is a concentration which causes at least one symptom of an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist, e.g. the concentration which causes at least one symptom of an inflammatory response in at least 70% of individuals, preferably at least 85% of individuals, from a randomly selected cohort of healthy individuals representative for the worldwide age distribution at the priority date.

An inflammatory response is expected upon administration of a sufficient amount of a TLR3 and/or TLR7 agonist. Symptoms of the expected inflammatory response upon administration of imiquimod are described in Stockfleth E et al. and include development of a sensation at the site of administration, for example a tingle, a prickle, an itch and/or a sensation of pressure.

A “lack of the expected inflammatory response” as used herein means that there is no sign of the development of a sensation at the site of administration, for example no sign of a tingle, a prickle, an itch and/or a sensation of pressure, within 24 hours after topical administration of the TLR agonist.

Alternatively there is no sign of an erythema at the site of administration 24 hours after topical administration of the TLR agonist, and in particular after administration in the context of a skin prick assay.

„Severe disease” as used herein is disease from infection with an RNA virus that leads to hospitalization, artificial ventilation (e.g. in the case of a virus affecting lung function like SARS-CoV-2) or even death.

As used herein, the term "subject" or “individual” as used herein denotes a mammal, but preferably a human being. In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disease or condition to which such term applies, or one or more symptoms of such disease or condition. Preferably it means reversing, alleviating, inhibiting the progress of, or preventing the disease or condition.

The term "diagnosing" as used herein refers to assessing the risk, i.e., the probability according to which a subject will develop severe disease from an infection with an RNA-virus as referred to in this specification. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant portion of subjects can be correctly diagnosed to develop the said severe disease from an infection with an RNA-virus upon infection with said virus. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 . Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows that the diagnosis will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.

“Non-invasive” as used herein refers to a diagnostic method which does not comprise a step, where the dermis and/or an endothelium is breached. A skin-prick test, similar to the allergy tests which are in current use, is considered non-invasive. In particular “Non-invasive” as used herein refers to a diagnostic method which does not comprise a step, where an epithelium is breached. For example a diagnostic method which relies on the taking of blood with a needle would include at least one step wherein an endothelium is breached and is thus an invasive diagnostic method as used herein.

“Paxlovid” as used herein relates to a combination of the two antiviral drugs nirmatrelvir and ritonavir. PAXLOVID is comprised of nirmatrelvir, a SARS-CoV-2 main protease (Mpro: also referred to as 3CLpro or nsp5 protease) inhibitor, co-packaged with ritonavir, an HIV-1 protease inhibitor and CYP3A inhibitor. Ritonavir, which has no activity against SARS-CoV-2 on its own, is included to inhibit the CYP3 A-mediated metabolism of nirmatrelvir and consequently increase nirmatrelvir plasma concentrations to levels anticipated to inhibit SARS-CoV-2 replication.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

It is to be understood that this invention is not limited to the particular materials and methods described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention - unless defined otherwise herein - and ranked in increasing order of priority: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rd ed. 2006); The Glossary of Genomics Terms (JAMA. 2013; 309(14): 1533-1535), Janeway’s Immunobiology, 9th edition and “Practical Flow Cytometry”, 4th edition by H.M. Shapiro. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The invention further relates to the following embodiments

1. A method for diagnosing an individual as having an increased risk of developing severe disease from an infection with an RNA-virus, comprising the steps of a) administering a TLR3 and/or a TLR7 agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist, and b) detecting the lack of a symptom of the expected inflammatory response or the delay of a symptom of the expected inflammatory response to identify an at-risk individual.

2. The method of item 1, wherein the administration in step a) is topical administration.

3. The method of any one of items 1 or 2, wherein administration in step a) is administration to the skin.

4. The method of item 3, wherein administration is to the forearm, the upper arm, the leg or the back of the hand.

5. The method of any one of items 1 to 4, wherein the expected symptom is the response of a sensory neuron.

6. The method of any one of items 2 to 5, wherein the expected symptom is a sensation at the site of administration.

7. The method of item 6 in combination with item 3, wherein the sensation is a tingle, a prickle, an itch and/or a pressure.

8. The method of item 6, wherein administration is to the nasal mucosa and wherein the sensation is a loss of smell.

9. The method of any one of items 1 to 4, wherein the expected symptom is skin erythema.

10. The method of any one of items 1 to 4 and 9, wherein the at-risk individual lacks all skin- related symptoms of the expected inflammatory response selected from skin erythema, blisters of the skin, burning of the skin, flaking of the skin, pain of the skin, swelling at the place of application, scabs on the skin, scaling and skin rash.

11. The method of any one of items 1 to 4, and 9 to 10, wherein the at-risk individual lacks all systemic symptoms of the expected inflammatory response selected from chills, cough, difficulty with breathing, ear congestion, loss of voice, sneezing, runny nose, flu-like symptoms, joint pain, fever or unusual tiredness.

12. The method of any one of items 1 to 11, wherein the at-risk individual shows no symptom of the expected inflammatory response.

13. The method according to any one of items 5 to 12 wherein the at-risk individual does not show the symptom or symptoms of the expected inflammatory response within 24 hours after administration.

14. The method of item 13, wherein the at-risk individual does not feel a sensation at the site of administration within 3 hours after administration.

15. The method of any one of items 1 to 14, wherein the TLR agonist is a TLR3 agonist.

16. The method of item 15 wherein the TLR3 agonist is selected from a poly(I:C)-based drug, like rintatolimod, PGV-001 and MCT465. The method of items 15 or 16, wherein the TLR3 agonist is a small molecule. The method of any one of items 1 to 14, wherein the TLR agonist is a TLR7 agonist. The method of item 18, where the TLR7 agonist is selected from the group consisting of imiquimod, R848, CV-8102, BNT-411, vesatolimod, guretolimod, AL-034, NKTR-262 and RG-7854. The method of items 18 or 19, wherein the TLR7 agonist is a small molecule. The method of item 18, wherein the TLR7 agonist is imiquimod. The method of item 21, wherein imiquimod is administered topically to the skin. The method of item 22, wherein the expected inflammatory response is a sensation at the site of administration. The method of item 23, wherein the sensation is a tingle, a prickle, an itch and/or a pressure The method of item 22, wherein the expected inflammatory response is skin irritation. The method of item 25, wherein the skin irritation is erythema. The method of any one of items 21 to 26, wherein imiquimod is administered as a cream. The method of any one of items 23 to 24, wherein imiquimod is administered as a cream comprising 5% imiquimod. The method of item 28, wherein the cream comprising 5% imiquimod is the FDA approved cream “Aldara” or a generic equivalent thereof. A TLR3 agonist and/or a TLR7 agonist for use as a diagnostic agent. The TLR3 agonist and/or TLR7 agonist according to item 30 for use in the diagnosis of increased risk towards developing severe symptoms upon infection with an RNA virus. The TLR3 agonist and/or TLR7 agonist for use according to items 30 or 31, wherein the method of diagnosing is not invasive. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 30 to 32, wherein the RNA virus can enter a human cell via ACE2 and/or Neuropilin- 1, preferably via ACE2 and Neuropilin- 1. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 30 to 33, wherein the RNA virus encodes viral proteins which are capable of interfering with RIG-1- and/or MDA-5 -mediated interferon signaling. The TLR3 agonist and/or TLR7 agonist for use according to item 34, wherein the RNA virus encodes a viral protein which shares at least 50% sequence homology at the protein level with any one of the SARS-CoV-2 proteins selected from the group consisting of S, M, N, NsplO, Nspl4, Nspl5, Nspl6, ORF9b, ORF3b, ORF6 and PLP. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 35, wherein the RNA virus is a single stranded RNA virus. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 36, wherein the RNA virus is an enveloped RNA virus. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 37, wherein the RNA virus is a positive strand RNA virus. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 38, wherein the RNA virus is a member of the virus order nidovirales. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 39, wherein the RNA virus is a member of the virus family coronaviridae. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 40, wherein the RNA virus is a betacoronavirus. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 41, wherein the RNA virus is SARS-CoV-2. The TLR3 agonist and/or TLR7 agonist for use according to any one of items 31 to 42, wherein the diagnostic method is a method according to items 1 to 29. The TLR3 agonist and/or TLR7 agonist according to any one of items 30 to 43, wherein the TLR agonist is comprised in a formulation in an amount appropriate for a one-time diagnostic administration. A unit dose formulation comprising a TLR3 agonist and/or TLR7 agonist in an amount and at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist, wherein the unit dose formulation is for use as a diagnostic agent. The unit dose formulation according to item 45, wherein the TLR agonist is a TLR7 agonist. The unit dose formulation according to item 46, wherein the TLR7 agonist is imiquimod. The unit dose formulation according to item 47, wherein the imiquimod concentration is at least 2% w/w and at most 10% w/w. The unit dose formulation according to any one of items 47 to 48, wherein the formulation is a 5% w/w cream of imiquimod. The unit dose formulation according to any one of items 47 to 49, wherein the amount of imiquimod contained in the unit dose formulation is at least 0. 1 milligram and at most 4 milligrams. A kit of parts comprising a first formulation comprising a TLR3 and/or a TLR7 agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist and a second formulation not comprising the TLR3 and/or TLR7 agonist, but being otherwise identical. The kit of parts according to item 51, wherein the first formulation is a unit dose formulation according to any one of items 45 to 50. The kit of parts according to item 52, wherein the first formulation is a 5% w/w imiquimod cream. The kit of parts according to any one of items 51 to 53 for diagnostic use, in particular for a diagnostic use according to any one of items 32 to 44. A method of preventing severe disease from an infection with an RNA-virus, wherein an individual having an increased risk of developing severe disease from an infection with an RNA-virus is identified by a method according to any one of items 1 to 29 and wherein an antiviral drug is then administered to said identified high-risk individual. An antiviral drug for use in the treatment of a viral infection in an individual having an increased risk of developing severe disease from the viral infection, wherein the individual having an increased risk of developing severe disease from an infection with a virus was identified by a diagnostic method according to any one of items 1 to 29. The antiviral drug for use according to item 56, wherein the antiviral drug is pavloxid and the viral infection is infection with SARS-CoV-2. The antiviral drug for use according to item 57, wherein imiquimod is the TLR7 agonist used in the diagnostic method according to any one of items 1 to 29. A method for diagnosing an individual as not having an increased risk of developing severe disease from an infection with an RNA-virus, comprising the steps of a) administering a TLR3 and/or a TLR7 agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR3 and/or a TLR7 agonist, and b) detecting the expected inflammatory response or a symptom of the expected inflammatory response to identify a not at-risk individual. The method of item 59, as further specified in any one of items 2 to 29. A non-invasive method of diagnosing an individual as having defective TLR signaling, in particular due to having a defective TLR, comprising the steps of a) non-invasively administering a TLR agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of the expected inflammatory response or the delay of the expected inflammatory response in an individual having a defective TLR signaling pathway, for example due to having a defective TLR. The method of item 61, wherein the administration in step a) is topical administration. The method of any one of items 61 or 62, wherein administration in step a) is administration to the skin. The method of item 63, wherein administration is to the forearm, the upper arm, the leg or the back of the hand. The method of any one of items 61 to 64, wherein the expected symptom is the response of a sensory neuron. The method of any one of items 62 to 65, wherein the expected symptom is a sensation at the site of administration. The method of item 66 in combination with item 63, wherein the sensation is a tingle, a prickle, an itch and/or a pressure. The method of item 66, wherein administration is to the nasal mucosa and wherein the sensation is a loss of smell. The method of any one of items 61 to 64, wherein the expected symptom is skin erythema. The method of any one of items 61 to 64 and 69, wherein the individual having a defective TLR signaling pathway lacks all skin-related symptoms of the expected inflammatory response selected from skin erythema, blisters of the skin, burning of the skin, flaking of the skin, pain of the skin, swelling at the place of application, scabs on the skin, scaling and skin rash. The method of any one of items 61 to 64, and 69 to 70, wherein the individual having a defective TLR signaling pathway lacks all systemic symptoms of the expected inflammatory response selected from chills, cough, difficulty with breathing, ear congestion, loss of voice, sneezing, runny nose, flu-like symptoms, joint pain, fever or unusual tiredness. The method of any one of items 61 to 71, wherein the at-risk individual shows no symptom of the expected inflammatory response. The method according to any one of items 65 to 72 wherein the at-risk individual does not show the symptom or symptoms of the expected inflammatory response within 24 hours after administration. The method of item 73, wherein the at-risk individual does not feel a sensation at the site of administration within 3 hours after administration. The method of any one of items 60 to 74, wherein the TLR agonist is a TLR7 agonist and wherein the individual showing the lack of the expected inflammatory response or the delay of the expected inflammatory response is diagnosed as having a defective TLR7 signaling pathway, in particular due to having a defective TLR. A unit dose formulation comprising a TLR agonist in an amount and at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR agonist, wherein the unit dose formulation is for use as a diagnostic agent. The unit dose formulation according to item 76, wherein the TLR agonist is a specific TLR agonist. The unit dose formulation according to any one of items 76 to 77 and 45 to 50, wherein the diagnostic use is diagnosing an individual as having defective TLR signaling, in particular due to having a defective TLR. A kit of parts comprising a first formulation comprising a TLR agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR agonist and a second formulation not comprising the TLR agonist, but being otherwise identical. The kit of parts according to item 79, wherein the first formulation is a unit dose formulation according to any one of items 76 to 78. The kit of parts according to any one of items 79 to 80, wherein the TLR agonist is a TLR7 agonist. The kit of parts according to item 81, wherein the TLR7 agonist is imiquimod. The kit of parts according to item 82, wherein the first formulation is a 2% to 10% w/w imiquimod cream. The kit of parts according to any one of items 79 to 83 for use in diagnosing an individual as having defective TLR signaling, in particular due to having a defective TLR. A TLR agonist for use in therapy, wherein the patients to be treated exclude individuals who have been diagnosed as having a defective TLR-signaling pathway corresponding to said TLR agonist by a non invasive diagnostic method. The TLR agonist for use according to item 85, wherein the TLR agonist is a specific TLR agonist. The TLR agonist for use according to any one of items 85 to 86, wherein the TLR agonist is a TLR7 agonist. The TLR agonist for use according to any one of items 85 to 87, wherein the non-invasive diagnostic method is a method according to any one of items 61 to 75. The TLR agonist for use according to any one of items 85 to 88, wherein the treatment is administration of a vaccine comprising said TLR agonist. The TLR agonist for use according to any one of items 85 to 88, wherein the treatment is cancer treatment by administration of a pharmaceutical composition comprising said TLR agonist. The TLR agonist for use according to item 90, wherein the TLR agonist is a TLR7 agonist and wherein the cancer is melanoma. A method for diagnosing an individual as having functional TLR signaling, comprising the steps of a) administering a TLR agonist at a concentration sufficient to cause an inflammatory response in individuals who are responsive to said TLR agonist, and b) detecting the expected inflammatory response or a symptom of the expected inflammatory response to identify an individual having functional TLR signaling. A non-invasive method of diagnosing an individual as having defective signaling upstream of NFKB, the steps of a) intradermally administering an agonist of a PAMP- and/or DAMP-receptor at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of an inflammatory response, such as local reddening, at the administration site or the delay of an inflammatory response, such as local reddening, at the administration site in an individual having a defective signaling pathway upstream of NFKB. The method of item 93, wherein the administration in step a) is by a skin prick test. The method of item 94, wherein administration is to the forearm, the upper arm, the leg or the back of the hand. The method of any one of items 93 to 95, wherein the PAMP-receptor is capable of activating NFkB and can be activated by biomolecules of viral origin. The method of item 96, wherein the biomolecule is viral RNA. The method of any one of items 93 to 95, wherein the PAMP-receptor is capable of activating NFkB and can be activated by biomolecules of bacterial origin. The method of item 98, wherein the biomolecule is a bacterial cell wall component. . The method of any one of items 93 to 95, wherein the PAMP-receptor is capable of activating NFkB and can be activated by biomolecules of fungal origin. . The method of item 100 wherein the biomolecule is a fungal cell wall component.. The method of any one of items 93 to 101, wherein the PAMP-receptor is RIG-1 or MDA-5. . An agonist of a PAMP-receptor or an agonist of a DAMP-receptor or an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB, for use as a diagnostic agent.. The agonist of a PAMP-receptor or an agonist of a DAMP-receptor or an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB, for use according to 103, wherein the method of diagnosing is not invasive. . The agonist for use according to 104 to 105, wherein the method of diagnosing is a skin prick assay. . The agonist for use according to any one of 103 to 105, wherein said agonist is a TLR-agonist, an agonist of a RIG-I-like receptor, an agonist of a NOD-like receptor, an agonist of a C-type-lectin-like receptor or an agonist of a cytosolic DNA sensor. . The agonist for use according to 106, wherein said agonist is a TLR-agonist. . The agonist for use according to 106, wherein said agonist is an agonist of a RIG-I- like receptor. . The agonist for use according to 106, wherein said agonist is an agonist of a NOD- like receptor. . The agonist for use according to 106, wherein said agonist is an agonist of a C-type- lectin-like receptor. . The agonist for use according to 106, wherein said agonist is an agonist of a cytosolic DNA sensor. . The agonist for use according to 107, wherein said TLR-agonist is a TLRl-agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR2 -agonist. . The agonist for use according to 107, wherein said TLR-agonist is a TLR3 -agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR4-agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR5 -agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR6-agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR7 -agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR8-agonist.. The agonist for use according to 107, wherein said TLR-agonist is a TLR9-agonist.. The agonist for use according to 108, wherein said agonist of a RIG-I-like receptor is a RIG-I agonist. . The agonist for use according to 108, wherein said agonist of a RIG-I-like receptor is a MDA5 agonist. . The agonist for use according to 109, wherein said agonist of a NOD-like receptor is a NODI agonist. . The agonist for use according to 109, wherein said agonist of a NOD-like receptor is a N0D2 agonist. . The agonist for use according to 109, wherein said agonist of a NOD-like receptor is a NLRP3 agonist. . The agonist for use according to 110, wherein said agonist of a C-type-lectin-like receptor is a CLEC7A agonist. . The agonist for use according to 110, wherein said agonist of a C-type-lectin-like receptor is a CLEC6A agonist. . The agonist for use according to 110, wherein said agonist of a C-type-lectin-like receptor is a CLEC4E agonist. . The agonist for use according to 111, wherein said agonist of a cytosolic DNA sensor is an agonist of the cGAS-STING pathway. . The agonist for use according to any one of 103 to 105, wherein said agonist is an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB. . The agonist for use according to 130, wherein said signal transduction protein is MYD88. . The agonist for use according to 130, wherein said signal transduction protein is IRAKI. . The agonist for use according to 130, wherein said signal transduction protein is IRAK4. . The agonist for use according to 130, wherein said signal transduction protein is TRAF6. . A collection of at least two different agonists, wherein the agonists are an agonist of a PAMP-receptor or an agonist of a DAMP-receptor or an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB, for use as a diagnostic agent, and wherein the at least two agonist are selective for different receptors. . The collection of at least two different agonists for use according to 135, wherein the method of diagnosing is not invasive. 137. The collection of at least two different agonists for use according to 135 to 136, wherein the method of diagnosing is a skin prick assay.

138. The collection of at least two different agonists for use according to 135 to 137, for diagnosing IRAK4-deficiency.

139. The collection of at least two different agonists for use according to 135 to 137, for diagnosing MyD88-deficiency.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Handbook of Experimental Immunology" (Weir, 1997); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques may be considered in making and practicing the invention.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Materials and Methods

Aldara 5% creme was used, as described in detail in the Aldara Summary of product characteristics available from the EMA, version as online on January 18, 2022. https://www.ema.europa.eu/en/documents/product-information/aldara-epar-product- information_en.pdf

Aldara was applied to the inside of the lower forearm, about half way between the inside of the elbow and the wrist joint. Before applying Aldara, the treatment area was washed with soap and water and dried thoroughly. An amount of cream the size of a lentil was applied to form a 3cm stripe in the treatment area, The cream was rubbed into the treatment area until the cream vanished. The cream remained on the skin for approximately 8 hours. During this period, showering and bathing was avoided. After 8 hours imiquimod cream was removed with mild soap and water. Hands were washed carefully before and after application of the cream.

Results

Individual 1: 54 year old male, moderately severe COVID-19 12 months prior to test, no hospitalization Erythema did not develop within 24 hours. After about one hour a sensation best described as a mild pressure developed at the site of administration. This sensation was transient.

Individual 2: 53 year old female, no confirmed COVID-19 prior to test, but asymptomatic disease possible (wife of individual 1 ; contact with individual 1 during infection)

Erythema did not develop within 24 hours. Already after 10 to 15 minutes a clear sensation best described as a tingle or a prickle developed at the site of administration. This sensation was transient.

Example 2

In an immunological prick test increasing concentrations of LPS, a toll-like receptor (TLR4)-agonist, were administered intracutaneously by a trained physician in order to identify a concentration of LPS that would result in an observable inflammatory response. By an initial up-titration experiment a concentration of lOpg/mL LPS diluted in phosphate buffered saline (PBS) was determined as being sufficient for producing a small red dot, appearing after about 90 minutes.

In the experiment shown in Figure 1, triplicates of lOpg/ml LPS samples (in PBS) were tested. Histamine (0.01%) or physiological saline solution (0.9%) served as a positive and negative control, respectively. Briefly, one drop of the control or TLR4-agonist solutions as indicated above was layered onto the skin, and an intracutaneous lancet was used to gently prick the skin and allow intracutaneous penetration of the assay solution. The order of administration on the arm in the experiment shown in figure Iwas as follows: for the left six samples LPS and PBS alternate. The order, from top to bottom, is LPS, PBS control, LPS, PBS control, LPS, PBS control. The right three samples are histamine controls. The numbers below the photos indicate the time when the photo was taken.

As expected, the positive histamine control caused an immediate itch and resulted in a transient peripuncture edema, with edema appearing after a few minutes and being well visible after about 20 minutes (see 15. 18h).

The reddening of the skin caused by the histamine solution faded after about 60 minutes, and also the small irritation at the site of the puncture for the LPS and PBS samples was no longer visible then (see circles at 16.02h). Overtime a small and rather persistent red dot developed at the point of the puncture site in the case of the three LPS samples. This reaction was neither observed at the sites of the histamine control nor of the saline control. This clear difference between the LPS samples and the two other types of samples became observable already about 90 minutes after the administration, becoming clearer at 3 to 4 hours after administration and persisted until even 24 hours after the administration. The three photos taken 8.5 hours after the start of the experiment show the red dot, which had developed at the site of administration for the three LPS samples (see circles bottom left). The center and right photos show the bottom and center sample, respectively, in more detail.

This example demonstrates that a TLR-agonist causes observable signs of inflammation when administered intracutaneously, such as in a standard immunological prick test. This observation can be explained by the activation of the TLR4 signaling pathway, resulting in the downstream activation of NFKB, which in turn results in the secretion of pro-inflammatory cytokines, such as IL6 and TNFa. The activation of NFKB is a feature shared by TLR signaling pathways and further DAMP and PAMP receptors..

Abbreviations:

TLR: toll-like receptor

RLR: RIG-1 like receptor

EMA: European Medicines Agency

FDA: US Food and Drug Administration

RNA: ribonucleic acid

Claims

1. A non-invasive method of diagnosing an individual as having defective TLR signaling, in particular due to having a defective TLR, comprising the steps of a) non-invasively administering a TLR agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the lack of an inflammatory response or the delay of an inflammatory response in an individual having a defective TLR signaling pathway.

2. The method of claim 1, wherein the administration in step a) is topical administration.

3. The method of any one of claims 1 or 2, wherein administration in step a) is intradermal administration to the skin.

4. The method of any one of claims 1 to 3, wherein the inflammatory response is skin erythema.

5. The method according to any one of claims 1 to 4 wherein the individual having defective TLR signaling does not show the symptom or symptoms of the inflammatory response within 24 hours after administration.

6. The method of any one of claims 1 to 5, wherein the TLR agonist is a TLR4 or a TLR7 agonist and wherein the individual showing the lack of the inflammatory response or the delay of the inflammatory response is diagnosed as having a defective TLR4 or a defective TLR7 signaling pathway.

7. The method of any one of claims 1 to 6 wherein the TLR agonist is a specific TLR7 agonist, wherein administration is intradermal administration to the skin and wherein the individual having defective TLR7 signaling does not show a reddening of the skin at the site of administration within 24 hours after administration, in particular wherein the individual is diagnosed as having an increased risk of developing severe disease from an infection with an RNA-virus.

8. An agonist of a PAMP -receptor or an agonist of a DAMP-receptor or an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB, for use as a diagnostic agent, wherein the diagnostic method is non-invasive.

9. The agonist for use according to claim 8, wherein the method of diagnosing is a skin prick assay.

10. The agonist for use according to any one of claims 8 to 9, wherein said agonist is a TLR- agonist, an agonist of a RIG-I-like receptor, an agonist of a NOD-like receptor, an agonist of a C-type -lectin-like receptor or an agonist of a cytosolic DNA sensor.

I L A collection of at least two different agonists, wherein the agonists are an agonist of a PAMP- receptor or an agonist of a DAMP-receptor or an agonist of a signal transduction protein, wherein said signal transduction protein is part of a signaling cascade between a PAMP- and/or DAMP-receptor and NFkB, for use as a diagnostic kit, and wherein the at least two agonists are selective for different receptors and wherein the diagnostic method is non- invasive.

12. The collection of agonists of claim 11, wherein the diagnostic method is a skin prick assay.

13. A non-invasive method of diagnosing an individual as having functional TLR signaling, in particular due to having a functional TLR, comprising the steps of a) non-invasively administering a TLR agonist at a concentration sufficient to cause an inflammatory response, and b) detecting the expected inflammatory response in an individual having a functional TLR signaling pathway corresponding to said TLR agonist.