CN113495155A

CN113495155A - Triage method

Info

Publication number: CN113495155A
Application number: CN202010265531.9A
Authority: CN
Inventors: J.V.米卡莱夫; M.E.埃克莱斯顿
Original assignee: Belgian Volition SPRL
Current assignee: Belgian Volition SPRL
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-10-12

Abstract

The present invention relates to a method of assigning a risk of adverse consequences to a subject suffering from an infection, in particular a respiratory tract infection.

Description

Triage method

Technical Field

The present invention relates to cell-free nucleosomes as biomarkers in a sample of bodily fluid for infecting a patient, in particular for identifying high risk patients who have an adverse reaction to infection.

Background

Influenza outbreaks occur once a year, all over the world, resulting in about three to five million cases of serious illness and about 290,000 to 650,000 deaths. Recently, the emergence and rapid development of a new infection, COVID-19, has escalated to a pandemic state. Some infections lead to Acute Respiratory Syndrome (ARS) or Severe Acute Respiratory Syndrome (SARS), a potentially fatal disease progression requiring drug treatment. Infection outbreaks and pandemics have placed extreme strain on the international healthcare services (international healthcare services), and therefore methods of triaging patients to identify those most likely to require hospital intervention are critical to helping healthcare providers (healthcare providers) prioritize patients, save lives, and more effectively handle the higher demands of healthcare services.

HoldenriederWait forInt. J Cancer (2001) 95: 114-. Circulating cell-free cells have been investigated in terms of their histone modifications, histone variants, DNA modifications and adduct contentThe epigenetic composition of the nucleosomes is a blood-based biomarker in cancer, see WO 2005/019826, WO 2013/030577, WO 2013/030579, and WO 2013/084002.

There remains a need in the art to provide a simple, cost-effective method to identify and prioritize individuals who may have a poor prognosis at the time of infection.

Brief description of the drawings

FIG. 1 immunoassay results of Neutrophil Extracellular Trap (NET) -derived nucleosomes in EDTA plasma and heparin plasma samples taken from two healthy volunteers. The EDTA sample contained a small amount of NET-derived nucleosome material. In contrast, heparin induced NET formation, and heparin plasma samples contained high levels of induced NET-derived nucleosomes.

Fig. 2 bioanalyzer electrophoresis results of NET-derived nucleosomes in EDTA plasma and heparin plasma samples taken from two healthy volunteers. The EDTA sample contained low levels of mononucleosomes and NET-derived nucleosome material. In contrast, heparin induced NET formation, and heparin plasma samples contained low levels of mononucleosomes (peak at about 60 seconds), but high levels of induced NET-derived nucleosomes (broad peak at about 110 seconds). Narrow peaks at about 43 seconds and about 110 seconds represent the DNA sample added for reference purposes.

Summary of The Invention

According to a first aspect, there is provided a method of assigning a risk of adverse outcome to a subject suffering from an infection, comprising:

(i) contacting a sample of bodily fluid obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof; and

(ii) using the detected cell free nucleosome levels to assign a likelihood of adverse outcome for the subject.

According to a further aspect, there is provided a method of assigning a risk of development or progression of a medical complication in a subject suffering from an infection, comprising:

(ii) using the detected cell free nucleosome levels to assign a likelihood of development or progression of a medical complication in said subject.

According to a further aspect, there is provided a method of selecting a subject suffering from an infection in need of medical treatment for a medical complication of the infection, comprising:

(ii) using the detected cell free nucleosome level to indicate the presence, progression or development of a medical complication in need of treatment in said subject.

In a preferred embodiment, the infection is a respiratory influenza or coronavirus infection and the medical complication is ARS or SARS or pneumonia. Accordingly, in one embodiment, there is provided a method of detecting a subject in need of medical treatment for pneumonia, ARS or SARS, comprising:

(ii) the level of cell free nucleosomes is used as an indicator that the subject is in need of medical treatment for pneumonia, ARS or SARS.

In a preferred embodiment, the infection is a respiratory influenza or coronavirus infection and the medical complication is ARS or SARS or pneumonia.

According to a further aspect of the invention there is provided a method of monitoring infection in a subject, comprising:

(i) contacting a sample of bodily fluid obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof;

(ii) repeating the detecting or measuring one or more times of the level of cell free nucleosomes or components thereof in a sample of bodily fluid obtained from the subject;

(iii) any change in the level of cell free nucleosomes or components thereof is used to monitor the progression of infection in a subject.

Detailed Description

Nucleosomes are released into the circulation upon chromatin fragmentation upon cell death. Many infections, such as viral infections, trigger cell death by multiple mechanisms (cell binding and entry, endosomal TLR3 activation and gene expression), thereby increasing the number of circulating nucleosomes in the blood (Danthi et al, annu. rev. virol. (2016) 3: 533-53). In addition, infection can induce NETosis, whereby posttranslational histone modifications, such as supercitrullination of histones H3 and H4 (Wang Y et al, j. Cell biol. (2009)184(2): 205-. However, extracellular nucleosomes and Neutrophil Extracellular Traps (NETs) can lead to serious complications if not cleared rapidly. For example, nucleosome binding to the mesangium is associated with renal injury in lupus (Kalaaji et al, Kidney int. (2007) 71(7): 665-. Indeed, host-directed NET toxicity is associated with respiratory distress, narrow airway obstruction, epithelial injury, inflammatory response and thrombosis (Marcos et al, nat. Med. (2010) 16: 1018-23; Hoeksema et al, Future Microbiol. (2016) 11: 441-53).

Most subjects infected with influenza or coronavirus experience mild illness. However, some subgroups of the population, including elderly people over the age of 60 years and people with underlying diseases such as diabetes, chronic lung disease, particularly chronic heart disease, are at risk of serious effects including ARS, SARS, pneumonia and death. The exact mechanism by which influenza or coronavirus infection causes complications including pneumonia is not known, but is thought to be caused by a hyperimmune response to the viral infection, where excess NETs cause acute lung injury, resulting in pneumonia and, in the worst case, death.

The present invention utilizes elevated levels of cell free nucleosomes (including NETs) to predict the severity of disease and the consequences of infectious disease.

Thus, according to a first aspect, there is provided a method of assigning a risk of adverse outcome to a subject suffering from an infection, comprising:

In one embodiment, there is provided a method of assigning a risk of adverse outcome to a subject suffering from an infection, comprising:

(i) contacting a bodily fluid sample obtained from the subject with a binding agent to detect or measure the level of neutrophil extracellular trap material or a component thereof; and

(ii) assigning a likelihood of adverse outcome to the subject using the detected level of neutrophil extracellular trap material.

Nucleosomes are the basic unit of chromatin structure and consist of a protein complex of eight highly conserved core histones (comprising a pair of each histone H2A, H2B, H3 and H4). Around this complex is wrapped approximately 146 base pairs of DNA. Another histone (H1 or H5) acts as a linker and is involved in the chromatin compaction process. DNA is wrapped around successive nucleosomes in what is commonly referred to as a "bead-like" structure, which forms the basic structure of open or euchromatin. In dense or heterochromatin, the string curls and supercoils into a closed and complex structure (Herranz and Esteller, Methods mol. (2007) 361: 25-62).

When detected in a body fluid sample, reference to "nucleosomes" may refer to "cell-free nucleosomes". It is to be understood that the term cell-free nucleosomes is intended throughout this document to include any cell-free chromatin fragment, which includes one or more nucleosomes.

It is understood that cell-free nucleosomes can be detected by binding to their components. The term "component thereof" as used herein refers to a portion of the nucleosome, i.e. it is not necessary to detect the entire nucleosome. Cell free nucleosome components may be selected from: histone (i.e., histone H1, H2A, H2B, H3, or H4), a histone post-translational modification, a histone variant or isoform, a nucleosome-bound protein (i.e., nucleosome-protein adduct), a nucleosome-associated DNA fragment, and/or a nucleosome-associated modified nucleotide. For example, its component may be histone (isoform) H3.1 or histone H1 or DNA.

The methods and uses of the invention allow the level of (cell-free) nucleosomes per se to be measured. Reference to "nucleosomes themselves" refers to the level or concentration of total nucleosomes present in a sample, without regard to any epigenetic features that the nucleosomes may or may not include. Detection of total nucleosome levels typically involves detection of histones common to all nucleosomes, such as histone H4. Thus, nucleosomes themselves can be measured by detecting core histones, such as histone H4. As described herein, histones form building blocks known as nucleosomes, which are used to package DNA in eukaryotic cells. As previously reported in WO 2016/067029 (incorporated herein by reference), specific histone variants, such as histone H3.1, H3.2 or H3t, can be used to isolate cell-free nucleosomes derived from tumor cells. Thus, the total level of cell-free nucleosomes of tumor origin can be detected.

Normal cell turnover in adults involves approximately 10 cells produced by cell division each day¹¹Individual cells, and similar numbers die (primarily by apoptosis). During apoptosis, chromatin is broken down into mononucleosomes and oligonucleosomes that can be released from the cell. Under normal conditions, the levels of circulating nucleosomes found in healthy subjects are reported to be low. Elevated levels (Holdenreider) are found in subjects suffering from a variety of conditions, including many cancers, autoimmune diseases, inflammatory conditions, stroke and myocardial infarction& Stieber, Crit. Rev. Clin. Lab. Sci. (2009) 46(1): 1–24）。

Previous nucleosome ELISA methods have been used primarily in cell culture, generally as a method of detecting apoptosis (Salgame et al, Nucleic Acids Res. (1997)25(3): 680-681; Holdenrieder et al (2001)supra(ii) a van Nieuwenhuijze et al, Ann. Rheum. Dis. (2003) 62: 10-14), but may also be usedCirculating cell-free nucleosomes were measured in serum and plasma (Holdenrieder et al (2001)). Cell-free serum and plasma nucleosome levels released into the circulation by dead cells have been measured by ELISA methods in a number of different cancer studies to assess their use as potential biomarkers.

The cell free nucleosomes may be mononucleosomes, oligonucleosomes, components of larger chromatin fragments or components of NETs or mixtures thereof.

Mononucleosomes and oligonucleosomes can be detected by enzyme-linked immunosorbent assays (ELISA) and several methods have been reported (e.g., Salgame et al (1997); Holdenrieder et al (2001); van Nieuwenhuijze et al (2003)). These assays typically use anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3, and H4) as capture antibodies and anti-DNA or anti-H2A-H2B-DNA complex antibodies as detection antibodies.

Circulating nucleosomes are not a homologous group of protein-nucleic acid complexes. Rather, they are heterogeneous sets of chromatin fragments derived from chromatin digestion upon cell death, and include a large number of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be apparent to those skilled in the art that an increase in nucleosome levels is associated with an increase in certain subsets of circulating nucleosomes containing specific epigenetic signals, including nucleosomes comprising specific histone isoforms (or variants), comprising specific post-translational histone modifications, comprising specific nucleotides or modified nucleotides and comprising specific protein adducts. Assays for these types of chromatin fragments are known in the art (see, e.g., WO 2005/019826, WO 2013/030579, WO 2013/030578, WO 2013/084002, which are incorporated herein by reference).

There are many proteins in NET that are directly or indirectly attached to nucleosomes. These proteins include, but are not limited to, Myeloperoxidase (MPO), Neutrophil Elastase (NE), lactoferrin, azuridin (azurocidin), cathepsin G, leukocyte protease 3, lysozyme C, neutrophil defensin 1, neutrophil defensin 3, myeloid cell nuclear differentiation antigen, S100 calbindin A8, S100 calbindin A9, S100 calbindin A12, actin β, actin γ, α -actin, plastin-2 (plastin-2), cytokeratin-10, catalase, α -enolase, and transketolase (Urban et al, PLOS Pathologs (2009) 10: e 1000639). Any nucleosome-protein adduct present in NET is an adduct that can be used in the method of the invention to detect elevated NET levels. C-reactive protein (CRP) may also be added to nucleosomes in NET and nucleosome-CRP adducts are thus adducts that can be used to detect elevated NET levels in the methods of the invention.

In a preferred embodiment of the invention, the adduct used is an MPO-nucleosome adduct or an NE-nucleosome adduct.

In one embodiment, the component of the cell free nucleosome comprises an epigenetic feature of the cell free nucleosome.

The biomarkers used in the methods of the invention may be the level of cell free nucleosomes themselves and/or the epigenetic characteristics of the cell free nucleosomes. It is to be understood that the terms "epigenetic signal structure" and "epigenetic signature" are used interchangeably herein. They refer to specific features of the nucleosome that can be detected. In one embodiment, the epigenetic characteristic of the nucleosome is selected from the group consisting of: post-translational histone modifications, histone isoforms, modified nucleotides, and/or proteins that bind to nucleosomes in nucleosome-protein adducts.

In one embodiment, the epigenetic feature of the nucleosome comprises one or more histone variants or isoforms. The epigenetic characteristic of the cell-free nucleosome may be a histone isoform, such as a histone isoform of the core nucleosome, in particular histone H3 isoform. The terms "histone variant" and "histone isoform" are used interchangeably herein. The structure of the nucleosome can also be altered by including alternative histone isoforms or variants which are different genes or splice products and have noThe same amino acid sequence. Many histone isoforms are known in the art. Histone variants can be classified into families, which are subdivided into separate classes. The nucleotide sequence of a large number of Histone variants is known and can be found, for example, in The NHGRI Histone Database of The national human genome institute (Mari ñ o-Ramfirez et al The Histone Database: an integrated resources for histones and Histone fold-associating proteins.DatabaseVol.2011 and http:// genome.nhgri.nih.gov/histones/complete.shtml), GenBank (NIH genetic sequence) database, EMBL nucleotide sequence database, and Japanese DNA Database (DDBJ). For example, variants of histone H2 include H2a1, H2a2, mH2a1, mH2a2, H2AX, and H2 AZ. In another example, histone isoforms of H3 include H3.1, H3.2, and H3 t.

In one embodiment, the histone isoform is H3.1.

Nucleosome structure can be altered by post-translational modification (PTM) of histones. PTM of histones usually occurs in the tail of core histones, and common modifications include acetylation, methylation, or ubiquitination (ubiquitination) of lysine residues, methylation of arginine residues, phosphorylation of serine residues, and the like. Many histone modifications are known in the art, increasing in number as new modifications are recognized (Zhao and Garcia (2015) Cold Spring Harb Perspect Biol, 7: a 025064). Thus, in one embodiment, the epigenetic feature of the cell-free nucleosome may be a histone post-translational modification (PTM). The histone PTM may be a core nucleosome, for example a histone PTM of H3, H2A, H2B or H4, in particular H3, H2A or H2B. In particular, the histone PTM is histone H3 PTM. Examples of such PTMs are described in WO 2005/019826.

For example, the post-translational modifications may include acetylation, methylation (which may be monomethylation, dimethylation or trimethylation), phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamylation and/or isomerization (see Ausio (2001) Biochem Cell Bio 79: 693). In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is H3 citrulline (H3 cit) or H4 citrulline (H4 cit). In a still further embodiment, the histone PTM is H3 cit.

In one embodiment, the histone PTM is ribosylation, also known as ADP-ribosylation. The posttranslational histone ADP-ribosylation of the nucleosomes occupying the inflammatory response marker promoter in macrophages is stimulated by exposure to lipopolysaccharide, leading to increased transcription, and may have antiviral properties. Furthermore, all members of the coronavirus family contain a highly conserved macro-domain in the non-structural protein 3 (nsp 3) that regulates post-translational ADP-ribosylation by enzymatic removal of covalently linked ADP-ribose from the protein target. Recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with a mutant macrodomain with reduced nsp3 ADP-ribosylation activity are hypoinfectious and cause early-enhancing Interferon (IFN), interferon-stimulated genes (ISG), and pro-inflammatory cytokine responses. Thus, changes in the levels of circulating ADP-ribosylating nucleosomes released from macrophages are contemplated for use in the methods of the invention.

One group or class of related histone posttranslational modifications (rather than a single modification) can also be detected. Typical examples relate, without limitation, to 2-site immunoassays that use an antibody or other selective binding agent that binds targeted to the nucleosome, and an antibody or other selective binding agent that binds targeted to the relevant histone modification group. By way of illustration and not limitation, examples of such antibodies that are directed to bind a histone modification group include anti-pan-acetylated antibodies (e.g., pan-acetyl H4 antibody [ H4panAc ]), anti-citrullinated antibodies, or anti-ubiquitin antibodies.

In one embodiment, the epigenetic characteristic of the nucleosome comprises one or more DNA modifications. In addition to epigenetic signaling mediated by nucleosome histone isoforms and PTM composition, nucleosomes also differ in their nucleotide and modified nucleotide composition. Some nucleosomes may contain more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

In one embodiment, the epigenetic characteristic of the nucleosome comprises one or more protein-nucleosome adducts or complexes. Another type of subset of circulating nucleosomes is the nucleosome protein adduct. Chromatin has been known for many years to contain a large number of non-histone proteins that bind to its constituent DNA and/or histones. These chromatin-associated proteins are of a very wide variety of types and have many functions, including transcription factors, transcription enhancers, transcription repressors, histone modification enzymes, DNA damage repair proteins, and the like. These chromatin fragments, including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins, are described in the prior art.

In one embodiment, the protein that is adducted to the nucleosome (which can therefore be used as a biomarker) is selected from: transcription factors, high mobility group proteins or chromatin modifying enzymes. Reference to a "transcription factor" refers to a protein that binds to DNA and regulates gene expression by promoting (i.e., activator) or inhibiting (i.e., repressor) transcription. Transcription factors contain one or more DNA Binding Domains (DBDs) that are linked to specific sequences of DNA adjacent to the genes they regulate.

All circulating nucleosomes and nucleosome moieties (nucleosome moieties), types or subgroups described herein may be used in the present invention.

It is to be understood that more than one epigenetic feature of cell-free nucleosomes can be detected in the methods and uses of the invention. Multiple biomarkers can be used as a combination biomarker. Thus, in one embodiment, the use comprises more than one epigenetic feature of the cell-free nucleosome as a combined biomarker. The epigenetic features may be of the same type (e.g., PTM, histone isoforms, nucleotide or protein adducts) or of different types (e.g., PTM-binding histone isoforms). For example, posttranslational histone modifications and histone variants can be detected (i.e., more than one type of epigenetic signature detected). Alternatively or additionally, more than one type of post-translational histone modification is detected, or more than one type of histone isoform is detected. In one aspect, the use comprises post-translational histone modification and histone isoforms as a combined biomarker in a sample for diagnosis or detection of infection. In one embodiment, the combination biomarker is H3.1 and H3 cit. In an alternative embodiment, the combination biomarker is H3.1 and H4 cit.

The term "biomarker" refers to a unique biological or biologically derived indicator of a process, event or condition. Biomarkers can be used in diagnostic methods, such as clinical screening and prognostic evaluation, as well as for monitoring treatment outcome, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and their use are valuable for identifying new drug therapies and for discovering new targets for drug therapies.

The methods of the invention involve assigning a patient risk of adverse outcome. Adverse consequences include death and/or acute events requiring immediate medical care, such as hospitalization (i.e., hospital treatment) and/or surgery. For many patients, the infection is overcome by their own immune system without the need for medical intervention. However, in many patients, the infection may progress or increase in severity without being overcome by the immune system, or the patient's own immune response to the infection may lead to adverse consequences. For example, adverse consequences may include the onset of an acute coronary or cardiac event (such as myocardial infarction and/or stroke), acute multi-or single-organ failure (such as renal failure, liver failure, and/or heart failure), debilitating acute disease and/or acute respiratory disease (such as pneumonia, hypoventilation/hypopnea, Acute Respiratory Distress Syndrome (ARDS), Severe Acute Respiratory Syndrome (SARS), bronchiolitis, and/or bronchitis). Thus, in one embodiment, the methods described herein prescribe a risk of the patient or subject to develop an acute respiratory disorder. In a further embodiment, the acute respiratory disorder is pneumonia. In a further embodiment, the acute respiratory disorder is hypoventilation/hypopnea. In still further embodiments, the acute respiratory disorder is Acute Respiratory Distress Syndrome (ARDS) and/or Severe Acute Respiratory Syndrome (SARS).

A given patient's risk of poor outcome may specify a near-term or short-term risk, or may specify an intermediate-term risk. Recent or short-term risk includes where a patient may develop adverse outcomes within 30 days of the occurrence of symptoms or a positive diagnosis, such as within 2 weeks or 14 days, within 1 week or 7 days, or within 5 days or less. Such recent or short-term risk may also include where the patient may develop adverse outcomes within 30 days of practicing the methods described herein, such as within 2 weeks or 14 days, within 1 week or 7 days, or within 5 days or less. Intermediate risk includes where a patient may develop adverse outcomes more than 30 days after symptoms appear, a positive diagnosis, and/or the practice of the methods described herein. Thus, in one embodiment, the methods described herein assign a patient or subject at risk of developing an adverse outcome within 2 weeks or 14 days of the appearance of symptoms or a positive diagnosis. In further embodiments, the methods described herein specify a risk of developing an adverse outcome within 1 week or 7 days of the occurrence of symptoms or a positive diagnosis. In yet a further embodiment, the methods described herein specify a risk of developing an adverse outcome within 5 days of the occurrence of symptoms or a positive diagnosis.

Thus, in another aspect of the invention, there is provided a method of identifying a subject having an infection in need of hospital treatment, comprising:

(ii) the detected level of cell free nucleosomes is used to determine whether the subject requires hospitalization.

It will be appreciated that the method of the invention may also be used to identify patients who do not require hospital treatment, i.e. to use the detected levels of cell-free nucleosomes to determine whether a subject should not be admitted to hospital treatment. This mode of the invention will help to identify patients who can be discharged early if they have been admitted.

The methods and uses described herein may be tested in a bodily fluid sample, particularly a blood, serum or plasma sample. Preferably, a plasma sample is used. Plasma samples may be collected in collection tubes containing one or more anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin or sodium citrate, particularly EDTA.

Infection with viral infection

The methods of the invention are particularly useful in controlling infectious outbreaks. Infection can be caused by different pathogens and environmental factors. In one embodiment, the infection is a viral, bacterial, fungal or microbial infection. Bacterial infections may include mycobacterial, pneumococcal and influenza infections such as those caused by Streptococcus pneumoniae (Streptococcus pneumoniae), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Haemophilus influenzae (Haemophilus influenzae) and Staphylococcus aureus (Staphylococcus aureus) (e.g. pneumonia). In a further embodiment, the infection is a viral infection. Viral infections may include infections caused by Respiratory Syncytial Virus (RSV), influenza a, influenza b, and coronaviruses (e.g., COVID-19).

Infection may be defined by the tissue affected by the disease. For example, the disease may affect the heart, brain, kidney, liver, pancreas, lung, and/or blood, and the infection may be a bacterial, viral, fungal, or microbial infection known to commonly affect such tissues or organs. In one embodiment, the infection is a respiratory infection. According to this embodiment, the infection affects the lungs, the upper respiratory tract and/or the lower respiratory tract.

Other tissues that may be affected by the disease include peripheral tissues such as limbs, hands and feet, and the infection may be a bacterial infection (e.g., gangrene). In one embodiment, the infection and/or disease may affect multiple tissues or organs simultaneously. For example, the infection may be a bacterial infection of the limbs, hands or feet, and the disease may also affect the blood (e.g. sepsis). In another example, the disease may be heart or coronary failure, and other tissues or organs affected by the disease may include the kidneys and the renal system and/or brain (e.g., stroke). In yet another example, the disease may affect the lungs, or the infection may be a respiratory tract infection, and the other tissue or organ affected may include the heart, the coronary artery system, and/or the brain (e.g., heart failure, myocardial infarction, and/or stroke).

In one embodiment, the respiratory infection is selected from: influenza, pneumonia and Severe Acute Respiratory Syndrome (SARS). SARS is a respiratory infection caused by the SARS coronavirus (SARS-CoV), other related coronaviruses are known (e.g., COVID-19 (also known as SARS-CoV-2, previously known as 2019-nCoV)). It is known to cause fever flu-like symptoms, coughing and lethargy, and can lead to pneumonia (e.g. direct viral pneumonia or secondary bacterial pneumonia).

The emergence and rapid growth of COVID-19 into a pandemic state has placed severe pressure on international healthcare services. The infectivity prediction range is up to 70-80% of the national population. It appears that although most people will experience mild symptoms, the two-digit percentage of infected people may be severely affected.

Identifying COVID-19 positive individuals at high risk for serious reactions or complications, including pneumonia, will enable triage and facilitate distribution of stressful medical resources, including intensive care beds and ventilators, until community immunity is established, protecting the community from future widespread outbreaks. Thus, in a preferred embodiment, there is provided a method of identifying a subject infected with an influenza or coronavirus infection in need of medical treatment, comprising:

(i) contacting a sample of bodily fluid obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof; and are

(ii) The detected level of cell free nucleosomes is used to determine whether the subject is in need of medical treatment.

Diagnostic and monitoring method

According to a further aspect, there is provided a method of monitoring the severity of infection in a subject, comprising:

(ii) repeating the detecting or measuring one or more times of the level of cell free nucleosomes or components thereof in a bodily fluid obtained from the subject;

According to a further aspect, there is provided a method of monitoring the progression of an infection in a subject having, suspected of having, or susceptible to having an infection, comprising the steps of:

(i) contacting a sample obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes; and

(ii) the detected levels of cell free nucleosomes are compared to an early sample taken from the subject to monitor the progression of infection.

If the subject is determined to have no infection or a mild infection, the present invention may still be used for the purpose of monitoring the progression of a future medical complication of the disease. For example, if the method comprises a sample from a subject determined to have a mild infection, biomarker level measurements may be repeated at another time point to determine whether the biomarker level has changed.

Detection and/or quantification may be performed directly on purified or enriched nucleosome samples, or indirectly on extracts or dilutions thereof. Quantifying the amount of the biomarker present in the sample may comprise determining the concentration of the biomarker present in the sample. The uses and methods of detection, monitoring and diagnosis according to the invention described herein can be used to confirm the presence of disease, monitor disease progression by assessing onset and progression, or assess improvement or regression of disease. The uses and methods of detection, monitoring and diagnosis may also be used in methods of assessing clinical screening, prognosis, treatment selection, assessing therapeutic benefit, i.e. in drug screening and drug development.

Detection or measurement may include immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor methods. In particular, the detection and/or measurement may comprise a 2-site immunoassay method of nucleosome moieties. Such methods are preferably used for in situ measurement of nucleosomes or nucleosomes that bind an epigenetic feature, using two anti-nucleosome binding agents or anti-nucleosome binding agents in combination with an anti-histone modification or anti-histone variant or anti-DNA modification or anti-addition protein detection binding agent. In addition, the detection and/or measurement may include a 2-site immunoassay, for example using a labeled or immobilized combination of: anti-nucleosomes, anti-histone modifications, anti-histone variants/isoforms, anti-DNA modifications, or anti-adduct protein binders.

In one embodiment, the method of detecting or measuring comprises contacting a body fluid sample with a solid phase comprising a binding agent for detecting cell-free nucleosomes or components thereof, and detecting binding to the binding agent.

In one embodiment, a method of detection or measurement comprises: (i) contacting the sample with a first binding agent that binds to an epigenetic feature of the cell-free nucleosome; (ii) (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent that binds to cell-free nucleosomes; and (iii) detecting or quantifying binding of the second binding agent in the sample.

In another embodiment, a method of detecting or measuring comprises: (i) contacting the sample with a first binding agent that binds to cell free nucleosomes; (ii) (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent that binds to an epigenetic feature of the cell-free nucleosome; and (iii) detecting or quantifying binding of the second binding agent in the sample.

Detecting or measuring biomarker levels may be performed using one or more reagents, such as a suitable binding agent. For example, the one or more binding agents may comprise a ligand or binding agent specific for a desired biomarker (e.g., nucleosomes or components thereof, epigenetic features of nucleosomes, structure/shape mimics of nucleosomes or components thereof), optionally in combination with one or more interleukins.

It will be apparent to those skilled in the art that the terms "antibody", "binding agent" or "ligand" as used herein are not limiting, but are intended to encompass any binding agent capable of binding a particular molecule or entity, and that any suitable binding agent may be used in the methods of the invention. It will also be apparent that the term "nucleosome" is intended to include mononucleosomes, oligonucleosomes, NET and any protein-DNA chromatin fragments that can be analysed in a fluid medium.

Methods of detecting biomarkers are known in the art. The agent may comprise one or more ligands or binding agents, such as naturally occurring or chemically synthesized compounds, capable of specifically binding to the desired target. The ligand or binding agent may comprise a peptide, antibody or fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide, capable of specifically binding to the desired target. The antibody may be a monoclonal antibody or a fragment thereof. It is to be understood that if an antibody fragment is used, it retains the ability to bind to the biomarker, such that the biomarker (according to the invention) can be detected. The ligand/binding agent may be labeled with a detectable label such as a luminescent, fluorescent, enzymatic or radioactive label; alternatively or additionally, the ligands of the invention may be tagged with an affinity tag, such as a biotin, avidin, streptavidin, or His (e.g. hexa-His) tag. Alternatively, ligand binding can be determined using label-free techniques, such as the technique of ForteBio corporation.

The term "detecting" or "diagnosing" as used herein includes the identification, confirmation and/or characterization of a disease state. The detection, monitoring and diagnostic methods of the invention can be used to confirm the presence of a disease, monitor the progression of a disease by assessing onset and progression, or assess the amelioration or regression of a disease. The methods of detection, monitoring and diagnosis may also be used in methods of assessing clinical screening, prognosis, selection of therapy, assessing therapeutic benefit, i.e. for drug screening and drug development

The methods of the invention may comprise normalization of the marker levels. For example, the level of cell free nucleosomes containing a particular epigenetic signature can be normalized to the level of the nucleosomes themselves (or some other type of nucleosome or parameter) in order to express that level as the proportion of nucleosomes containing that signature. For example, citrullinated nucleosome levels are expressed as a proportion of citrullinated nucleosomes.

In one embodiment, the methods described herein are repeated on multiple occasions. This embodiment provides the advantage of being able to monitor the results of the detection over a period of time. Such an arrangement would provide the benefit of monitoring or assessing the efficacy of treatment of the disease state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilization, amelioration, recurrence and/or remission.

In the monitoring method, the test sample may be collected on two or more occasions. The method can further comprise comparing the level of the biomarker present in the test sample to one or more controls and/or to one or more previous test samples taken from the same test subject earlier (e.g., prior to initiation of treatment) and/or from the same test subject at an earlier stage of treatment. The method may comprise detecting a change in the nature or amount of the biomarker in a test sample taken at different occasions.

A change in the level of the biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject may indicate a beneficial effect, e.g., stabilization or improvement, of the therapy on the disease or suspected disease. In addition, once treatment is complete, the methods of the invention can be repeated periodically to monitor for recurrence of the disease.

Methods of monitoring treatment efficacy can be used to monitor the effectiveness of treatment of existing and new therapies in human subjects and non-human animals (e.g., animal models). These monitoring methods can incorporate screens for new drug substances and substance combinations.

In further embodiments, faster changes caused by fast-acting therapies can be monitored over shorter time intervals of hours or days.

A diagnostic or monitoring kit (or panel) for carrying out the methods of the invention is provided. Such kits will suitably comprise one or more ligands for detecting and/or quantifying a biomarker of the invention, and/or a biosensor as described herein, and/or an array, optionally together with instructions for use of the kit.

Another aspect of the invention is a kit for detecting the presence of an infection comprising a biosensor capable of detecting and/or quantifying one or more biomarkers as defined herein. The term "biosensor" as used herein refers to any substance capable of detecting the presence of a biomarker. Examples of biosensors are described herein. The biosensor may comprise a ligand binding agent or ligand capable of specifically binding to a biomarker as described herein. Such biosensors may be used to detect and/or quantify the biomarkers of the invention.

Suitably, the biosensor for detecting one or more biomarkers combines biomolecular recognition with appropriate means to convert the detection or quantification of the presence of a biomarker in a sample into a signal. Biosensors may be suitable for "alternative site" diagnostic testing, for example in wards, out subjects' departments, surgery, home, field and work sites. Biosensors that detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic, biolayer interferometry (BLI), and micro-engineered sensors. Print recognition elements, thin film transistor technology, magneto-acoustic resonator devices, and other novel acousto-electric systems may be used in biosensors to detect one or more biomarkers.

Biomarkers for detecting the presence of disease are a fundamental goal in the discovery of new targets and drug molecules that slow or stop disease progression. Since the levels of biomarkers are indicative of disease and drug response, the biomarkers can be used to identify novel therapeutic compounds in vitro and/or in vivo assays. The biomarkers described herein can be used in methods of screening for compounds that modulate the activity of the biomarkers.

Thus, in a further aspect of the invention there is provided the use of a binding agent or ligand as described, which may be a peptide, antibody or fragment thereof or aptamer or oligonucleotide directed against a biomarker of the invention; or use of a biosensor, or array, or kit according to the invention, for identifying a substance capable of promoting and/or inhibiting biomarker production.

The immunoassays described herein include any method that employs one or more antibodies or other specific binding agents that bind directly to the biomarkers defined herein. Immunoassays include two-site immunoassays or immunoassays using enzyme detection methods (e.g., ELISA), fluorescence label immunoassays, time-resolved fluorescence label immunoassays, chemiluminescence immunoassays, immunoturbidimetry, particle label immunoassays and immunoradiometric assays, as well as single-site immunoassays, reagent-limited immunoassays, competitive immunoassays, including labeled antigen and labeled antibody single antibody immunoassays with multiple label types including radioactive, enzyme, fluorescent, time-resolved fluorescence, and particle label. All such immunoassay methods are well known in the art, see, e.g., Salgale et al (1997) and van Nieuwenhuijze et al (2003).

The identification, detection and/or quantification may be performed by any method suitable for identifying the presence and/or amount of a particular protein in a biological sample from a subject or a purified or extracted biological sample or dilution thereof. In particular, quantification may be performed by measuring the concentration of the target in one or more samples. Biological samples that can be tested in the methods of the invention include those defined above. The samples may be prepared, for example, with appropriate dilution or concentration, and stored in a conventional manner. The invention finds particular use in plasma samples obtainable from a subject.

Identification, detection and/or quantification of the biomarker may be performed by detecting the biomarker or a fragment thereof, e.g., a fragment having a C-terminal truncation or having an N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Of particular note, peptides that are identical or related to the tail sequence of histones are particularly useful histone fragments.

For example, detection and/or quantification may be performed by one or more methods selected from the group consisting of: immunoassays, immunochromatography, SELDI (-TOF), MALDI (-TOF), 1-D gel based analysis, 2-D gel based analysis, Mass Spectrometry (MS), Reverse Phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC, and other LC or LC-MS based techniques. Suitable LC-MS techniques include ICAT (Applied Biosystems, CA, USA) or iTRAQ (Applied Biosystems, CA, USA). Liquid chromatography (e.g., High Pressure Liquid Chromatography (HPLC) or Low Pressure Liquid Chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy can also be used.

Methods involving detection and/or quantification of one or more biomarkers of the invention may be performed on a bench-top instrument, or may be incorporated into a disposable diagnostic or monitoring platform that may be used in a non-laboratory environment, such as at a physician's office or at the bedside of a subject. Biosensors suitable for practicing the methods of the invention include "credit" cards with optical or acoustic readers. Biosensors may be configured to be able to electronically transmit collected data to a physician for interpretation, and thus may form the basis of electronic medicine.

Identification of biomarkers of disease states allows integration of diagnostic procedures and treatment protocols. Biomarkers provide a means to indicate treatment response, response failure, adverse side effect profile, degree of drug compliance, and to obtain adequate serum drug levels. Biomarkers can be used to provide warning of adverse drug reactions. Biomarkers are useful in the development of personalized therapies because assessment of response can be used to fine-tune the dosage, minimize the number of prescribed drugs, reduce the delay in obtaining effective therapy, and avoid adverse drug reactions. Thus, by monitoring the biomarkers of the invention, subject care can be tailored precisely to match the needs determined by the disease and pharmacogenomic profiles of subjects, and the biomarkers can thus be used to titrate optimal dosages, predict positive treatment responses, and identify those subjects at high risk of serious side effects.

Biomarker-based testing provides a first-line assessment of "new" subjects and provides objective measurements for accurate and rapid diagnosis that cannot be achieved using current measurement approaches.

Biomarker monitoring methods, biosensors and kits are also of critical importance as subject monitoring tools to enable physicians to determine whether a relapse is due to worsening of the disease. If the pharmacological treatment is assessed as inadequate, the treatment may be restored or increased; the treatment may be altered, if appropriate. Since biomarkers are sensitive to disease states, they provide an indication of the effect of drug treatment.

Reference to "subject" or "patient" is used interchangeably herein. The subject may be a human or animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. The groups and methods described herein can be performed in vitro, in vivo, or ex vivo.

Detection and/or quantification may be compared to a cut-off level. The cutoff value may be predetermined by analyzing the results from multiple patients and controls and determining a suitable value for classifying a subject as having or not having a disease. For example, for a disease in which the level of the biomarker is higher in a patient with the disease, if the detected level is above the cut-off value, it is indicative that the patient has the disease. Alternatively, for a disease in which the biomarker level is lower in a patient with the disease, if the detected level is below a cut-off value, it is indicative that the patient has the disease. Advantages of using a simple cutoff include that the clinician can easily understand the test and that no software or other aids are required in interpreting the test results. Cut-off levels can be determined using methods known in the art.

Detection and/or quantification may also be compared to a control. It will be apparent to those skilled in the art that the control subject may be selected on a variety of bases, which may include, for example, subjects known to be free of the disease, or may be subjects with different diseases (e.g., studies for differential diagnosis). A "control" can include a healthy subject, a non-diseased subject, and/or a subject without infection. The control may also be an infected subject that shows no symptoms or shows mild symptoms, such as a subject infected with a respiratory virus that shows no symptoms or mild symptoms. Mild symptoms may include tractable symptoms that do not require hospital intervention and/or invasive medical treatment.

In one embodiment, a subject that tests positive by the methods of the invention may be infected with a viral disease and additionally suffer or continue to suffer from further medical complications. In contrast, control subjects may also be infected with viral disease, but do not suffer and continue to suffer from medical complications. Comparison with controls is well known in the diagnostic arts. The range of values found in the control group can be used as a normal or healthy or reference range to which values found for the test subject can be compared. For example, if the reference range is < 10 units, a test value of 5 units will be considered normal, or no treatment required, but a value of 11 units will be considered abnormal and indicate the need for treatment.

Thus, in one embodiment, the method additionally comprises comparing the level of cell free nucleosomes, or a component thereof, in a sample of bodily fluid from the subject with one or more controls. For example, the method may comprise comparing the level of cell free nucleosomes present in a sample obtained from the subject to the level of cell free nucleosomes present in a sample obtained from a normal subject. The control may be a healthy subject.

In one embodiment, the level of cell free nucleosomes or components thereof is increased compared to a control.

It is to be understood that the control level need not be measured for comparison purposes in every instance. For example, for healthy/non-diseased controls, once the "normal range" is established, it can be used as a benchmark for all subsequent tests. By obtaining samples from multiple uninfected control subjects and testing the levels of biomarkers, a normal range can be established. The results (i.e., biomarker levels) of a subject suspected of having an infection can then be examined to see if they fall within or exceed the respective normal ranges. The use of "normal range" is the standard practice for disease detection.

In one embodiment, the method additionally comprises determining at least one clinical parameter of the patient. This parameter may be used to interpret the results. The clinical parameters may include any relevant clinical information such as, but not limited to, body temperature, gender, weight, Body Mass Index (BMI), smoking status, and eating habits. Thus, in one embodiment, the clinical parameter is selected from: body temperature, age, sex, and Body Mass Index (BMI).

In one embodiment, the methods of the invention are performed to identify subjects at high risk of developing a severe response to infection and therefore in need of medical intervention.

Additional biomarkers

As one of the measurement sets, cell free nucleosome levels can be detected or measured. This panel may comprise different epigenetic characteristics of the nucleosomes as described above (e.g., histone isoforms and PTMs). Biomarkers useful in the panel of tests for the detection of severe respiratory infections requiring medical intervention include, but are not limited to, cytokine moieties (in particular interleukins), C-reactive protein, myeloperoxidase, D-dimer, factor VII activating protease (FSAP), fibrinogen and fibrin/fibrinogen breakdown products. In one embodiment, the set comprises one or more cytokines, such as one or more interleukins.

Interleukins (ILs) are a group of cytokines that act as signaling molecules, usually secreted by leukocytes. They have a key role in stimulating immune responses and inflammation. They were first identified in the 1970 s and are numerically named as more interleukin types were discovered. Examples of interleukins include, but are not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15.

In one embodiment, the one or more interleukins are selected from: interleukin-6 (IL-6) and interleukin-12 (IL-12).

The interleukin may be IL-6. Interleukin-6 (IL-6) is a cytokine with multiple biological functions. It is a potent inducer of fever and acute phase reactions. The sequence of human IL-6 is known in the art and is described at UniProt accession number P05231. In a particular embodiment, the interleukin may be IL-6.

Alternatively, or in addition, the interleukin may be IL-12. Interleukin-12 (IL-12) is a T cell stimulating factor because it stimulates the growth and function of T cells. It is a heterodimeric cytokine consisting of IL-12A and IL-12B. The sequence of human IL-12A is known in the art and is described at UniProt accession number P29459, and the sequence of human IL-12B is also known and is described at UniProt accession number P29460. In a particular embodiment, the interleukin may be IL-12.

In one embodiment, the panel comprises cell free nucleosomes or epigenetic characteristics thereof and an interleukin. In another embodiment, the panel comprises an epigenetic feature of the cell free nucleosome and two interleukins. For example, cell free nucleosome measurements can be combined with more than one interleukin measurement (e.g., IL-6 and IL-12). In a further embodiment, the epigenetic characteristic of the cell-free nucleosome is selected from the group consisting of histone isoforms, such as H3.1, and post-translationally modified histones, such as H3 cit. In still further embodiments, the measurement set is H3.1, H3cit, H4cit, and IL-6.

In one embodiment, the panel comprises C-reactive protein (CRP). CRP is a pentameric protein found in plasma, and CRP (not added to nucleosomes) levels increase in plasma in response to inflammation (as in bacterial, viral, fungal, and microbial infections). After IL-6 secretion by macrophages and T cells, CRP levels are elevated and their physiological role is to bind lysophosphatidylcholine expressed on the surface of dead or dying cells in order to activate the complement system via C1 q. It also binds to phosphorylcholine on the surface of some bacteria and enhances phagocytosis. Measurement of CRP levels can be used to determine the progression of disease and the effectiveness of treatment, and elevated CRP levels have been shown in patients with elevated risk for diabetes, hypertension, and cardiovascular disease. Elevated CRP levels are also found in patients with renal failure and inflammatory bowel disease (IBD, including crohn's disease and ulcerative colitis), and are roughly associated with coronary heart disease, although they are not specific prognostic markers since elevated CRP is not directly associated with heart disease. Viral infections such as SARS or coronaviruses (e.g., COVID-19) may also result in increased CRP levels in plasma due to elevation of CRP during inflammation.

In one embodiment, the set comprises Myeloperoxidase (MPO). MPO is expressed in neutrophils and produces hypohalous acid to achieve its antimicrobial activity. It is stored in azurophilic granules (azurolitic granules) and released into the extracellular space during degranulation. The level of MPO (unmeasured addition to nucleosomes) has been shown to be a useful predictor of myocardial infarction, and has been combined with the measurement of CRP (unmeasured addition to nucleosomes) to improve the accuracy of predicting the risk of myocardial infarction in patients.

The biomarkers of the invention can be used to derive models. Methods for deriving models or algorithms are well known in the art, and suitable software packages are available. Typical software tools for this purpose include SPSS (statistical Package for the Social sciences) and "R". These software packages provide linear and non-linear data modeling of clinical data.

It will be apparent to one skilled in the art that any combination of biomarkers disclosed herein can be used in sets and algorithms for detecting infection, and that other markers can be added to the set including those markers.

According to one aspect of the invention there is provided the use of a panel test to detect a patient suffering from an infection, wherein the panel test comprises reagents to detect the measurement of nucleosomes, or components thereof, and one or more interleukins in a sample obtained from the patient.

Method of treatment

According to a further aspect, there is provided a method of treating an infection in a subject, comprising the steps of:

(i) detecting or measuring the level of cell free nucleosomes in a sample obtained from the subject;

(ii) (ii) using the level measured in step (i) as an indication of the presence and/or severity and/or medical complications of said infection in the subject; and

(iii) (iii) administering a therapeutic agent if it is determined in step (ii) that the subject has a severe infection or medical complication.

According to a further aspect, there is provided a method of treating an infection in a subject in need thereof, comprising the step of administering a therapeutic agent to a subject identified as having a different level of cell free nucleosomes in a sample obtained from the subject when compared to the level of cell free nucleosomes in a sample obtained from a control subject.

In one embodiment, the treatment is selected from one or more of the following: antibiotic therapy (e.g. penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides, clindamycin, sulfonamides, trimethoprim, metronidazole, tinidazole, quinolones and/or nitrofurantoin), antimicrobial therapy (e.g. ethambutol, isoniazid, pyrazinamide, rifampin, aminoglycosides (amikacin, kanamycin), polypeptides (capreomycin, erythromycin, enramycin), fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), thioamides (ethionamide, prothioconazole), cycloserine (closerin), terizidone, rifabutin, macrolides (clarithromycin), linezolid, thiohydrazone (thioacetazone), methidazine, arginine, vitamin D and/or R207910), antiviral therapy (e.g. neuraminidase inhibitors, tetracycline, antibiotics, nitrofurantoin, nitrones, nitrofurantoins, etc.), and combinations thereof, cap-dependent endonuclease inhibitors, adamantanes, peramivir, zanamivir, oseltamivir phosphate, and baroxavir), and antifungal therapy (e.g., clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, and amphotericin).

The method can comprise the following steps:

(i) measuring the level of cell-free nucleosomes (optionally in combination with one or more interleukin levels) in a sample obtained from the subject;

(ii) identifying the subject as having an infection in need of treatment based on a higher level of cell-free nucleosomes compared to a control; and

(iii) administering a treatment to the subject.

In a preferred embodiment, there is provided a method of identifying a subject infected with a respiratory virus, said subject having or at risk of developing a medical complication in need of treatment, said method comprising the steps of:

(ii) identifying the subject as at risk of developing a medical complication in need of treatment based on a higher level of cell-free nucleosomes compared to a control; and

(iii) administering a treatment to the subject.

In a preferred embodiment, the respiratory infection is influenza or coronavirus and the medical complication is pneumonia. Suitable therapies may include, but are not limited to, respiratory support using a medical ventilator designed to provide mechanical ventilation of air into and out of the lungs of a patient who is functionally unable to breathe sufficiently independently, and/or providing oxygen and/or antiviral, antibacterial, or anti-inflammatory drugs.

The therapeutic antibody is administered intravenously to neutralize a moiety that causes injury or disease in a subject. Therapeutic antibodies and other similar or derived therapeutic binding agents such as Fab and Fv fragments are typically human or humanized in nature with respect to their heavy and light chain amino acid sequences. Therapeutic antibodies and methods for their development and production are well known in the art. Accordingly, in a further aspect of the invention there is provided anti-nucleosome antibodies for use in the treatment of severe hyperimmune reactions, including pneumonia.

Thus, in a further embodiment, there is provided a method of treating a subject suffering from a respiratory viral infection with a medical complication, comprising the steps of:

(ii) identifying the subject as having a medical complication based on a higher level of cell-free nucleosomes compared to a control; and

(iii) administering to the subject a therapeutic anti-nucleosome antibody.

According to another aspect of the invention there is provided a method of treating an infection comprising identifying a patient in need of treatment for said infection using a panel test comprising reagents to detect a measurement (measurement) of nucleosomes or components thereof, and providing said treatment. Infected patients are expected to have higher levels of cell free nucleosomes compared to controls.

It is to be understood that the embodiments described herein may be applied to all aspects of the invention, i.e. the embodiments described for use may be equally applicable to the claimed methods, etc.

The invention will now be illustrated with reference to the following non-limiting examples.

Example 1

We induced NET formation in leukocytes in fresh healthy whole blood samples by addition of heparin and subsequently confirmed the detection of NET material produced in plasma (lellott et al, International Immunology (2019) pii: dxz 084). We collected whole blood samples from two healthy volunteers, who were expected to have low circulating NET material levels, in EDTA plasma blood collection tubes and in heparin plasma blood collection tubes. Both EDTA plasma blood collection tubes were immediately centrifuged to separate the cell and plasma fractions, thereby minimizing contamination with large chromatin (including NET material), and the plasma was transferred to a cryotube and frozen. Two heparin plasma blood collection tubes were incubated at room temperature for 1 hour, and the tubes were gently rotated. The tubes were then centrifuged and plasma was transferred to a cryopreservation tube and frozen.

The samples were assayed in duplicate for nucleosomes containing histone isoform H3.1 using an ELISA procedure. Briefly, 20 μ l of sample was added to a microtiter well containing magnetic particles pre-coated with anti-histone H3.1 antibody. The sample is incubated, the magnetic particles are separated and washed. Anti-nucleosome antibodies that bind to a conformational nucleosome epitope conjugated to horseradish peroxidase are added to the magnetic particles. The particles were incubated, then separated and washed. Bound anti-nucleosome antibodies were measured using a coloured substrate reaction. The results are shown in fig. 1 and indicate that NET material levels are higher in heparin tubes but lower in EDTA tubes. This clearly shows that elevated levels of circulating NET material can be detected in a simple low cost immunoassay.

Example 2

DNA was extracted from two heparin and plasma samples described in example 1 and applied to a chip-based capillary electrophoresis apparatus (Agilent Bioanalyzer) to analyze DNA by fragment size. The size of the DNA fragment corresponding to the mononucleosomes has a retention time of about 60 seconds. As shown in fig. 2, the mononucleosome levels were low (as expected for healthy volunteers). The DNA fragment size corresponding to NET material has a longer retention time of about 110 seconds. As shown in fig. 2, the level of NET material in EDTA plasma was low (as expected for healthy volunteers), but was high in heparin plasma tubes where NET formation was stimulated by exposure to heparin. This result confirms that the increased nucleosome levels observed in heparin plasma in example 1 above are NET-derived rather than mononucleosomes.

Example 3

EDTA plasma samples were collected from 100 subjects who tested positive for coronavirus infection, including 50 control subjects with mild symptoms and 50 test subjects with respiratory complications and requiring ventilator support. Circulating NET material was measured using the nucleosome immunoassay method as described in example 1. All 50 control subjects were found to have low NET nucleosome levels, which were used to establish the control range. 50 test subjects were found to have higher NET nucleosome levels, which were used as an indicator that subjects had respiratory complications in need of treatment in addition to viral infection.

Example 4

The experiment performed in example 3 was repeated, but the immunoassay performed measures citrullinated nucleosomes. 50 control subjects were found to have low citrullinated nucleosome levels, and these levels were used to establish a control range. 50 test subjects were found to have higher citrullinated nucleosome levels, and these high levels were used as an indicator that subjects had respiratory complications in need of treatment in addition to viral infection.

Example 5

The experiment performed in example 3 was repeated, but the immunoassay performed measures myeloperoxidase-nucleosome adduct levels. 50 control subjects were found to have low myeloperoxidase-nucleosome levels, and these levels were used to establish a control range. 50 test subjects were found to have higher myeloperoxidase-nucleosome levels, and these high levels were used as an indicator that the subjects had respiratory complications in need of treatment in addition to viral infection.

Example 6

The experiment performed in example 3 was repeated, but an immunoassay was performed to measure the neutrophil elastase-nucleosome adduct levels. 50 control subjects were found to have low neutrophil elastase-nucleosome levels, and these levels were used to establish a control range. 50 test subjects were found to have higher neutrophil elastase-nucleosome levels, and these high levels were used as an indicator that subjects had respiratory complications in need of treatment in addition to viral infection.

Example 7

The experiment performed in example 5 was repeated, but in addition CRP and IL6 levels were measured in EDTA plasma samples or serum samples from the same subjects. Algorithms were developed using Logistic regression analysis of the results to maximize clinical sensitivity and specificity for identifying test subjects.

Claims

1. Use of a binding agent in the manufacture of a kit for use in a method of assigning a risk of adverse outcome to a subject suffering from an infection, the method comprising:

2. The use as defined in claim 1, wherein the infection is a viral, bacterial, fungal or microbial infection.

3. The use as defined in claim 1 or claim 2, wherein the infection is a respiratory tract infection.

4. The use as defined in claim 3, wherein the respiratory tract infection is selected from the group consisting of: influenza, pneumonia and Severe Acute Respiratory Syndrome (SARS).

5. The use as defined in any one of claims 1 to 4, wherein the body fluid sample is a blood, serum or plasma sample.

6. The use as defined in any one of claims 1 to 5, wherein the cell free nucleosome is part of or derived from a neutrophil extracellular trap.

7. The use as defined in any one of claims 1 to 6, wherein the component of the cell free nucleosomes comprises an epigenetic feature of the cell free nucleosomes.

8. The use as defined in claim 7, wherein the epigenetic feature is a histone isoform, such as a histone isoform of the core nucleosome, in particular a histone H3 isoform.

9. The use as defined in claim 8, wherein the histone isoform is H3.1.

10. The use as defined in claim 7, wherein the epigenetic feature is a histone post-translational modification (PTM), such as a histone PTM of the core nucleosome, in particular a histone H3 or H4 PTM.

11. The use as defined in claim 10, wherein the histone PTM is selected from citrullination or ribosylation.

12. The use as defined in any one of claims 1 to 11, wherein the level of cell free nucleosomes or components thereof is detected or measured using an immunoassay, immunochemical, mass spectrometry, chromatographic, chromatin immunoprecipitation or biosensor method.

13. The use as defined in any one of claims 1 to 12, wherein the method of detecting or measuring comprises contacting the body fluid sample with a solid phase comprising a binding agent for detecting cell-free nucleosomes or components thereof, and detecting binding to the binding agent.

14. The use as defined in any one of claims 1 to 13, wherein the method of detecting or measuring comprises: contacting the sample with a first binding agent that binds to an epigenetic feature of the cell-free nucleosome; (ii) (ii) contacting the sample bound by the first binding agent in step (i) with a second binding agent that binds to cell-free nucleosomes; and (iii) detecting or quantifying binding of the second binding agent in the sample.

15. The use as defined in any one of claims 1 to 14, wherein the subject is a human or animal subject.

16. The use as defined in any one of claims 1 to 15, additionally comprising comparing the level of cell-free nucleosomes or components thereof in the sample of bodily fluid of the subject with one or more controls.

17. The use as defined in claim 16, wherein the control is a healthy subject.

18. The use as defined in claim 17, wherein the control is an infected subject showing no symptoms or showing mild symptoms.

19. The use as defined in any one of claims 1 to 18, wherein the level of cell free nucleosomes or components thereof is increased relative to a control.

20. The use as defined in any one of claims 1 to 19, wherein as one of the measurement groups, cell free nucleosome levels are detected or measured.

21. The use as defined in claim 20, wherein said set comprises one or more interleukins.

22. The use as defined in claim 21, wherein the one or more interleukins are selected from the group consisting of: IL-6 and IL-12.

23. A use as defined in any one of claims 20 to 22, wherein the panel comprises C-reactive protein (CRP), Myeloperoxidase (MPO), D-dimer and/or factor VII activating protease (FSAP).

24. The use as defined in any one of claims 20 to 23, wherein the set comprises MPO.

25. Use of a binding agent in the manufacture of a kit for use in a method of detecting a subject in need of medical treatment for pneumonia, Acute Respiratory Syndrome (ARS) or Severe Acute Respiratory Syndrome (SARS), the method comprising:

(ii) using the cell free nucleosome level as an indicator that the subject is in need of medical treatment for pneumonia, ARS or SARS.