EA044128B1 - ANALYTICAL AND THERAPEUTIC METHODS AND COMPOSITIONS AND THEIR APPLICATION - Google Patents

Description

Field of technology

The invention generally relates to compositions and methods for treating, detecting and assisting in the diagnosis of Streptococcus pyogenes infection, rheumatism or post-streptococcal glomerulonephritis (PSGN), as well as compositions and methods for assessing the tendency to develop rheumatism or

P.S.G.N.

State of the art

Group A streptococci (GAS, Streptococcus pyogenes) cause or are associated with a range of diseases ranging in severity from mild skin infections and pharyngitis to severe invasive disease and post-infectious immune complications such as rheumatism or post-streptococcal glomerulonephritis (PSGN). Acute rheumatic fever and associated rheumatic heart disease are the leading cause of acquired heart defects in developing countries.

Streptococcal serology is critical for the diagnosis of postinfectious immune complications because these complications occur several weeks after GAS infection, when diagnosis of the causative bacteria is usually no longer possible. Typically, current clinical practice involves measuring antibody titers to two antigens, streptolysin-O (SLO) and deoxyribonuclease B (DNase B). The serological tests are called anti-streptolysin-O (ASO) and anti-deoxyribonuclease-B (ADB), respectively. ASO titers are typically measured using a nephelometric or turbidimetric assay, and values are usually reported in international units per milliliter (IU/mL). ADB tests are less standardized because, unlike ASO, there are no reference sera for DNase B. ADB titers are typically measured using an enzyme inhibition assay, where inhibition of DNase B activity by sera is detected using a color dye.

It is interesting to note that there is significant variability in the immunokinetics of antibody responses in the ASO and ADB tests, with the majority of children with GAS pharyngitis showing an increase in ASO and ADB titers (compared to pre-infection levels) for more than one year after infection [1 ]. Therefore, there is a significant risk of a false-positive diagnosis.

There remains a need for more effective ways to identify subjects with current or recent GAS infection, and subjects with a predisposition to develop rheumatic fever or PSGN, as well as ways to more effectively detect and diagnose rheumatic fever or PSGN.

The objective of the invention is to solve the above problems or to provide methods and compositions for identifying and assisting in the diagnosis of rheumatism or post-streptococcal glomerulonephritis or for assessing the susceptibility to developing rheumatism or post-streptococcal glomerulonephritis, and/or at least providing the public with a useful choice.

All references, including patents or patent applications, cited herein are incorporated herein by reference. No reference represents prior art. Discussion of references implies that the authors provide evidence, and applicants reserve the right to challenge the accuracy and relevance of the cited documents. It should be clearly understood that while reference is made herein to a number of prior art publications, reference to them does not constitute an admission that any of these documents form part of the general knowledge of the art in New Zealand or any other country.

The essence of the invention

These and other objects are achieved in accordance with one or more aspects of the present invention.

Accordingly, in one aspect, the present invention provides a method for detecting recent exposure to Streptococcus pyogenes in a subject, comprising obtaining a biological sample from the subject that may contain or is suspected of containing antibodies specific for one or more Streptococcus pyogenes antigens;

bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen is capable of binding antigen-specific antibodies present in the biological sample to form two or more populations of antigen:antigen-specific complexes antibody, if antigen-specific antibodies are present in the biological sample; and detection of said complexes, with an increase in detection of one or more complexes above a threshold indicating recent exposure to Streptococcus pyogenes in the subject.

In another aspect, the present invention provides a method for detecting or diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN), including acute post-streptococcal glomerulonephritis (APSGN) in a subject, or an increased likelihood of developing rheumatic fever or PSGN in a subject, the method comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more Streptococcus pyogenes antigens;

ii) bringing said biological sample into contact with two or more Streptococcus pyogenes antigen populations, wherein each of said two or more Streptococcus antigen populations is

- 1 044128 cus pyogenes is capable of binding antigen-specific antibodies present in a biological sample to form two or more populations of antigen:antigen-specific antibody complexes if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein an increase in detection of one or more complexes above a threshold value indicates an increased likelihood of developing rheumatic fever or APSGN or recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever or PSGN in the subject;

iv) assessment of one or more of the diagnostic criteria for rheumatism or PSGN;

v) wherein an increase in detection of one or more of these complexes above a threshold value in combination with one or more other diagnostic criteria for rheumatism or APSGN indicates rheumatism or PSGN in the subject.

In yet another aspect, the present invention provides a method for detecting the presence of Streptococcus pyogenes infection in a subject, comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more Streptococcus pyogenes antigens;

ii) bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen binds antigen-specific antibodies in the biological sample to form two or more populations of antigen:antigen-specific antibody complexes, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein increased detection of one or more of the complexes indicates the presence of Streptococcus pyogenes in the subject or recent exposure to Streptococcus pyogenes in the subject.

In yet another aspect, the present invention provides a method for detecting antigen-specific antibodies against Streptococcus pyogenes in a biological sample, wherein the antigen-specific antibodies against Streptococcus pyogenes specifically bind to a Streptococcus pyogenes antigen, the method comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more Streptococcus pyogenes antigens;

ii) bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen binds antigen-specific antibodies in the biological sample to form two or more populations of antigen:antigen-specific antibody complexes, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein increased detection of one or more of the complexes indicates that the biological sample contains antibodies specific for a Streptococcus pyogenes antigen.

In various embodiments, an increase in detection of one or more of the complexes represents detection of the presence of one or more of the complexes.

Accordingly, in one embodiment, a method for detecting or diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN), including acute post-streptococcal glomerulonephritis (APSGN) in a subject, or an increased likelihood of developing rheumatic fever or PSGN in a subject, includes:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more Streptococcus pyogenes antigens;

ii) bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen is capable of binding antigen-specific antibodies present in the biological sample to form two or more populations of antigen:antigen complexes -specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein the presence of one or more of the antigen:antigen-specific antibody complexes indicates an increased likelihood of developing rheumatic fever or APSGN or recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever or PSGN in the subject;

iv) assessment of one or more of the diagnostic criteria for rheumatism or PSGN;

v) wherein the presence of one or more of these antigen:antigen-specific antibody complexes in combination with one or more other diagnostic criteria for rheumatic fever or APSGN indicates rheumatic fever or PSGN in the subject.

- 2 044128

In yet another embodiment, a method for detecting the presence of a Streptococcus pyogenes infection in a subject includes:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more antigens

Streptococcus pyogenes;

ii) bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen binds antigen-specific antibodies in the biological sample to form two or more populations of antigen:antigen-specific antibody complexes, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein the presence of one or more of the antigen:antigen-specific antibody complexes indicates the presence of Streptococcus pyogenes in the subject or recent exposure to Streptococcus pyogenes in the subject.

In yet another embodiment, a method of detecting antigen-specific antibodies against Streptococcus pyogenes in a biological sample, wherein the antigen-specific antibodies against Streptococcus pyogenes specifically bind to a Streptococcus pyogenes antigen, includes:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for one or more Streptococcus pyogenes antigens;

ii) bringing said biological sample into contact with two or more populations of Streptococcus pyogenes antigen, wherein each of said two or more populations of Streptococcus pyogenes antigen binds antigen-specific antibodies in the biological sample to form two or more populations of antigen:antigen-specific antibody complexes, if antigen-specific antibodies are present in the biological sample; and iii) detecting said complexes, wherein the presence of one or more of the antigen:antigen-specific antibody complexes indicates that the biological sample contains antibodies specific for a Streptococcus pyogenes antigen.

In one aspect, the present invention relates to a method of treating a patient suffering from rheumatic fever or PSGN, the method comprising the following steps:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes; and ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen complexes: antigen-specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iv) moreover, the presence of complexes specific to the SpnA antigen. Streptococcus pyogenes, or a count of Streptococcus pyogenes SpnA antigen-specific complexes above a threshold indicates recent exposure to SpnA. Streptococcus pyogenes per subject;

v) wherein the presence of one or more other diagnostic criteria for rheumatism or PSGN together with the absence of Streptococcus pyogenes SpnA antigen-specific complexes, or a determination of the number of Streptococcus pyogenes SpnA antigen-specific complexes below a threshold value indicates exposure to Streptococcus pyogenes SpnA antigen on the subject in the past; and vi) if the subject has recently been exposed to Streptococcus pyogenes, then treat for recent rheumatic fever or acute PSGN; and if the subject has a history of exposure to Streptococcus pyogenes, treat established or subsequent Streptococcus pyogenes infection or treat rheumatism or PSGN.

In one aspect, the present invention provides a method of treating a patient suffering from rheumatic fever or PSGN with an antibiotic effective against Streptococcus pyogenes, the method comprising the following steps:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes; and ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen complexes: antigen-specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iv) moreover, the presence of complexes specific to the SpnA Streptococcus pyogenes antigen, or determination of the number of complexes specific to the SpnA Strep antigen -

- 3 044128 tococcus pyogenes, above the threshold indicates recent exposure of the subject to SpnA Streptococcus pyogenes;

v) wherein the presence of one or more other diagnostic criteria for rheumatism or PSGN together with the absence of Streptococcus pyogenes SpnA antigen-specific complexes, or a determination of the number of Streptococcus pyogenes SpnA antigen-specific complexes below a threshold value indicates exposure to Streptococcus pyogenes SpnA antigen on the subject in the past; and vi) if the subject has recently been exposed to Streptococcus pyogenes, then administer an antibiotic effective against acute or current Streptococcus pyogenes infection; and if the subject has been previously exposed to Streptococcus pyogenes, then an antibiotic effective against established or subsequent Streptococcus pyogenes infection is administered.

In one aspect, the present invention provides a method of treating a patient suffering from rheumatic fever or PSGN with an antibiotic effective against Streptococcus pyogenes, the method comprising the following steps:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes;

ii) bringing said biological sample into contact with one or more Streptococcus pyogenes SpnA antigen populations and one or more Streptococcus pyogenes anti-DNase B antigen populations and/or one or more Streptococcus pyogenes anti-SLO antigen populations, wherein said one or more Streptococcus pyogenes antigen populations capable of binding antigen-specific antibodies present in a biological sample to form one or more populations of antigen:antigen-specific antibody complexes if antigen-specific antibodies are present in the biological sample; and iii) detection of these complexes, and:

a) the presence of Streptococcus pyogenes SpnA-specific complexes or a detection of Streptococcus pyogenes SpnA-specific complexes above a threshold indicates recent exposure of the subject to Streptococcus pyogenes SpnA; And

b) the presence of Streptococcus pyogenes DNase B-specific complexes and/or Streptococcus pyogenes SLO-specific complexes and the absence of Streptococcus pyogenes SpnA-specific complexes or detection of Streptococcus pyogenes SpnA-specific complexes , below the threshold indicates the subject's past exposure to SpnA Streptococcus pyogenes, iv) if the subject has recently been exposed to Streptococcus pyogenes, then administer an antibiotic effective against acute Streptococcus pyogenes infection; and if the subject has previously been exposed to Streptococcus pyogenes, then administer an antibiotic effective against established or subsequent Streptococcus pyogenes infection.

In one aspect, the invention relates to a method of treating a patient suffering from rheumatism or PSGN, the method comprising the steps of:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes;

ii) bringing said biological sample into contact with one or more Streptococcus pyogenes SpnA antigen populations and one or more Streptococcus pyogenes anti-DNase B antigen populations and/or one or more Streptococcus pyogenes anti-SLO antigen populations, wherein said one or more Streptococcus pyogenes antigen populations capable of binding antigen-specific antibodies present in a biological sample to form one or more populations of antigen:antigen-specific antibody complexes if antigen-specific antibodies are present in the biological sample; and iii) detection of these complexes, and:

a) the presence of Streptococcus pyogenes SpnA-specific complexes or a detection of Streptococcus pyogenes SpnA-specific complexes above a threshold indicates recent exposure of the subject to Streptococcus pyogenes SpnA; And

b) the presence of Streptococcus pyogenes DNase B-specific complexes and/or Streptococcus pyogenes SLO-specific complexes and the absence of Streptococcus pyogenes SpnA-specific complexes or detection of Streptococcus pyogenes SpnA-specific complexes , below the threshold indicates the subject's past exposure to SpnA Streptococcus pyogenes, iv) if the subject has recently been exposed to Streptococcus pyogenes, then treat for recent onset rheumatism or acute PSGN; and if the subject has a history of exposure to Streptococcus pyogenes, then treatment for rheumatic fever or PSGN is given.

In yet another aspect, the present invention provides a method for treating rheumatism or PSGN in a subject in need thereof, the method comprising the following steps:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes; And

- 4 044128 ii) bringing said biological sample into contact with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of complexes antigen: antigen-specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iv) wherein the presence of complexes specific for the Streptococcus pyogenes SpnA antigen, or a determination of the number of complexes specific for the Streptococcus pyogenes SpnA antigen above a threshold value, indicates recent exposure of the subject to SpnA Streptococcus pyogenes;

v) wherein the presence of one or more other diagnostic criteria for rheumatism or PSGN together with the absence of Streptococcus pyogenes SpnA antigen-specific complexes, or a determination of the number of Streptococcus pyogenes SpnA antigen-specific complexes below a threshold value indicates exposure to Streptococcus pyogenes SpnA antigen on the subject in the past; and vi) if the subject has recently been exposed to Streptococcus pyogenes, then treat for recent rheumatic fever or acute PSGN; and if the subject has a history of exposure to Streptococcus pyogenes, then treatment for rheumatic fever or PSGN is given.

In yet another aspect, the present invention provides a method for treating rheumatism or PSGN in a subject in need thereof, the method comprising the following steps:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) optionally assessing one or more of the subject's other diagnostic criteria for rheumatic fever or PSGN;

iii) predicting that the subject is suffering from recent onset rheumatism or acute PSGN if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in an amount exceeding a threshold value; and iv) administering to said subject a therapeutically effective amount of an antibiotic effective against a recent Streptococcus pyogenes infection.

In one embodiment, a method of treating rheumatism or PSGN in a subject in need thereof includes the steps of:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iii) predicting that the subject is suffering from recent onset rheumatism or acute PSGN if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in an amount exceeding a threshold value; and the subject has one or more of the other diagnostic criteria for rheumatic fever or PSGN; and iv) administering to said subject a therapeutically effective amount of an antibiotic effective against acute Streptococcus pyogenes infection.

In yet another aspect, the present invention provides a method for treating rheumatism or PSGN in a subject in need thereof, the method comprising the following steps:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iii) predicting that the subject has rheumatic fever or PSGN if the sample contains one or more of the antibodies specific for Streptococcus pyogenes SpnA in an amount below the threshold value and the subject has one or more of the other diagnostic criteria for rheumatic fever or PSGN; and iv) administering to the subject a therapeutically effective amount of an antibiotic effective against established or subsequent Streptococcus pyogenes infection.

In various embodiments, the presence, absence, or amount of one or more antibodies specific for Streptococcus pyogenes SpnA is determined by contacting said biological sample with one or more Streptococcus pyogenes SpnA antigen populations, wherein said one or more Streptococcus pyogenes SpnA antigen populations pyogenes is capable of binding antigen-specific antibodies present in a biological sample to form one or more populations of antigen:antigen-specific antibody complexes if antigen-specific antibodies are present in the biological sample; and in various embodiments, an amount of one or more antibodies specific for Streptococcus pyogenes SpnA greater than a threshold value is an antibody titer associated with or indicative of recent exposure to Streptococcus pyogenes in the subject.

In various embodiments, an amount of one or more antibodies specific for Streptococcus pyogenes SpnA that is less than a threshold value is an antibody titer associated with or indicative of the subject's past exposure to Streptococcus pyogenes.

In various embodiments, the threshold value is the amount of SpnA-specific antibodies that separates the range of antibody titers or mean antibody titers observed in a population of rheumatic fever or PSGN patients within 20 days of their hospitalization from the range of antibody titers or mean antibody titers observed in a population of patients with rheumatism or PSGN 20 days after their hospitalization.

In one embodiment, the threshold value is the upper limit of normal (ULN), being the 80th percentile of the corresponding healthy population.

In one aspect, the present invention provides a method of treating a patient with an antibiotic effective against Streptococcus pyogenes, wherein the patient suffers from or has been exposed to a Streptococcus pyogenes infection, the method comprising the steps of:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes; and ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen complexes: antigen-specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) evaluating one or more other diagnostic criteria for the presence of Streptococcus pyogenes in the subject or rheumatic fever or PSGN in the subject;

iv) wherein the presence of complexes specific for the Streptococcus pyogenes SpnA antigen, or a determination of the number of complexes specific for the Streptococcus pyogenes SpnA antigen above a threshold value, indicates recent exposure of the subject to SpnA Streptococcus pyogenes;

v) wherein the presence of one or more other diagnostic criteria for rheumatism or PSGN together with the absence of Streptococcus pyogenes SpnA antigen-specific complexes, or a determination of the number of Streptococcus pyogenes SpnA antigen-specific complexes below a threshold value indicates exposure to Streptococcus pyogenes SpnA antigen on the subject in the past; and vi) if the subject has recently been exposed to Streptococcus pyogenes, then administer an antibiotic effective against acute Streptococcus pyogenes infection; and if the subject has previously been exposed to Streptococcus pyogenes, then administer an antibiotic effective against established or subsequent Streptococcus pyogenes infection.

In one embodiment, a method of treating a patient with an antibiotic effective against Streptococcus pyogenes includes the steps of:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes;

ii) bringing said biological sample into contact with one or more Streptococcus pyogenes SpnA antigen populations and one or more Streptococcus pyogenes anti-DNase B antigen populations and/or one or more Streptococcus pyogenes anti-SLO antigen populations, wherein said one or more Streptococcus pyogenes antigen populations capable of binding antigen-specific antibodies present in a biological sample to form one or more populations of antigen:antigen-specific antibody complexes if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein

a) the presence of Streptococcus pyogenes SpnA-specific complexes or a detection of Streptococcus pyogenes SpnA-specific complexes above a threshold indicates recent exposure of the subject to Streptococcus pyogenes SpnA; And

b) the presence of Streptococcus pyogenes DNase B-specific complexes and/or Streptococcus pyogenes SLO-specific complexes and the absence of Streptococcus pyogenes SpnA-specific complexes or detection of Streptococcus pyogenes SpnA-specific complexes , below the threshold indicates the subject's past exposure to SpnA Streptococcus pyogenes, iv) if the subject has recently been exposed to Streptococcus pyogenes, then administer an antibiotic effective against acute Streptococcus pyogenes infection; and if the subject has previously been exposed to Streptococcus pyogenes, then administer an antibiotic effective against established or subsequent Streptococcus pyogenes infection.

In various embodiments, treatment of recent onset rheumatism or acute PSGN, or treatment of acute or current Streptococcus pyogenes infection, is the administration of an antibiotic effective against acute Streptococcus pyogenes infection, for example, administration in accordance with a treatment regimen. For example,

- 6 044128 a regimen effective against acute Streptococcus pyogenes infection, or a regimen effective against new-onset rheumatism or acute PSGN, includes a 10-day course of one or more antibiotics, for example, a 10-day course of bicillin. Other suitable treatment regimens will be known to those skilled in the art taking advantage of the present invention, including certain exemplary examples described herein.

In various embodiments, treatment of rheumatic fever or PSGN, or treatment of an established or subsequent Streptococcus pyogenes infection, is the administration of an antibiotic effective against an established or subsequent Streptococcus pyogenes infection, for example, administration in accordance with a treatment regimen, such as a prophylactic treatment regimen. For example, a dosage regimen effective against established or subsequent Streptococcus pyogenes infection, or a dosage regimen effective for the treatment of rheumatism or PSGN, includes monthly administration of one or more antibiotics, for example, monthly administration of bicillin. Other suitable treatment regimens will be known to those skilled in the art taking advantage of the present invention, including certain exemplary examples described herein.

In one example, treatment of rheumatic fever or PSGN or treatment of established or subsequent Streptococcus pyogenes infection includes bed rest and/or hospitalization.

In one example, administering treatment for recent onset rheumatic fever or acute PSGN involves administering to the subject an antibiotic effective against acute Streptococcus pyogenes infection.

In one example, administering treatment for rheumatic fever or PSGN involves administering to the subject an antibiotic effective against an established or subsequent Streptococcus pyogenes infection.

In one example, administering treatment for rheumatism or PSGN includes hospitalizing the subject and/or administering or requiring the subject to bed rest.

In one aspect, the present invention provides a method for predicting the sensitivity of a patient suffering from rheumatic fever or PSGN to treatment with an antibiotic, the method comprising the following steps:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iii) predicting the likely response of a subject to treatment with an antibiotic if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in an amount below a threshold value and the subject has one or more of the other diagnostic criteria for rheumatic fever or PSGN; and iv) administering to the subject a therapeutically effective amount of an antibiotic effective against established or subsequent Streptococcus pyogenes infection.

In one aspect, the present invention provides a method for predicting the sensitivity of a patient suffering from rheumatic fever or PSGN to treatment with an antibiotic, the method comprising the steps of:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) optionally assessing one or more of the subject's other diagnostic criteria for rheumatic fever or PSGN;

iii) predicting the likely response of a subject to treatment with an antibiotic effective for the treatment of recent onset rheumatic fever or acute PSGN if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in excess of a threshold value, and optionally if the subject has one or more of the other diagnostic criteria for rheumatic fever or PSGN; and iv) administering to the subject a therapeutically effective amount of an antibiotic effective against acute Streptococcus pyogenes infection.

In yet another aspect, the present invention relates to a method for determining a treatment regimen for a patient suffering from rheumatic fever or PSGN, the method comprising the steps of:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

iii) determining that the subject should be treated according to a regimen suitable for the treatment of rheumatic fever or PSGN if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in an amount below the threshold value and the subject has one or more of other diagnostic criteria for rheumatism or PSGN; and iv) treating the subject with a treatment regimen appropriate for the treatment of rheumatism or PSGN.

In one embodiment, the treatment of rheumatic fever or PSGN is the treatment of chronic rheumatic heart disease.

In one embodiment, treatment of rheumatic fever or PSGN includes administering to the subject a therapeutically effective amount of an antibiotic effective against an established or subsequent Streptococcus pyogenes infection, for example, a prophylactically effective amount of such antibiotic.

In one embodiment, a treatment regimen suitable for the treatment of rheumatic fever or PSGN is monthly administration of an antibiotic, such as monthly administration of bicillin (benzathine benzylpenicillin).

In yet another aspect, the present invention relates to a method for determining a treatment regimen for a patient suffering from rheumatic fever or PSGN, the method comprising the steps of:

i) determining the presence, absence or amount of one or more antibodies specific for Streptococcus pyogenes SpnA in a biological sample from the subject; and ii) optionally assessing one or more of the subject's other diagnostic criteria for rheumatic fever or PSGN;

iii) determining that the subject should be treated according to a regimen appropriate for the treatment of recent onset rheumatic fever or acute PSGN if the sample contains one or more antibodies specific for Streptococcus pyogenes SpnA in an amount above a threshold value, and optionally the subject has one or more of the other diagnostic criteria for rheumatic fever or PSGN; and iv) optionally, administering to the subject a therapeutically effective amount of an antibiotic effective against acute Streptococcus pyogenes infection, in accordance with the treatment regimen.

In one embodiment, if the subject has recently been exposed to Streptococcus pyogenes, an antibiotic effective against acute Streptococcus pyogenes infection is administered at a dose effective against acute Streptococcus pyogenes infection.

In one embodiment, if the subject has recently been exposed to Streptococcus pyogenes, an antibiotic effective against acute Streptococcus pyogenes infection is administered at a dose greater than the dose for the subject suffering from established or subsequent Streptococcus pyogenes infection or rheumatic fever or PSGN.

In one embodiment, if the subject has recently been exposed to Streptococcus pyogenes, an antibiotic effective against acute Streptococcus pyogenes infection is administered in combination with one or more other therapeutic agents, e.g., an anti-inflammatory agent, e.g., aspirin, glucocorticoids, e.g., prednisone, antipsychotics , for example, haloperidol, positive inotropic agents, for example, digoxin, or in combination with one or more other treatments, for example, prolonged hospitalization, bed rest, etc.

In one embodiment, the risk of an adverse reaction or consequence from administering an antibiotic to a patient exposed to, but not recently exposed to, Streptococcus pyogenes is lower following administration of an antibiotic effective against chronic Streptococcus pyogenes infection than if said antibiotic were an antibiotic administered to the subject suffering from acute Streptococcus pyogenes infection or recent onset of rheumatism or acute PSGN.

In one embodiment, the risk of an adverse reaction or consequence from administering an antibiotic to a patient exposed to, but not recently exposed to, Streptococcus pyogenes is lower following administration of an antibiotic effective against Streptococcus pyogenes infection in an amount effective after an established or chronic Streptococcus pyogenes infection. than if said antibiotic were administered in an amount effective to treat acute Streptococcus pyogenes infection or recent onset rheumatic fever or acute PSGN.

In one embodiment, if the subject has recently been exposed to Streptococcus pyogenes, then administration of an antibiotic effective against acute Streptococcus pyogenes infection includes parenteral administration of the antibiotic.

In various embodiments, the antibiotic effective against Streptococcus pyogenes is selected from the group consisting of penicillin, amoxicillin, oxacillin, erythromycin, azithromycin, clarithromycin, cephalothin, cefoxitin, cefixime, cefuroxime, cefotaxime, ceftriaxone, vancomycin, clindamycin, rifampicin, ciproflox cin, tetracycline, cotrimoxazole and chloramphenicol.

In various embodiments, the antibiotic effective against Streptococcus pyogenes is selected from the group consisting of β-lactams, for example, penicillin, amoxicillin (amoxicillin), cefixime, cefpodoxin, cefotaxime, ceftriaxone, oxacillin; macrolides, for example, erythromycin, spiramycin, azithromycin; lincosamines, such as clindamycin; streptogramins, for example, pristinamycin; ketolides, such as telithromycin; phenicols, eg chloramphenicol; glycopeptides, for example, teicoplanin, vancomycin; fluoroquinolones, such as levofloxacin; and tetracyclines, eg tetracycline.

In one embodiment, the antibiotic effective against acute Streptococcus pyogenes infection is a penicillin, such as benzylpenicillin, including benzylpenicillin novocaine (e.g., Christicillin) and benzathine benzylpenicillin (e.g., bicillin, bicillin L-A), phenoxymethylpenicillin (e.g., Beepen-VK, Betapen-VK, Robicillin VK, Veetids), erythromycin, eg E-Mycin, Ery-Tab, erythrocin, or sulfadiazine, eg microsulfone.

In one embodiment, if the patient has recently been exposed to Streptococcus pyogenes, then administration of an antibiotic effective against acute Streptococcus pyogenes infection includes parenteral administration of benzylpenicillin, erythromycin, or sulfadiazine, such as intravenous or intramuscular administration of benzylpenicillin, erythromycin, or sulfadiazine. In yet another embodiment, oral administration replaces parenteral administration.

In various embodiments, an antibiotic effective against acute Streptococcus pyogenes infection is administered according to a dosage regimen. In various examples, the dosage regimen includes administering a loading dose of said antibiotic, for example, during the acute period of treatment.

In some embodiments, the acute treatment period is from about 5 days to about 20 days, such as from about 8 days to about 15 days, or from about 10 days to about 12 days, including about 10 days.

In one example, a dosage regimen effective against acute Streptococcus pyogenes infection, or dosage regimens effective against new-onset rheumatism or acute PSGN, comprises a 10-day course of one or more antibiotics, for example, a 10-day course of bicillin. For example, some typical dosage regimens effective against acute Streptococcus pyogenes infection, or dosage regimens effective against new-onset rheumatism or acute PSGN, include:

i) for benzathine benzylpenicillin (bicillin): approximately 900 mg as a single dose, usually administered by deep IM injection, for adults and children weighing more than 30 kg, and approximately 450-675 mg as a single dose for children weighing up to 30 kg, or 600,000 IU as a single IM dose for patients up to 20 kg and 1.2x106 IU as a single IM dose for patients weighing more than 20 kg; all of these regimens are usually for 10 days ii) for phenoxymethylpenicillin: 125 to 250 mg twice daily, orally if IM administration is not possible, usually for 10 days, or 10 mg/kg to 500 mg twice once a day for 10 days;

iii) for erythromycin: 10 mg/kg to a maximum of 500 mg twice daily for 10 days;

iv) for erythromycin ethyl succinate: 40 mg/kg/day in 2-4 divided doses, up to a maximum dose of 1 g/day in children.

In various embodiments, an antibiotic effective against established or chronic Streptococcus pyogenes infection is administered according to an administration schedule. For example, some exemplary dosage regimens effective against established or subsequent Streptococcus pyogenes infection, or dosage regimens effective for the treatment of rheumatic fever or PSGN, including chronic rheumatic heart disease, include:

i) for benzathine benzylpenicillin (bicillin): approximately 900 mg as a single dose, usually given by deep IM injection, for adults and children weighing more than 30 kg every 4 weeks, and approximately 450-675 mg for children weighing up to 30 kg every 4 weeks, or 600,000 IU intramuscularly every 4 weeks for patients up to 20 kg and 1.2x106 IU intramuscularly every 4 weeks for patients weighing more than 20 kg;

ii) for phenoxymethylpenicillin: 125 to 250 mg twice daily, orally if IM administration is not possible, usually for 10 days;

iii) for erythromycin: 250 mg twice daily;

iv) for erythromycin ethyl succinate: 400 mg twice daily.

In yet another aspect, the present invention provides a method for detecting recent exposure to Streptococcus pyogenes in a subject, comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes;

ii) bringing said biological sample into contact with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antibodies specific to the SpnA antigen present in the biological sample to form one or more populations complexes SpnA antigen:antibody specific to the SpnA antigen, if the biological sample contains antibodies specific to the SpnA antigen; a and iii) detecting the presence or absence of complexes, iv) optionally, assessing one or more of the other diagnostic criteria for the presence of Streptococcus pyogenes or rheumatic fever or PSGN in the subject;

v) wherein the presence of complexes specific for the Streptococcus pyogenes SpnA antigen, or the number of complexes specific for the Streptococcus pyogenes SpnA antigen above a threshold value, indicates recent exposure of the subject to SpnA Streptococcus pyogenes;

vi) wherein the presence of one or more other diagnostic criteria for rheumatism or PSGN together with the absence of complexes specific for the Streptococcus pyogenes SpnA antigen, or the number of complexes specific for the Streptococcus pyogenes SpnA antigen,

- 9 044128 below the threshold indicates the subject's past exposure to Streptococcus pyogenes SpnA.

In yet another aspect, the present invention provides a method for detecting or diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN), including acute post-streptococcal glomerulonephritis (APSGN) in a subject, or an increased likelihood of developing rheumatic fever or PSGN in a subject, the method comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes;

ii) bringing said biological sample into contact with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antibodies specific to the SpnA antigen present in the biological sample to form one or more populations SpnA antigen:antibody specific to SpnA antigen complexes, if the biological sample contains antibodies specific to the antigen; and iii) detecting the presence or absence of complexes;

iv) assessing one or more other diagnostic criteria for rheumatic fever or PSGN in the subject;

v) wherein the presence of one or more of the other diagnostic criteria for rheumatism or PSGN together with the presence of Streptococcus pyogenes SpnA-specific complexes, or the detection of a number of Streptococcus pyogenes SpnA-specific complexes exceeding a threshold value, indicates an increased likelihood of developing rheumatism or APSGN or evidence of recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever or PSGN in the subject;

vi) and wherein the presence of one or more of the other diagnostic criteria for rheumatism or PSGN together with the absence of complexes specific for Streptococcus pyogenes SpnA, or the number of complexes specific for Streptococcus pyogenes SpnA less than a threshold value, indicates rheumatism or PSGN in subject or evidence of the subject's past exposure to Streptococcus pyogenes as one of the criteria for the subject having rheumatic fever or PSGN, such as an increased risk of subsequent Streptococcus pyogenes infection or sequelae thereof.

In one embodiment, one or more of the other diagnostic criteria is the presence or absence of antibodies specific for one or more Streptococcus pyogenes antigens other than SpnA. For example, one or more of the other diagnostic criteria is the presence or absence of Streptococcus pyogenes DNase B-specific antibodies or Streptococcus pyogenes SLO-specific antibodies.

Any of the embodiments described herein may relate to any of the aspects set forth herein. To avoid confusion, it will be apparent from the description herein that any of the aspects described herein, for example, any of the methods described herein, in some embodiments, may use one or more of the Streptococcus pyogenes nuclease A (SpnA) polypeptides , for example, one or more truncated SpnA polypeptides, or one or more SpnA fragments described herein. For example, in various embodiments, one or more of the antigens or one or more of the Streptococcus pyogenes SpnA antigen populations are present as one or more SpnA polypeptides, e.g., one or more truncated SpnA polypeptides or one or more of the SpnA fragments described in this document.

Likewise, when used in any of the aspects described herein, for example, any of the methods described herein, one or more of the Streptococcus pyogenes DNase B antigens or polypeptides, in some embodiments, may be one or more of the DNase B polypeptides , for example, one or more of the DNase B antigenic fragments described herein. For example, in various embodiments, one or more of the antigens or one or more of the Streptococcus pyogenes DNase B antigen populations are present as one or more DNase B polypeptides, such as one or more of the DNase B fragments described herein.

Likewise, when used in any of the aspects described herein, for example, any of the methods described herein, one or more of the Streptococcus pyogenes SLO antigens or polypeptides, in some embodiments, may be one or more of the DNase B polypeptides. for example, one or more of the SLO antigenic fragments described herein. For example, in various embodiments, one or more of the antigens or one or more of the Streptococcus pyogenes SLO antigen populations are present as one or more of the SLO polypeptides, such as one or more of the SLO fragments described herein.

In one embodiment, the presence of two or more populations of antigen:antigen-specific antibody complexes indicates the presence of Streptococcus pyogenes in the subject, or indicates recent exposure to Streptococcus pyogenes in the subject, or indicates that the biological sample contains antibodies specific for two or more antigens Streptococcus pyogenes.

- 10 044128

In one embodiment, the increase in detection of one or more complexes represents an increase relative to a control level of antigen established for each study population.

In one embodiment, the one or more antibodies specific for one or more Streptococcus pyogenes antigens is one or more serum antibodies.

In one embodiment, one or more of the serum antibodies is one or more IgG antibodies.

In one embodiment, one or more of the serum antibodies is one or more IgA antibodies or one or more IgM antibodies.

In one embodiment, one or more of the other diagnostic criteria is the presence or absence of one or more clinical symptoms associated with rheumatic fever or PSGN.

In one embodiment, said one or more of the clinical symptoms is selected from migratory polyarthritis, carditis, hematuria, erythema rheumatica, subcutaneous nodules, Sydenham's chorea, or pyoderma.

In one embodiment, one or more of the Streptococcus pyogenes antigens is an antigen of one of the following proteins:

i) Streptococcus pyogenes nuclease A (SpnA), ii) deoxyribonuclease B (DNase B) or iii) streptolysin-O (SLO).

In one embodiment, one or more of the Streptococcus pyogenes antigens is selected from the group consisting of:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 8, or iv) deoxyribonuclease B (DNase B) or

v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, or vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic fragment an SLO comprising, essentially consisting of, or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, or

x) any combination of two or more of i)-ix) above.

In one embodiment, the biological sample is contacted with a population of each of the following Streptococcus pyogenes antigens:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 adjacent amino acids from the amino acid sequence SEQ ID NO: 8, and iv) deoxyribonuclease B (DNase B) or

v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, and vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic an SLO fragment containing, essentially consisting of, or consisting of at least 10 contiguous amino acids from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the biological sample is contacted with a population of each of the following Streptococcus pyogenes antigens:

i) nuclease A (SpnA) of Streptococcus pyogenes,

- 11 044128 ii) deoxyribonuclease B (DNase B) and iii) streptolysin-O (SLO).

In one embodiment, the biological sample is contacted with a population of each of the following Streptococcus pyogenes antigens:

i) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, ii) an antigenic fragment of DNase B containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, iii) an antigenic an SLO fragment containing, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, two or more populations of Streptococcus pyogenes antigen are present in the composition.

In one embodiment, one or more of the Streptococcus pyogenes antigens is labeled with a detectable label and/or associated with a microparticle, bead, or detectable agent.

In one embodiment, one or more of the Streptococcus pyogenes antigen populations are covalently linked to the beads or microparticles.

In one embodiment, each of the Streptococcus pyogenes antigen populations is covalently associated with the beads or microparticles, and each of the different populations of beads or microparticles are not necessarily different from each other.

In one embodiment, the beads are polystyrene beads, magnetic beads, carboxylated beads, functionalized beads, or the microparticles are polystyrene microbeads, magnetic beads, carboxylated microparticles, or functionalized microparticles. In various examples, the beads are suitable for use in multiplex assays, for example, assays comprising two or more populations of beads or microparticles, where each population is conjugated to a different antigen. In various examples, the beads or microparticles are suitable for use in immunoassays, such as CBA, luminex assays, and the like.

In one embodiment, detection of antigen:antibody complexes involves exposing the complexes to a specific binding partner bearing a detectable label and detecting the label signal in the presence of antigen-specific antibodies in the biological sample.

In one embodiment, the specific binding partner comprises an antibody or fragment thereof.

In one embodiment, the specific binding partner is an anti-IgG antibody, an anti-IgG-PE antibody, or a fragment thereof.

In one embodiment, antigen:antibody complexes are detected using a flow instrument, an immunoassay, such as an immunoplate assay, electrophoresis and/or immunoblotting, immunochromatographic strips, an electronic biosensor, a resonance biosensor, or a microfluidic device or sensor.

In one embodiment, the immunoassay, for example, a plate-based immunoassay, is an ELISA or luminex assay.

In one embodiment, antigen:antibody complexes are detected by a luminex assay, such as the luminex assay exemplified herein.

In one embodiment, the presence of one or more complexes or one or more antigen-specific antibodies is detected using a secondary antibody labeled with a detectable label.

In one embodiment, the detectably labeled secondary antibody is an anti-IgG-PE antibody.

In one embodiment, the Streptococcus pyogenes antigens are labeled with a detectable label.

In one embodiment, the detectable label is a fluorophore.

In one embodiment, the biological sample is obtained from a mammalian species.

In one embodiment, the biological sample is a biological fluid sample.

In one embodiment, the subject is a human.

In yet another aspect, the present invention provides an isolated, purified or recombinant SpnA polypeptide, wherein said SpnA polypeptide:

i) truncated at the N-terminus;

ii) truncated at the C-terminus or iii) truncated at both the N-terminus and the C-terminus;

refers to wild-type SpnA.

In one embodiment, the SpnA polypeptide:

i) is immunogenic; or ii) is capable of immunologically cross-reacting with wild-type SpnA; or

- 12 044128 iii) marked with a detectable mark; or iv) has improved stability when stored at room temperature compared to

Wild type SpnA; or

v) contains 10 or more contiguous amino acids from SEQ ID NO: 8; or vi) is any combination of two or more of i)-v) above.

In one embodiment, the SpnA polypeptide has an increased average Tagg value compared to wild-type SpnA, where Tagg is the temperature at which 50% of the protein molecules aggregate, for example, as measured by SDS-PAGE. For example, a SpnA polypeptide has an average Tagg value of at least about 50°C, such as an average Tagg value of at least about 50°C, as analyzed using SDS-PAGE.

In one embodiment, the SpnA polypeptide has increased thermal stability at a temperature of from about 35°C to about 60°C compared to the wild type SpnA polypeptide.

In one embodiment, the polypeptide has increased thermostability, increased immunogenic stability, or both increased thermostability and increased immunogenic stability.

Additionally, for the avoidance of doubt, those skilled in the art will appreciate that any of the aspects described herein, for example, any of the methods described herein, in some embodiments, may use one or more of the truncated polypeptides Streptococcus pyogenes nuclease A (SpnA), for example, one or more of the truncated SpnA polypeptides described above, including one or more of the SpnA fragments described herein. For example, in various embodiments, one or more of the antigens or one or more of the Streptococcus pyogenes SpnA antigen populations are present as one or more truncated SpnA polypeptides, e.g., one or more N-terminally truncated SpnA polypeptides or one or more truncated SpnA polypeptides. The C-terminus of SpnA polypeptides or one or more of the SpnA fragments described herein.

In yet another aspect, the present invention provides a composition comprising an isolated, purified or recombinant SpnA polypeptide described herein.

In an additional aspect, the present invention provides a composition comprising SpnA tagged with a detectable label.

In one embodiment, the detectably tagged SpnA is a truncated SpnA polypeptide, e.g., one or more N-terminally truncated SpnA polypeptides, or one or more C-terminally truncated SpnA polypeptides, or one or more SpnA fragments described in this document.

In yet another further aspect, the present invention provides a kit for detecting or diagnosing rheumatism or PSGN in a subject, for detecting the presence of Streptococcus pyogenes infection in a subject, or for detecting antigen-specific antibodies to Streptococcus pyogenes in a biological sample, said kit comprising a composition comprising: at least one Streptococcus pyogenes antigen selected from the group consisting of:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 8, or iv) deoxyribonuclease B (DNase B) or

v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, or vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic fragment An SLO comprising, consisting essentially of or consisting of at least 10 contiguous amino acids from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, optionally at least one composition containing a control sample of an antibody, wherein the control sample of an antibody contains an antibody specific for one of the Streptococcus pyogenes antigens present in the kit, optionally, one or more reagents for creating an environment favorable for contact of one or more antigens with a biological sample, optionally, one or more reagents for detecting the complex formed

- 13 044128 between one or more antigens and one or more antibodies that bind a Streptococcus pyogenes antigen present in a biological sample, and instructions for use.

In one embodiment, at least one of the Streptococcus pyogenes antigens is covalently linked to the bead or microparticle.

In one embodiment, the composition includes a population of:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 adjacent amino acids from the amino acid sequence SEQ ID NO: 8.

In one embodiment, the composition contains a population of each of the following Streptococcus pyogenes antigens:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 adjacent amino acids from the amino acid sequence SEQ ID NO: 8, and iv) deoxyribonuclease B (DNase B) or

v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, and vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic an SLO fragment containing, essentially consisting of, or consisting of at least 10 contiguous amino acids from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In various embodiments of the methods presented herein, the method, prior to step ii), further comprises obtaining the kit described herein.

In various embodiments of the methods or kits provided herein, one or more of the Streptococcus pyogenes antigens is selected from the group consisting of antistreptolysin (ASO), antihyaluronidase (AHase), antistreptokinase (ASKase), antinicotinamide adenine dinucleotidase (anti-NAD).

In one embodiment, the method includes the use of either a composition or kit containing two or more populations of beads or microparticles, wherein each population of beads or microparticles contains a different Streptococcus pyogenes antigen and at least one of the populations of beads or microparticles contains a population of one of the following Streptococcus pyogenes antigens :

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least of at least 10 contiguous amino acids from the amino acid sequence of SEQ ID NO: 8, wherein the beads or microparticles can be used in a flow instrument, immunoassay, for example, plate immunoassay, electrophoresis and/or immunoblotting, immunochromatographic strips, electronic biosensor, resonance biosensor or microfluidic device or sensor, including, for example, solid phase ELISA, Luminex or CBA analysis.

One or more of the two or more populations of granules or microparticles optionally contain a population of one of the following Streptococcus pyogenes antigens:

i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 8, or iv) deoxyribonuclease B (DNase B) or

v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, or vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of amino-

- 14 044128 amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic fragment SLO containing, essentially consisting of or consisting of at least 10 adjacent amino acids from the amino acid sequence SEQ ID NO: 1 or

SEQ ID NO: 2.

In yet another aspect, the present invention provides a method for detecting recent exposure to Streptococcus pyogenes in a subject, comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes SpnA;

ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen:antigen complexes -specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, with an increase in detection of one or more complexes above a threshold indicating recent exposure to Streptococcus pyogenes in the subject.

In yet another aspect, the present invention provides a method for detecting or diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN), including acute post-streptococcal glomerulonephritis (APSGN) in a subject, or an increased likelihood of developing rheumatic fever or PSGN in a subject, the method comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes SpnA;

ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen:antigen complexes -specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein an increase in detection of one or more complexes above a threshold value indicates an increased likelihood of developing rheumatic fever or APSGN or recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever or PSGN in the subject;

iv) assessing one or more of the diagnostic criteria for rheumatic fever or PSGN in the subject;

v) wherein an increase in detection of one or more of these complexes above a threshold value in combination with one or more other diagnostic criteria for rheumatism or APSGN indicates rheumatism or APSGN in the subject.

In one embodiment, said one or more diagnostic criteria is the presence or absence of one or more clinical symptoms associated with rheumatic fever or PSGN.

In one embodiment, said one or more of the clinical symptoms is selected from migratory polyarthritis, carditis, hematuria, erythema rheumatica, subcutaneous nodules, Sydenham's chorea, or pyoderma.

In yet another aspect, the present invention provides a method for detecting Streptococcus pyogenes infection in a subject, comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes SpnA;

ii) contacting said biological sample with one or more populations of Streptococcus pyogenes SpnA antigen, wherein said one or more populations of Streptococcus pyogenes SpnA antigen are capable of binding antigen-specific antibodies present in the biological sample to form one or more populations of antigen:antigen complexes -specific antibody, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein increased detection of one or more of the complexes indicates the presence of Streptococcus pyogenes in the subject or recent exposure to Streptococcus pyogenes in the subject.

In yet another aspect, the present invention provides a method for detecting antigen-specific antibodies against Streptococcus pyogenes in a biological sample, wherein the antigen-specific antibodies against Streptococcus pyogenes specifically bind to SpnA of Streptococcus pyogenes, the method comprising:

i) obtaining a biological sample from a subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes SpnA;

ii) bringing said biological sample into contact with one or more Streptococcus pyogenes SpnA antigen populations, wherein said one or more Streptococcus pyogenes SpnA antigen populations are capable of binding antigen-specific antibodies present in the biological

- 15 044128 sample, with the formation of one or more populations of antigen:antigen-specific antibody complexes, if antigen-specific antibodies are present in the biological sample; and iii) detection of said complexes, wherein increased detection of one or more of the complexes indicates that the biological sample contains antibodies specific for a Streptococcus pyogenes antigen.

In one embodiment, the post-treptococcal glomerulonephritis is acute post-treptococcal glomerulonephritis (APSGN).

In various embodiments, an increase in detection of one or more of the complexes is an increase in detection of one or more of the complexes above a threshold.

In one embodiment, a reference threshold (also called a cutoff or ULN) is determined for a specific population. In one example, for new-onset rheumatic fever (ARF) in a given country, such as ARF in New Zealand, the threshold value is determined using a cohort of healthy, well-matched volunteers. The cut-offs (or reference levels) thus established apply to all cases of ARF in New Zealand. It should be appreciated that reference thresholds may vary across countries, ethnic groups, and populations, and thus, in certain embodiments, different countries or populations define their own reference thresholds.

In one embodiment, the reference threshold value for detection of Streptococcus pyogenes antigen is the average titer of said antibody observed in samples obtained from a population of rheumatic fever or PSGN patients within 20 days of their hospitalization. For example, a reference cutoff value for a Streptococcus pyogenes antigen for use in a method described herein is the average antigen-specific antibody titer observed using the methods described herein in samples obtained from a population of patients with rheumatic fever or PSGN, demographically comparable to the subject within 20 days of their hospitalization.

In one embodiment, a reference threshold for the detection of anti-SpnA:SpnA antibody complexes, e.g., a reference threshold for SpnA for use in determining recent exposure to Streptococcus pyogenes, is the average titer of anti-SpnA antibody observed in samples obtained from a population of patients with rheumatic fever or PSGN within 20 days of their hospitalization. For example, the reference cutoff value for SpnA for use in determining recent exposure to Streptococcus pyogenes is the mean titer of anti-SpnA antibodies observed using the methods described herein in samples obtained from a rheumatic fever or PSGN population demographically comparable to the subject within 20 days of their hospitalization.

Other objects, aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that while preferred embodiments of the invention are set forth in the detailed description and specific examples, they are provided for illustrative purposes only as various changes and modifications within the spirit of the present invention will become apparent to those skilled in the art from this detailed description. .

Brief description of the figures

Additional aspects of the present invention will become apparent from the following description, which is given by way of example only and with reference to the accompanying drawings, in which:

fig. 1 presents scatter plots showing the correlation between median fluorescence (MFI) values for single and multichannel assays determined using the Cytometric Bead Array for SLO (Figure 1A), DNase B (Figure 1B), and SpnA (Figure 1C ) in 10 serum samples. Linear regression analysis was performed to determine the following R ² values: SLO=0.999; DNase B=0.998; and SpnA=0.998;

in fig. Figure 2 shows the results of an ELISA of purified IgG against three group A Streptococcus antigens. Antibodies were purified from IVIG using affinity chromatography, resulting in IgG with specificity for SLO (A), DNase B (B), and SpnA (C). Scatter measures represent standard deviations;

in fig. 3 are scatter plots showing serum antibody concentrations determined using the Cytometric Bead Array for SLO (A), DNase (B), and SpnA (C). The ULN values for each antigen are shown (dotted line). One-way Kruskal-Wallis analysis of variance was performed to determine p values;

in fig. Figure 4 shows scatter plots showing the correlation between commercially available tests and the Cytometric Bead Array for SLO (A), DNase B (B). Linear regression analysis was performed to determine R ² values for SLO=0.968 and DNase B=0.934;

in fig. Figure 5 shows the standard curves for SLO, DNase B and SpnA for the Cytometric Bead Array, fitted with a five-parameter logistic formula using FCAP Array software. Purified IgG specific for SLO (500 ng/ml), DNase B (500 ng/ml), and SpnA (1500 ng/ml) was diluted twofold and incubated with antigen-coupled beads;

- 16 044128 in Fig. 6 depicts the amino acid sequence of the recombinant SLO fragment containing amino acids 34-571 (FIG. 6A), while the amino acid sequence of the detoxified SLO analog is shown in FIG. 6B. Substituted amino acids are highlighted and underlined;

in fig. 7 shows the amino acid sequence of a recombinant fragment of DNase B containing amino acids 43-271;

in fig. Figure 8 shows the amino acid sequence of the recombinant SpnA fragment containing amino acids 28-854;

in fig. Figure 9 shows three scatter plots comparing a single-channel (separately for each antigen) and a multi-channel luminex assay, in which beads with three antigens were mixed in equal parts and incubated with the test serum in one assay. In fig. 9A shows the correlation between MFI for SLO in single-channel and multi-channel luminex analysis; in fig. 9B shows the correlation between MFI for DNase B in single-channel and multi-channel luminex assays; and in fig. 9C shows the correlation between MFI for SpnA in single-channel and multi-channel luminex assays as described herein in Example 2;

in fig. 10 shows two scatter plots showing the correlation between commercially available assays and the luminex assay for SLO (FIG. 10A) and DNase B (FIG. 10B). Linear regression analysis was performed to determine R ² values for SLO=0.933 and DNase B=0.942;

in fig. 11 shows three graphs showing the levels of IgG antibodies in patient sera, determined using luminex analysis, and the sera were segregated depending on the day after admission. There were no significant differences in the concentration of anti-SLO antibody in sera collected <20 days after hospitalization compared with sera collected >20 days after hospitalization (Fig. 11, left panel), as well as in the concentrations of anti-DNase B antibody between the two groups were not observed (Fig. 11, middle panel). Conversely, in sera collected >20 days after hospitalization, there was a significant decrease in the concentration of anti-SpnA antibody compared to sera collected <20 days after hospitalization (Fig. 11, right panel), as described in Example 2 herein. ;

in fig. 12 shows three graphs showing the levels of IgG antibodies in patient sera, determined using luminex analysis, and the sera were segregated depending on the day after admission. There were no significant differences in the concentration of anti-SLO antibody in sera collected <20 days after hospitalization compared with sera collected >20 days after hospitalization (Fig. 12, left panel), as well as in the concentrations of anti-DNase B antibody between the two groups were not observed (Fig. 12, middle panel). Conversely, in sera collected >20 days after hospitalization, there was a significant decrease in the concentration of anti-SpnA antibody compared to sera collected <20 days after hospitalization (Fig. 12, right panel), as described in Example 3 herein. ;

in fig. 13 are three graphs showing the thermostability analysis of native SpnA and truncated SpnA polypeptide described herein as described in Example 4. FIG. 13A is a chromatogram of the analysis of the percentage of protein in a representative conformation at each temperature by SDS-PAGE. Fig. 13B is a graph showing Tagg (temperature at which 50% of the protein molecules aggregate) for each protein at each temperature. Fig. 13C is a plot of the average TAGG value for each polypeptide, which depicts the increased average Tagg values for the truncated construct of 51.0±0.6°C, which is significantly higher than for the native SpnA polypeptide (47.5±0.9 °C).

Detailed Description of the Invention

The present invention generally relates to methods and compositions for detecting the presence of GAS-specific antibodies in biological samples. Detection of such antibodies can be used to identify subjects at increased risk of experiencing immune sequelae following infection, and to identify subjects who may benefit from a particular treatment, in addition to other uses that will become apparent to one skilled in the art upon reading the following description .

Unless otherwise clearly required by context, throughout the specification and claims, the words contain, containing, etc. should be construed in a comprehensive and not in an exclusive or exhaustive sense, that is, in a sense that is inclusive but not limited.

In certain embodiments, the present invention provides a method for assisting in diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN) or assessing the susceptibility to developing rheumatic fever or PSGN in a subject, comprising determining the presence or amount of one or more antibodies specific for one or more antigens Streptococcus pyogenes in a biological sample of a subject, wherein an increased level of said antibodies in the biological sample relative to the level of said antibodies in a control sample indicates

- 17 044128 about rheumatism or PSGN or an increased tendency to develop rheumatism or PSGN.

In other embodiments, the present invention provides a method for determining the effectiveness of a treatment for rheumatism or PSGN in a subject, comprising determining the presence or amount of one or more antibodies specific for one or more Streptococcus pyogenes antigens from one or more biological samples obtained from the subject before or during a course of treatment, wherein a decrease in the level of one or more antibodies specific for one or more Streptococcus pyogenes antigens in samples obtained from the subject over time indicates the effectiveness of the treatment.

In a further embodiment, the present invention provides a method for selecting a subject for treatment of rheumatic fever or PSGN, comprising (a) determining the presence or amount of one or more antibodies specific for one or more Streptococcus pyogenes antigens in a biological sample obtained from the subject; (b) comparing the level of said antibodies in the biological sample with the level of said antibodies in a control sample; and (c) selecting a subject for treatment if the level of said antibodies in the biological sample exceeds the level of said antibodies in the control sample.

The present invention is based in part on the affinity of antibodies for antigenic components - the ability of antibodies to specifically recognize and bind to an antigen or epitope, and the determination of this specific recognition and binding. In other words, the present invention is based in part on the detection or identification of complexes formed upon recognition and binding of an antibody to a specific epitope.

The term affinity refers to the strength of binding of a particular epitope to the complementarity determining region (CDR) of the binding molecule; in the context of the present invention this is usually an immunoglobulin molecule. The term avidity refers to the overall stability of the complex between a population of immunoglobulins and the antigen(s), i.e. functional stability of the combination of a mixture of immunoglobulins with an antigen. Those skilled in the art will understand that avidity relates both to the affinity of individual immunoglobulin molecules in a population for specific epitopes and to the valence of the immunoglobulins and the antigen. For example, the interaction between a divalent monoclonal antibody and an antigen with a high content of epitope repeats, such as a polymer, should be characterized by high avidity. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method well known in the art, including some of the methods described herein. Common methods for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody:antigen interaction may vary when measured under different conditions, eg, salt concentration, pH, buffer, temperature. Thus, and particularly when data need to be compared, measurements of affinity and other antigen binding parameters, eg KD, IC ₅₀ , are typically performed using standardized antibody and antigen solutions, standardized buffer, and standardized assay conditions.

The terms specifically binding or specifically recognizing, used interchangeably herein, generally mean that a binding molecule, such as an antibody, binds to an epitope via its antigen binding domain, and that binding implies some complementarity between the antigen binding domain and the epitope. Accordingly, an antibody is said to bind specifically to an epitope if it binds to that epitope through its antigen-binding domain more readily than to a random, unrelated epitope. Such an antibody may be referred to herein as an antibody specific for the specified epitope or class of epitopes. The term specificity is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be said to have higher specificity for a given epitope than antibody B, or antibody A may be said to bind to epitope x with higher specificity than to the related epitope y.

The term definition as used herein includes estimating, quantifying, calculating, or otherwise obtaining the amount of specified material (eg, antibody, antigen, or biomarker) present in a particular sample. This can be achieved by measuring the reading of an endpoint parameter, which may be, for example, the appearance of a detectable product, any detectable change in, for example, the level of a substrate or any change in the rate of appearance of a product or disappearance of a substrate, or a measurement of the amount of antibody associated with an antigen, biomarker, complex or other reagent as described herein.

When present, the term immunological binding characteristics or other characteristics of binding of an antibody to an antigen in its various grammatical forms refers to the specificity, affinity, cross-reactivity and other characteristics of binding of an antibody.

- 18 044128

As used herein, the terms immunogenic stability, immunological stability and their grammatical equivalents imply the maintenance of one or more immunogenic or immunological characteristics, for example, the maintenance of the ability to induce a specified, including a specific immunological response, for example, the ability to bind or be recognized by an antibody or the ability to induce a cellular immunological response . For example, when used in relation to a particular antigen, polypeptide or other agent, immunogenic stability or immunological stability includes, for example, maintaining antibody-specific recognition, for example, the ability of the antigen, polypeptide or other agent to bind to a particular antibody. It should be understood that immunological stability can also refer to the stability of an antibody, eg, maintaining the ability to recognize and bind to an epitope.

As used herein, the term preferential binding means binding, for example, of an antibody to an epitope that occurs more readily than to another epitope, for example, a related, similar, homologous or similar epitope. Thus, an antibody that preferentially binds to a given epitope is more likely to bind to that epitope than to another epitope, even in cases where such antibody may exhibit some cross-reactivity with the other epitope.

For example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with a dissociation constant (KD) less than the antibody's _KD for the second epitope. In one embodiment, an antibody may be considered to preferentially bind a first antigen if it binds a first epitope with an affinity that is at least one order of magnitude less than the antibody's KD for the second epitope. In yet another embodiment, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least two orders of magnitude less than the KD of the antibody for the second epitope.

In yet another example, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with an association rate constant (association rate, k(on)) less than the antibody's k(on) for the second epitope. In one embodiment, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with a k(on) that is at least one order of magnitude greater than the antibody's k(on) for the second epitope. In yet another embodiment, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with a k(on) that is at least two orders of magnitude greater than the antibody's k(on) for the second epitope.

In other examples, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with a dissociation rate constant (dissociation rate, k(off)) less than the antibody's k(off) for the second epitope. In one embodiment, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with a k(off) that is at least one order of magnitude smaller than the antibody's k(off) for the second epitope. In yet another embodiment, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with a k(off) that is at least two orders of magnitude smaller than the antibody's k(off) for the second epitope.

Some methods that may be used herein assess the competitive binding of two or more antibodies, typically by assessing the ability of one antibody to inhibit the binding of one or more other antibodies to an epitope. In some embodiments, an antibody competitively inhibits the binding of a reference antibody to a given epitope if it preferentially binds to that epitope with such efficiency that it somewhat blocks the binding of the reference antibody to the given epitope. Competitive inhibition can be determined by any method known in the art, for example, using a competitive ELISA. Typically, an antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies or antigen-binding fragments, variants or derivatives thereof described herein may also be described or indicated in terms of their cross-reactivity. As used herein, the term cross-reactivity refers to the ability of an antibody specific for one antigen to recognize and bind to a second antigen. Typically, the observation of antibody cross-reactivity is taken as a measure of the affinity between two (or more) different antigenic substances that support such cross-reactivity. Thus, an antibody is cross-reactive if it binds to an epitope different from the epitope that induced its production.

It should be appreciated that in certain contexts epitopes may also be referred to or described as cross-reacting, with two or more different epitopes being recognized and/or bound by a particular antibody. The cross-reacting epitope typically contains many of the same additional structural features as the inducing or parent epitope.

- 19 044128

In some circumstances, binding to a cross-reacting epitope may have greater affinity than to the original epitope.

It should be appreciated that antibodies and antigens with little or no cross-reactivity are generally preferred for diagnostic purposes.

As used herein, the term polypeptide is intended to include the singular polypeptide as well as the plural polypeptides and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to designate a chain or chains of two or more amino acids are included in the definition of polypeptide, and the term polypeptide may be used instead of or interchangeably with any of these terms .

The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, modification with known protecting/blocking groups, proteolytic cleavage, or modification with unnatural amino acids. The polypeptide can be obtained from a natural biological source or through recombinant technology, but not necessarily by translation of the specified nucleotide sequence. It can be obtained by any method, including chemical synthesis.

The size of the polypeptide of the present invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more or 2000 or more amino acids. Polypeptides may be characterized by a specific three-dimensional structure, although they do not necessarily possess such a structure. The term glycoprotein refers to a protein associated with at least one carbohydrate moiety that is attached to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine residue or an asparagine residue.

By isolated polypeptide or fragment, variant or derivative thereof is meant a polypeptide that is not found in its natural environment. No special level of cleaning is required. For example, an isolated polypeptide can be recovered from its native or natural environment. For purposes of the invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides separated, fractionated, or partially or substantially purified by any suitable method.

In addition, the polypeptides of the present invention include fragments, derivatives, analogs or variants of the above polypeptides and any combinations thereof. The terms fragment, variant, derivative and analog when referring to antibodies or antibody polypeptides of the present invention include any polypeptides that retain at least some of the antigen binding properties of the corresponding native binding molecule, antibody or polypeptide. Fragments of the polypeptides of the present invention include proteolytic fragments as well as deletion fragments. Polypeptide variants include fragments described above, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may occur naturally or not occur in nature. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions or additions. In addition, variant polypeptides may also be referred to herein as polypeptide analogues. As used herein, polypeptide derivatives are defined as polypeptides that, through modification, have acquired additional properties not found in the native polypeptide. Examples include fusion proteins or functionally modified polypeptides, for example, PEGylated polypeptides, bead-linked polypeptides, and polypeptides covalently linked to one or more other agents or compounds. In one embodiment, a polypeptide derivative refers to a subject polypeptide containing one or more residues chemically modified by reaction of a functional side group. Derivatives also include peptides containing one or more of the naturally occurring derivatives of the twenty standard amino acids. For example, 4hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine.

In one embodiment, analogues of the specifically identified polypeptides may have approximately 70% sequence identity to the sequence of the specified polypeptide, for example, the amino acid sequence shown in the figures, or fragments thereof, for example, a sequence of 10 or more contiguous amino acids, i.e. 70% of the residues of each polypeptide are the same. In an additional embodiment, analogues

- 20 044128 specifically identified polypeptides can be characterized by an identity of more than 75%. In a further embodiment, analogues of the specifically identified polypeptides may have greater than 80% identity. In a further embodiment, analogues of the specifically identified polypeptides may have greater than 85% identity. In a further embodiment, analogues of the specifically identified polypeptides may have greater than 90% identity. In a further embodiment, analogues of the specifically identified polypeptides may have greater than 95% identity. In a further embodiment, analogues of the specifically identified polypeptides may have greater than 99% identity. In a further embodiment, analogues of the polypeptides of the present invention may contain less than about 20, for example, less than 10 amino acid substitutions, modifications or deletions compared to the sequence of the specified polypeptide.

In a further embodiment, the polypeptides may have greater than 70% homology. In an additional embodiment, the polypeptides may have homology greater than

75%. In an additional embodiment, the polypeptides may have homology greater than

80%. In an additional embodiment, the polypeptides may have homology greater than

85%. In an additional embodiment, the polypeptides may have homology greater than

90%. In an additional embodiment, the polypeptides may have homology greater than

95%. In an additional embodiment, the polypeptides may have homology greater than

99%. In a further embodiment, derivatives and analogs of the polypeptides of the present invention may contain less than about 20, more preferably less than 10 amino acid substitutions, modifications or deletions. Preferred substitutions are those known in the art to be conservative, i.e. Substituted residues share common physical or chemical properties, such as hydrophobicity, size, charge, or functional groups.

In addition, polypeptide analogues are contemplated that have a certain degree of identity within a range of a specified number of contiguous amino acid residues. For example, an analogue of the specified polypeptide in one embodiment has greater than 90% amino acid sequence identity within a range of 10 or more contiguous amino acids to the reference/specified sequence. In yet another embodiment, an analog of said polypeptide has greater than 90% amino acid sequence identity within a range of 20 or more contiguous amino acids to the reference/specified sequence

The term polynucleotide is intended to include the singular nucleic acid as well as the plural nucleic acids, and refers to an isolated nucleic acid molecule or nucleotide construct, including, for example, messenger RNA (mRNA) or plasmid DNA (pDNA). The polynucleotide may contain a conventional phosphodiester bond or a non-conventional bond (eg, an amide bond, for example, in peptide nucleic acids (PNA)). The term nucleic acid refers to any one or more nucleotide segments, such as DNA or RNA fragments, present in a polynucleotide. By isolated nucleic acid or polynucleotide is meant a nucleic acid molecule, DNA or RNA, extracted from its natural environment. For example, a recombinant polynucleotide encoding an antibody or antigen contained in a vector is considered to be isolated for purposes of the present invention. Additional examples of isolated polynucleotide include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides described herein. The isolated polynucleotides or nucleic acids described herein further include such synthetically produced molecules. In addition, the polynucleotide or nucleic acid may be or may contain a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

As used herein, the term sample refers to any biological material obtained from a subject or patient, although it should be appreciated that most embodiments (e.g., methods) described herein will favor samples that may contain one or more of antibodies. In one embodiment, the sample may comprise blood, plasma, serum, cerebrospinal fluid (CSF), or urine. For example, the sample may contain whole blood, plasma, B cells obtained by enrichment from blood samples, or cultured cells (eg, B cells from a subject). The sample may also include a biopsy or tissue sample, including mucosal or nerve tissue. In other embodiments, the sample may contain whole cells and/or cell lysate. Methods for collecting and/or obtaining samples are well known in the art.

By subject, individual, animal, patient or mammal is meant any subject, particularly a mammalian subject, such as a human patient, for whom diagnosis, prognosis, prevention or therapy is desired.

As used herein, the terms treat or treatment refer to both therapeutic treatment and prophylactic or preventive measures that aim to prevent

- 21 044128 mitigating or slowing (reducing) unwanted physiological changes or disorders, such as the development or progression of rheumatism or APSGN. Favorable or desirable clinical outcomes include relief of symptoms, reduction in disease severity, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or mitigation of disease state, clearance of the pathogen, and remission (partial or complete), detectable or undetectable, but not limited to them. Treatment may also mean increased survival compared to the predicted survival rate without treatment. Those in need of treatment include individuals with the specified condition or disorder, as well as individuals susceptible to the condition or disorder, or individuals in whom it is necessary to prevent the manifestation of the condition or disorder.

In some embodiments, the antigens described herein are used to quantitatively or qualitatively detect antibodies specific for Streptococcus pyogenes in a sample. This can be achieved using methods that provide a visually detectable signal, which can be any of fluorescence (immunofluorescence), chromogenic enzyme reaction product, precipitate formation, chemiluminescence or bioluminescence. Some embodiments use a fluorescently or color-tagged antibody in combination with light microscopy, flow cytometry, or fluorometric detection. Other techniques and labels that can be used to detect the antibody include colloidal gold, radiolabel, GFP (green fluorescent protein), etc., avidin/streptavidin-biotin, magnetic beads, and physical systems such as nanotechnology system, sensitive to the fact of binding, but are not limited to them.

Antigens, antibodies or fragments thereof can be used in histological staining, such as immunohistochemistry, immunofluorescence or immunoelectron microscopy, and for in situ detection of antibodies or proteins. Those skilled in the art will appreciate that any of a wide variety of histological techniques, such as staining procedures, can be modified to achieve such in situ detection.

One way in which an antibody or antigen described herein can be labeled and directly detected is by binding it to an enzyme and using it in an enzyme-linked immunosorbent assay (ELISA). This enzyme, in turn, upon subsequent exposure to a suitable substrate, reacts with this substrate in such a way that a chemical group is formed, which can be detected, for example, spectrophotometrically, fluorometrically or visually. Enzymes that can be used to detectably label an antibody or antigen include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribo nuclease, urease, catalase , glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase, but are not limited to. Detection can be performed by colorimetric methods that use a chromogenic enzyme substrate. In some embodiments, detection is accomplished by visually comparing the extent of enzymatic reaction of the substrate with similarly prepared standards, and this procedure is suitable for both soluble color products and insoluble color products, including, for example, those used on nitrocellulose or plastic substrates, test strips and so on.).

In some embodiments, detection of an antibody-antigen reaction may, where appropriate, be further assisted by the use of a secondary antibody or other binding partner that binds the antigen:antibody complex (by reacting with the antigen or, more commonly, the antibody). Typically, the secondary antibody or ligand is detectably labeled, with detection of the secondary antibody allowing the presence of an antigen:antibody complex to be inferred.

Specific binding partners, such as secondary antibodies, often react with a conserved region of the immunoglobulin specific to the animal species from which the sample is obtained. In the examples discussed in detail herein, the secondary antibody or specific binding partner has affinity for human immunoglobulins, such as IgG. The choice of secondary antibody depends at least in part on the source of the sample and, therefore, on the nature of the antibodies expected to be present in it.

A number of well-known detection methods are suitable for use in implementing the methods described herein. For example, enzyme-linked immunosorbent assays such as immunofluorescence assay (IFA), photometric assay, enzyme-linked immunosorbent assay (ELISA), ELISPOT assay, and immunoblotting can be easily adapted to detect specific antibodies.

Other detection systems that can also be used include those based on the use of protein A derived from Staphylococcus aureus strain Cowan I, protein G of group C Streptococcus (eg, strain 26RP66), or systems that use a biotinavidin coupling reaction.

Other enzyme-linked immunosorbent detection methods in which those described in

- 22 044128 herein, antigens and antibodies are Western blotting and dot blotting, in which the reagent is separated by electrophoresis and transferred to a nitrocellulose membrane or other suitable support. Then the test sample (for example, plasma) is brought into contact with the membrane, and the presence of the formed immune complexes is detected in the manner already described. In one embodiment of this method, purified antigen is applied to a membrane in lines or spots and allowed to bind to antibodies present in the sample, and the resulting immune complexes are detected using the techniques described herein.

The presence of antibody:antigen complexes can also be detected by agglutination. In one typical example of such a method, the antigens described herein are used to coat, for example, latex particles to form a homogeneous suspension. When mixed with a sample, such as serum, containing specific antibodies capable of recognizing antigens, the latex particles agglutinate and the presence of large aggregates can be visually detected.

Detection of the reaction of an antibody with an antigen can be facilitated by the use of an antibody or ligand labeled with a detectable group by methods known in the art.

Such a detectable group provides visual detection of a precipitate or color change, visual detection by microscopy, or automatic detection by spectrometry or radiometric measurement, or the like. Examples of detectable groups include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase and alkaline phosphatase (for light microscopy or electron microscopy and biochemical detection and for biochemical detection by color change), and biotin-streptavidin (for light or electron microscopy). Methods Used detections and groups can be selected, for example, from the above list or other suitable examples according to standard criteria applied to such samples, which are well known in the art.

Although radioisotope-based methods are now generally considered less preferred than in the past, there are methods for detecting radioactive substances that perform detection by radiolabeling antigens, antibodies or antibody fragments and using radioimmunoassay (RIA). The radioactive isotope can be detected by means such as the use of a gamma/beta counter or scintillation counter or autoradiography.

More common methods involve labeling the antibody or antigen to be detected with non-radioactive detectable labels, such as a fluorescent compound. After exposing a fluorescently labeled antibody or antigen to light of the appropriate wavelength, its presence can be detected by fluorescence. Some of the most commonly used fluorescent labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

The antibody or antigen can also be detectably labeled using metals that emit fluorescent radiation, such as ¹⁵² Eu or other lanthanide elements. These metals can be attached to the antibody or antigen using metal chelate groups such as diethylenetriaminepentaacetic acid (ETPA).

The antibody or antigen can also be detectably labeled by linking it to a chemiluminescent compound. The presence of a chemiluminescently tagged antibody or antigen is then determined by detecting the luminescence produced during the chemical reaction. Examples of compounds particularly useful as a chemiluminescent label are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound can be used to label an antibody or antigen. Bioluminescence occurs in biological systems, typically due to the activity of a catalytic protein that increases the efficiency of the chemiluminescent reaction. The presence of a given bioluminescent protein is determined by detecting luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

Specific examples of methods for detecting antibodies in biological samples, including methods using test strips or other immobilized analytical devices, are described, for example, in the following patents: US patent No. 5965356 (herpes simplex virus specific serological assay); US patent No. 6114179 (method and kit for detecting antigens and/or antibodies); US patent No. 6077681 (diagnosis of motor neuropathy by detection of antibodies); US patent No. 6057907 (marker for pathologies, including an autoimmune reaction and/or for inflammatory diseases); and US patent No. 5552285 (immunoassay methods, compositions and kits for detecting antibodies to oxidized DNA bases).

As an example, a microsphere assay (also called a flow bead assay) can be used to detect one or more antibodies specific for one or more Streptococcus pyogenes antigens in biological fluids (eg, a blood or serum sample from a subject). This technology, represented by systems developed by Luminex Corporation and other systems developed by Becton Dickinson, allows the processing of very small amounts of sample, typically 20 μL, to detect one or more analytes. Principle

- 23 044128 This assay is based on the combination of a capture antibody with microspheres containing certain amounts of red dye and infrared dye. After incubating these beads with a sample of the phycoerthrin (PE)-linked secondary detection antibody, the beads are analyzed using a flow cytometer. One laser detects the beads and the second laser detects the intensity of the PE associated with those beads. This technology is used for the detection of many biologically important molecules or agents, including cytokines in multichannel assays, serotyping of Streptococcus pneumoniae, simultaneous measurement of human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP), simultaneous detection of serum IgG against Toxoplasma gondii, rubella virus , cytomegalovirus and herpes simplex virus types 1 and 2 (see technical notes obtained from Luminex Corp., for example, on their website or in their catalog).

In some embodiments, one or more antibodies specific for one or more Streptococcus pyogenes antigens present in the biological sample bind to the antigenic protein associated with the microspheres. In some embodiments, the secondary detector antibody (an example of what is also referred to herein as a specific binding partner) is a monoclonal antibody, such as anti-human IgG. In other embodiments, the secondary detection antibody is a polyclonal antibody, such as anti-human IgG. Secondary antibodies used in such methods may be coupled to, for example, biotin, or otherwise detectably labeled.

Immunological methods suitable for the applications discussed herein include immunological methods based on the formation of antibody:antigen complexes, for example, using labeled antibodies or antibody fragments, including labeled secondary antibodies. Typical methods include ELISA-based methods, for example, indirect ELISA, competitive ELISA, sandwich ELISA, in addition to other immunological methods, agents or apparatus, for example, immunochromatographic strips, fluorescent immunomicroparticles, Western blotting, biosensors based on electrochemical reactions catalyzed by enzymes attached to antibodies, magnetic particles coated with antibodies, surface plasmon resonance and other techniques that detect the analyte associated with the antibody. There are various methods and technologies to implement these methods; these are well known to those skilled in the art and may include microfluidic, automated and/or high throughput technologies.

Suitable assays may be quantitative techniques, such as ELISA, or qualitative techniques, such as rapid immunochromatographic assays, immunoblotting, dipsticks, and the like. It should be appreciated that assays typically used as quantitative assays may, under certain circumstances, be used as qualitative assays.

Qualitative, not necessarily rapid, assays, such as immunochromatographic assays, especially those using immunochromatographic strips, are particularly suitable for use in areas where more sophisticated analytical technologies are not available or practical, such as in developing countries, or as first-line diagnostic tools to identify subjects who may benefit from further assessment.

For example, in one embodiment of a qualitative assay to determine the presence or amount of one or more antibodies specific for one or more Streptococcus pyogenes antigens, greater than or less than a certain amount, a qualitative ELISA using two or more antigens described in this document. This procedure is performed using a kit described herein, for example, a kit containing a composition, for example, a buffer solution or solutions to obtain a sample containing one or more Streptococcus pyogenes antigens, optionally a negative control containing no antibodies specific for Streptococcus pyogenes antigen, optionally a positive control containing one or more antibodies specific for one or more Streptococcus pyogenes antigens, and components of a solid-phase ELISA, e.g., a reaction vessel, e.g., a multiwell plate, and one or more secondary detector antibodies for detection forming a complex between one or more antibodies specific for one or more Streptococcus pyogenes antigens and one or more Streptococcus pyogenes antigens, and optionally one or more components for immobilizing the complex and/or for developing the assay. In one embodiment, a composition containing one or more Streptococcus pyogenes antigens is first introduced into a reaction vessel, such as an ELISA plate, under conditions where one or more antigens are immobilized on the vessel. In yet another embodiment, the reaction vessel, such as an ELISA plate, has one or more Streptococcus pyogenes antigens already immobilized thereon. In another example discussed in detail, one or more of the secondary detection antibodies are either already immobilized on or in the reaction vessel, or are contained in a composition and introduced into the reaction vessel under conditions to immobilize the detection antibodies therein. Those skilled in the art will appreciate that there are various alternative methods whereby formation, capture, and labeling of an analyte-dependent complex provide a detectable signal, and that detection capability in these methods typically depends on immobilization of the antibody:antigen complex.

In yet another embodiment of a qualitative assay that determines the presence of one or more antibodies specific for one or more Streptococcus pyogenes antigens, or that determines whether the amount of said antibodies is greater than or less than a certain threshold, immunochromatographic test strips are used. In one example, said strip contains two or more of the antigens described herein and is typically provided with one or more compositions, such as a sample preparation buffer or solutions, optionally a negative control containing no antibodies to the Streptococcus pyogenes antigen, optionally a positive control containing one or more antibodies specific for one or more antigens, a specific binding partner for detecting the formation of a complex between one or more antibodies specific for one or more Streptococcus pyogenes antigens, one or more Streptococcus pyogenes antigens and, optionally, one or more components for displaying the analysis. In one example, a separate immunochromatographic strip lacking Streptococcus pyogenes antigens is provided as a negative control. In other embodiments, one or more of the immunochromatographic strips comprises at least two different regions, one of which contains one or more Streptococcus pyogenes antigens, and the other optionally does not contain Streptococcus pyogenes antigens, and optionally contains one or more immobilized antibodies or other agents , capable of binding to a secondary detector antibody or specific binding partner, and thus acts as a positive control region.

In one particular embodiment, detection is performed using a capture ELISA. Capture solid-phase ELISA (also known as sandwich ELISA) is a sensitive assay for the quantification of very small (picogram to microgram) amounts of substances (e.g., hormones, cell signaling compounds, infectious disease antigens and cytokines, and, in the context of of the present invention, antibodies). This type of solid phase ELISA is typically considered when the analyte may be too dilute to bind to a carrier material such as a polystyrene microtiter plate (eg, a protein in a cell culture supernatant), or binds poorly to plastics (eg, a low molecular weight organic compound). Optimal dilution levels of the capture reagent (eg, capture antigen), samples, controls, and detection antibodies, as well as incubation times, are typically determined empirically and may require extensive titration. Ideally, an enzyme-labeled detector antibody or detector antigen can be used. However, if the detection antibody or antigen is unlabeled, the secondary antibody should not cross-react with the immobilized antibody or antigen.

As used herein, the terms detectable group, detectable label, and their grammatical equivalents refer to any atom, molecule or fragment thereof, reagent or agent, the presence, absence or level of which can be directly or indirectly monitored. One example involves radioactive isotopes. Other examples include (i) enzymes that can catalyze color or light-emitting (luminescent) reactions, and (ii) fluorophores. Detection of an outer group can be carried out directly, provided that the detectable group is itself detectable, such as in the case of fluorophores. Alternatively, detection of a detectable group can be accomplished indirectly. In the latter case, a second group is usually used which reacts with the detectable group and is itself directly detectable. The detectable group may be part of the antibody or labeled antigen, for example, attached to it by a covalent bond. For example, an antibody constant region can be used as an indirectly detectable group to which a secondary antibody containing the directly detectable group can specifically bind.

Thus, secondary antibodies are particularly suitable means for detecting antibodies in the methods described herein. This secondary antibody may itself be conjugated to a detectable moiety. One way in which an antibody of the present invention can be detectably labeled is by binding it to an enzyme. This enzyme, in turn, upon subsequent exposure to a suitable substrate, reacts with this substrate in such a way that a chemical group is formed, which can be detected, for example, spectrophotometrically, fluorometrically or visually. Enzymes that can be used to detectably label an antibody include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triosephosphate isomerase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-6 -phosphate dehydrogenase, glucoamylase and acetyl- 25 044128 cholinesterase, but are not limited to them.

Detection can be performed by colorimetric methods that use a chromogenic enzyme substrate. Detection can also be achieved by visually comparing the extent of the substrate's enzymatic reaction compared to similarly prepared standards.

In embodiments (eg, methods, assays, and kits) that utilize a solid support to which a reagent is bound, such as, as appropriate, an antigen or antibody, the solid support is any water-insoluble, water-resistant solid support. Examples of suitable solid supports include beads such as polystyrene, filter paper, test tubes, test strips and microtiter plates. The bound reagent may be bound to the solid support by covalent bonds or by adsorption. The advantage of using a solid support is that no centrifugation step is required to separate the solid and liquid phases, although in particular embodiments, such as bead-based assays, centrifugation may be used to facilitate processing.

The above solid support may include polymers, for example, polystyrene, agarose, sepharose, cellulose, glass beads and magnetizable particles of cellulose or other polymers. The solid support may take the form of large or small granules or particles, tubes, plates, strips or other shape.

The solid support is typically a tube or microtiter plate whose inner walls are coated with a first antibody or antigen, for example, antigens specific to detectable antibodies against Streptococcus pyogenes, or fragments or derivatives thereof, for example, the specific antigens described herein.

In addition, a kit is provided containing one or more components necessary to perform one or more of the methods described herein. The kit typically contains a composition containing at least one of the Streptococcus pyogenes antigens described herein, for example, a composition containing two or more of these antigens. The kit may include other components to perform the method in accordance with the present invention. For example, the kit may contain one or more reagents for creating an environment favorable for contact of one or more antigens with a biological sample. The kit may also contain sample collection equipment, such as a swab, pipette, or similar collection means, and equipment for performing one or more reaction steps during which the reagents come into contact, such as incubation means comprising a liquid or semi-solid carrier placed on a plate, test tube, glass or plastic surface, well or strip of absorbent paper, or similar means. Other components of the kit may include one or more reagents capable of detecting a complex formed between one or more antigens and one or more antibodies specific for a Streptococcus pyogenes antigen present in a biological sample.

The methods, assays, and kits discussed herein may, in certain embodiments, include or utilize contact of one or more samples with other assay reagents (e.g., one or more Streptococcus pyogenes antigens) with reagents located in a matrix, such as , enables rapid processing of multiple samples, including high-throughput implementations.

As used herein, the term array refers to the addressable spatial arrangement of recognition agent(s), for example, a combination of two or more Streptococcus pyogenes antigens. Each matrix address is a predefined specific spatial region containing one or more recognition agents. For example, the matrix can be a set of vessels (test tubes), plates, microwells in a microplate, each of which contains a set of antigens. There are various options for implementing matrices. In one implementation, each address contains similar or identical recognition agents, and multiple samples are analyzed, one for each address. In yet another embodiment, each address contains a different recognition agent or combination of recognition agents, and aliquots of one sample are introduced into each address. Other implementations use a combination of these approaches. The matrix may also be any solid support carrying known recognition agents in discrete areas (dots, lines, columns), for example, when different recognition agents are placed in different combinations at one or more different locations, or when different samples are brought into contact at different locations. In some embodiments, the array includes built-in appropriate controls, such as regions without sample, regions without antigen, regions without sample and antigen (e.g., containing only solvent and reagents), and regions containing synthetic or isolated antibodies coupled to one or more antigens, as a positive control.

Solid supports used herein, for example for an array or array, are typically substantially insoluble in liquid phases. The solid supports of the present invention are not limited to a particular type of support. On the contrary, a large number of substrates exist and are known to those skilled in the art. Thus, suitable solid supports include solid and semi-solid matrices, e.g., aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, silicon chip, multiwell plates (also called microtiter plates or microtiter plates), membranes, filters, conductive and non-conductive metals, glass (including microscope slides) and magnetic substrates. More specific examples of suitable solid supports include silica gels, polymer membranes, particles, modified plastic films, glass beads, cotton wool, plastic beads, alumina gels, polysaccharides such as Sepharose, nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic granules, starch, etc.

Antigens.

It will be appreciated that the methods described herein, and in particular the multi-channel methods described herein, can in principle use any Streptococcus pyogenes antigen to which the subject generates antibodies in vivo. As shown in the Examples section of this document, the clinically established antigens DNase B and SLO could be cross-linked to beads for use in a multichannel CBA assay. The present inventors have found that in certain cases, without being bound by theory, certain antigens, such as SpnA, can be effectively stabilized to facilitate their inclusion in the multichannel assays described herein. In the case of SpnA, shortening the full-length polypeptide improved immunological stability, especially when cross-linked or otherwise linked to a solid support (in this case, beads) used in embodiments of the methods described herein.

In one embodiment, two or more, and in the examples discussed in detail, three or more Streptococcus pyogenes antigens are used, for example, in a single multi-channel assay. In one example, each antigen is associated with beads or other support, and each population of beads associated with the antigen is different from the other populations. One example of such a multichannel assay is presented in the Examples section of this document, where each of the SLO, DNase B, and SpnA antigens is covalently linked to its bead population, so that each antigen-labeled bead population can be detected separately, and as a result, in one analysis can determine the amount of antibody specific to each antigen.

Some embodiments provide two or more Streptococcus pyogenes antigens in a single composition, e.g., in a single solution containing any necessary buffers or carriers, stabilizers, etc., e.g., when contacted with a sample, thereby Particularly suitable for multi-channel implementation. In one example, the Streptococcus pyogenes SpnA antigen is provided together with at least one of the other Streptococcus pyogenes antigens, for example, SpnA and at least one of the other Streptococcus pyogenes antigens in one composition, for example, upon contact with a sample. The example discussed in detail provides the SpnA antigen and the Streptococcus pyogenes SLO, for example, in the same composition, for example, when brought into contact with a sample. Another example discussed in detail provides the SpnA antigen and the Streptococcus pyogenes DNase B, for example, in a single composition, for example by contacting them with a sample. In another example discussed in detail, Streptococcus pyogenes SpnA, DNase B and SLO antigens are provided, for example, in a single composition, for example, when brought into contact with a sample.

SLO (AAK33267.1) is a secreted pore-forming cytolysin. In one embodiment, SLO is derived from GAS Streptococcus pyogenes M1 (strain: SF370, serotype: M1, gene locus tag: SPy_0167 (NCBI: NP_268546.1)). In one example, a recombinant SLO fragment is used, for example, a recombinant polypeptide containing amino acids 34-571 (see FIG. 6A, SEQ ID NO: 1). In one example, the recombinant polypeptide is labeled, for example, to facilitate purification, and the use of N-terminal or C-terminal His tags is particularly contemplated. In yet another embodiment, a detoxified analogue of SLO is used in which one or more amino acids identified as contributing to toxicity are replaced with other amino acids, resulting in a polypeptide that immunologically cross-reacts with wild-type SLO but exhibits reduced toxicity (e.g., to eliminate restrictions or requirements for production, handling or isolation). The amino acid sequence of a representative example of a detoxified SLO analog is shown in FIG. 6B and SEQ ID NO: 2. In this example of detoxified SLO, two point mutations in the coding nucleotide sequence result in substitutions P427L and W535F: the substituted amino acids are underlined in FIG. 6A and 6B.

DNase B (AAK34710.1) is a secreted enzyme that degrades DNA. In one embodiment, DNase B is obtained from GAS Streptococcus pyogenes M1 (strain: SF370, serotype: M1, gene locus tag: SPy_2043 (NCBI: NP_269989.1)). In one example, a recombinant DNase B fragment is used, for example, a recombinant polypeptide containing amino acids 43-271 (see FIG. 7, SEQ ID NO: 5). In one example, the recombinant polypeptide is labeled, for example, to facilitate purification, and in particular consider the use of N-terminal or C-terminal

His-tags.

SpnA (AAK33693.1) is a cell wall-attached enzyme that also degrades DNA. In one embodiment, SpnA is obtained from GAS Streptococcus pyogenes M1 (strain: SF370, serotype: M1, gene locus tag: SPy_0747 (NCBI: NP_268972.1)). In one example, a recombinant fragment of SpnA is used, for example, a recombinant polypeptide containing amino acids 28 to 854 (see FIG. 8, SEQ ID NO: 8). In one example, the recombinant polypeptide is tagged, for example, to facilitate purification, and the use of N-terminal or C-terminal His tags is particularly contemplated.

Those skilled in the art will understand that in certain embodiments of the invention described herein, including the methods, polypeptides, beads, compositions and kits described herein, and in particular, embodiments related to multi-channel diagnostic methods and The compositions described herein provide accurate and rapid diagnosis and/or identification of patients who require treatment of recent onset rheumatism or acute PSGN or acute Streptococcus pyogenes infection, but who do not require long-term treatment, including long-term prophylactic treatment of rheumatism, PSGN or persistent infection Streptococcus pyogenes, or only continuous monitoring is required, for example to quickly detect subsequent Streptococcus pyogenes infection.

In various embodiments, for example, when using the method described herein, a patient is identified who is or has recently been exposed to Streptococcus pyogenes, or has recently had a Streptococcus pyogenes infection; said patient is subject to both treatment appropriate for the treatment of recent Streptococcus pyogenes infection and ongoing treatment, for example, prophylactic treatment to prevent subsequent Streptococcus pyogenes infection or to alleviate one or more symptoms of rheumatic fever, PSGN, chronic rheumatic heart disease or to monitor disease status. In other embodiments, when applying the method described herein, a patient is identified who is or has been exposed to Streptococcus pyogenes, or has a history of infection with Streptococcus pyogenes; said patient is subject to ongoing treatment, for example, prophylactic treatment to prevent subsequent Streptococcus pyogenes infection or to alleviate one or more symptoms of rheumatic fever, PSGN, chronic rheumatic heart disease or to monitor disease status. Exemplary treatments include those described herein with respect to

The following examples, sequence listing and figures are provided to facilitate understanding of the present invention, the true spirit of which is set forth in the accompanying claims. It should be understood that modifications to the set forth procedures are possible without departing from the spirit of the invention.

Examples

Example 1.

This case study describes the development of a multichannel bead-based assay to determine the presence and quantity of GAS-specific antibodies in biological samples.

Ways.

Subjects under study.

In this study, human serum was obtained from four different sources. Patients with ARF were diagnosed according to the New Zealand modification of the Jones criteria [3] and included in the study during hospitalization at Starship Children's Hospital, Auckland, between 2004 and 2006. (n=8) and at Waikato Hospital in Hamilton from 2012-2015. (n=8). Serum from ethnically matched healthy children aged 6 years was obtained from the Children of Scope (CoS) study. In addition, serum was obtained from unmatched healthy volunteers aged 20 years or older recruited from the University of Auckland as an additional control group. Demographic characteristics are shown in Table. 1. All participants provided written informed consent, and appropriate ethics board approval was obtained for each of the four centers.

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Study participants*

Table 1

medical history. Data on the ethnicity of these participants was not available.

Obtaining antigens.

All recombinant antigens used in this study were prepared as mature forms without N-terminal signal sequences. SLO with a molecular weight of 64.4 kDa (amino acids 34-571, SEQ ID NO: 1) and an N-terminal His ₆ tag was purchased from Fitzgerald Industries International. The gene encoding DNase B was amplified from genomic DNA of S. pyogenes SF370 (ATCC 700294) using the following primers: forward 5'CACCATGCGACAAACACAGGTCTCAAATGATGTTG-3' [SEQ ID NO: 3] and reverse 5'TTTCTGAGTAGGTGTACCGTTATGGTAGTTAATGG-3' [SEQ ID NO: 4] for cloning into pET101/D TOPO (Life Technologies) using Toro cloning methodology. The resulting vector encoded DNase B (amino acids 43-271, amino acid sequence shown herein as [SEQ ID NO: 5]) with a C-terminal His ₆ tag and a total molecular weight of 29 kDa. The protein was expressed in Escherichia coli BL21ADE3 cells with induction with 1 mM IPTG at 37°C for 3 h in liquid Lennox medium (LB) supplemented with ampicillin and 0.1% glucose. SpnA was amplified from genomic DNA of S. pyogenes SF370 using primers containing KasI and XhoI restriction enzyme sites (underlined): forward 5'AAAGGCGCCCGCCAAAATTTGACTTATGCCAA-3' [SEQ ID NO: 6] and reverse 5'AAACTCGAGCTATTTGGAAAATGATAATTGAAGTAACA-3' [SEQ ID NO: 7]. The resulting spnA amplicon (encoding SpnA amino acids 28-854, [SEQ ID NO: 8]) was cloned into the pProExHta vector encoding an N-terminal His ₆ tag and transformed into E. coli BL21ADE3 cells. Protein expression was induced with 0.3 mM IPTG at 18°C for 16 h in LB medium containing ampicillin. Both rDNaseB and rSpnA were isolated from E. coli cell lysates using standard Ni ²⁺ -NTA affinity chromatography. The His ₆ tag was cleaved from rSpnA using recombinant tobacco etch virus protease (rTEV to recombinant protein ratio 1:100), yielding an 85 kDa protein. The purity of all antigens was confirmed by SDS-PAGE.

Purification of antigen-specific IgG.

IgG specific for SLO, DNase B and SpnA were purified from total human immunoglobulin (intravenous immunoglobulin, IVIG (Intragam® P)). Affinity columns for each of the GAS antigens were prepared by covalently linking the antigens to agarose resin through their primary amino groups using the AminoLink® coupling kit (Thermo Scientific). 5 ml of IVIG solution was diluted four times with phosphate buffered saline (PBS) (pH 7.4) and passed through the resin to allow antibody binding. The resin was washed four times with PBS to remove unbound antibody. Bound antibody was eluted using 0.2 M glycine-HCl buffer (pH 2.5-3.0) and immediately neutralized with 1 M Tris buffer (pH 9). Traces of IgA were removed using Melon spin columns (Thermo Scientific), and the resulting antigen-specific IgG was concentrated using a centrifugal filter (Merck Millipore). An enzyme-linked immunosorbent assay (ELISA) was performed to confirm the specificity of the eluted IgG for the antigen against which it was isolated. Antigen was immobilized onto the plates at a concentration of 5 μg/ml, and the plates were blocked with PBS supplemented with 0.1% Tween-20 and 5% nonfat dry milk (PBST-5% milk) for 1 h at room temperature (RT). Purified IgG was added for 1 h at room temperature, and binding was determined using a secondary antibody with anti-human horseradish peroxidase (HRP) (1:3000; Santa Cruz Biotechnology) as previously published [4].

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Cytometric Bead Array Analysis.

Each of the antigens was coupled to functional beads using the amine-sulfhydryl cross-linker sulfo-SMCC according to the manufacturer's instructions (Becton, Dickinson and Company). Briefly, color-coded 7.5-μm polystyrene beads were prepared for conjugation by adding 25 mM dithiothreitol (DTT). The target antigen (90 μg) was modified by adding 44 μg/ml sulfo-SMCC solution, and unreacted protein was removed using a Bio-Rad spin column (Biorad). The modified protein and functional beads were then mixed and incubated at room temperature for 1 h, then N-ethylmaleimide (44 μg/ml) was added and incubated for another 15 min. The washed conjugated beads were stored at 4°C, protected from light. Antigens were conjugated to functional beads containing different ratios of fluorophores (APCy7 and APC) to produce fluorescence at unique positions using two detectors (FL3 and FL4) on a flow cytometer. The positions of the granules that provided maximum separation between antigens were chosen as follows: rSLO - position E4; rDNaseB - position A4; and rSpnA - position A9.

Incubations for Cytometric Bead Array (CBA) experiments were performed in duplicate in 96-well U-bottom plates. Single-channel assays were performed by incubating rSLO, rDNaseB, and rSpnA-coated beads separately for 1 h at room temperature with serum samples diluted 1:10,000 in assay diluent. To determine serum antibody binding, R-phycoerythrin-conjugated donkey anti-human Fcy IgG antibody (Jackson ImmunoResearch) was added at a concentration of 1:100 for 2 h at room temperature. The median fluorescence intensity (MFI) for each reaction was read on a flow cytometer (BD Accuri C6). Multichannel bead assays were performed following the same protocol, except that beads bound to rSLO, rDNaseB, and rSpnA were mixed in equal proportions before adding serum samples diluted 1:10,000. In multichannel detection assays, donkey anti-human Fcy IgG antibody diluted 1:30 was used for detection.

A seven-point standard curve was generated for each antigen by mixing known starting concentrations of antibodies specific for SLO, DNase B, and SpnA in one tube and performing a series of two-fold dilutions. The starting concentration for SLO and DNase B was 500 ng/ml and for SpnA 1500 ng/ml. The beads were incubated with each of the diluted standards for 1 hour, followed by detection of donkey anti-human Fcy IgG as described above. MFI values were converted to concentration (μg/mL) and a five-parameter logistic regression equation was used to generate a standard curve for each antigen using the Array Flow Cytometric Analysis Program (FCAP) version 3 (BD). When using sera from study subjects in a multichannel assay along with seven-point standard curves for each antigen, MFI was converted to concentration (μg/mL) using FCAP Array software. The lower detection limit of each antigen was defined as the minimum concentration on the standard curve for which the MFI was 3 standard deviations above the blank sample reading (where the blank sample was secondary antibody beads), as previously published [5].

Analysis and statistical processing of data.

Upper limit of normal (ULN) values were calculated for each antigen by ranking the antibody concentrations determined for each of the CoS serum samples and determining the 80th percentile in Microsoft Excel (version 15.24). Statistical analyzes and graphs were generated using GraphPad Prism (version 7a). All correlations were analyzed using linear regression.

Obtaining ASO and ADB titers using commercial test systems.

ASO and ADB titers were determined at Labtests Pathology, Auckland, New Zealand. ASO titers (IU/mL) were measured turbidimetrically using a human anti-streptolysin-O kit on a SPAplus analyzer (The Binding Site, California, USA). ADB titers (U/mL) were measured using an enzyme inhibition assay (bioMerieux, Marcy l'Etoile, France). This assay provides imprecise values for low titers <100 U/mL; the average value for samples falling within this range was 50 U/mL.

Results.

Bead binding and multichannel analysis.

To generate GAS antigen-bound beads for analysis in this study, highly purified preparations of each of the three antigens were prepared using recombinant technologies in E. coli. Secreted proteins (SLO and DNaseB) were expressed without an N-terminal signal sequence, and SpnA was expressed without an N-terminal signal sequence and in a form truncated at residue K854 upstream of the sortase motif to improve protein stability. Each of the three proteins was associated with a functional CBA granule that fluoresced at a unique position: rSLO (position E4), rDNaseB (position A4), and rSpnA (position A9). The positions of the granules were chosen

- 30 044128 to ensure maximum separation in the two-color fluorescence graph. Various serum dilutions and anti-IgG detection reagent concentrations were examined to determine the linear range and saturation point of the assay. This process of trial and error determined that the 1:10,000 serum dilution was within the linear range for all three antigens.

To evaluate the feasibility of simultaneously measuring titers of three antigens, the results of single-channel assays were compared with multichannel assays. Single-channel assays were performed in which each bead was incubated with sera from 10 participants diluted 1:10,000. These 10 sera were a mixture of ARF and control samples selected based on a previously performed ELISA, which showed that the reactivity of these participants' sera against the three antigens varied from low to high, providing a good MFI distribution in the CBA assay (Fig. 1 ). These same 10 sera were then tested in multichannel assays in which three antigen beads were mixed in equal parts and incubated with the test serum in one assay well. The MFI in these multichannel assays showed an extremely strong correlation with the single-channel MFI for each antigen, as shown in Fig. 1 (R ² values: SLO = 0.999; DNase B = 0.998; and SpnA = 0.998). This indicates no interference or IgG cross-reactivity between beads and demonstrates the feasibility of a multichannel assay including three streptococcal antigens.

Standardization and accuracy of multichannel SVA analysis.

To determine the concentration of antibodies binding the beads to the attached antigens and to allow comparison between assay runs, standard curves were generated for each of the three antigens. IgG specific for SLO, DNase B, and SpnA were purified from IVIG by affinity chromatography. The specificity of the purified antibodies was confirmed using solid-phase ELISA. As shown in FIG. 2, antibodies specific for SLO, DNase B, and SpnA were only reactive toward their own antigen and had no detectable reactivity toward the other two antigens. Purified IgG was used to generate seven-point standard curves for each antigen. Known concentrations of purified IgG were diluted twofold, starting at concentrations of 500 ng/ml for anti-SLO and anti-DNase B antibodies and 1500 ng/ml for anti-SpnA. These diluted standards were incubated with antigen-coupled beads in a multichannel format and standard curves were fitted using a five-parameter logistic formula. An example of standard curves is shown in Fig. 5. The standard curves were highly reproducible, with at least 98% accuracy of fit, demonstrating the utility of affinity purified polyclonal antibodies as reference standards for these antigens.

Purified antibody standards were used to determine the lower limits of detection of these three antigens in the CBA assay: anti-SLO antibody, 1 ng/ml; anti-DNase B antibody 0.1 ng/ml; and anti-SpnA antibody - 0.1 ng/ml. The coefficient of variation (CV) for the multichannel assay was estimated using the same 10 sera as in the single/multichannel assay comparison above. IgG concentrations in these 10 sera were measured in an assay that included IgG standard curves; and the mean intra-assay and inter-assay CV values for each antigen were <4% and <15%, respectively (Table 2). These CVs demonstrate good accuracy and precision of multichannel bead-based analysis. The reproducibility of both the standard curve and the assay results means that these reagents can be used to test the coupling efficiency and integrity of future batches of antigen-coupled beads.

table 2

Coefficient SLO you are variations DNase B SpnA CV within the analysis (%) 3.05 3.44 3.24 CV between assays (%) 11.17 12.32 12.03

Measurement of antibody titers in sera of patients with ARF.

To evaluate the utility of SpnA antigen and bead-based technology in clinical streptococcal serology, a multi-channel assay was performed for all subjects (Table 1). Concentrations of IgG specific for SLO, DNase B, and SpnA could be determined for all 47 subjects in a single experiment performed by a single operator over 1 day. As shown in FIG. 3, the mean antibody titers for each of the three antigens in the ARF samples were significantly higher than the mean titers in both healthy children and healthy adult controls. Consistent with observations in previous studies [6], titers for ASO and ADB were elevated and showed greater prevalence in healthy children than in healthy adults, as indicated by the higher CIs in Table 1. 3. It is noteworthy that SpnA titers in healthy children were similar to those in healthy adults and were characterized by narrower confidence intervals compared with those for ASO and ADB in the group of healthy children (Table 1). This confirms previous observations of low background SpnA titers in healthy individuals [2].

The ability of antigens to detect past GAS exposure to diagnose ARF was assessed using ULN values. The calculated ULN, or 80th percentile, in the healthy group was 644, 360, and 170 μg/mL for SLO, DNase B, and SpnA, respectively. The lower ULN for SpnA reflects the reduction in titers observed in healthy children compared to SLO and DNase B. These experimentally determined thresholds, shown by the dotted line in Fig. 3 was then applied to the ARF samples to determine the sensitivity of each antigen. This is the number of true positives detected depending on whether the observed titer was above the ULN. DNase B provided the lowest sensitivity and detected only 9 of 16 ARF samples (56.25%). SLO provided intermediate sensitivity and detected 12 of 16 ARF samples (75%). SpnA provided the highest sensitivity, detecting 14 of 16 ARF samples (87.5%).

Table 3

Summary statistics for antibody concentrations (μg/mL) specific to SLO, DNase B, and SpnA determined using the Cytometric Bead Assay

Antigen Acute rheumatism (n = 16) Healthy children (n = 13) Healthy adults (n = 18) Streptolysin-0 Mean (95% CI) Median (95% CI) 1539 (859.9-2218.0) 1225 (601.4-1948.0) 559 (115.2-1003.0) 326 (123.7-757.9) 173 (89.6-256.2) 123 (53.6-201.4) DNase B Mean (95% CI) Median 718.5 (387.4-1050) 439.5 (240.9-1071) 180.9 (56.2-305.5) 89.01 (0-383.7) 41.7 (24.7-58.7) 29.38 (22.8-49.6) value (95% CI) SpnA Mean (95% CI) Median (95% CI) 1029 (304.9-1752) 422.2 (229.1-1156) 119.8 (36.7-202.9) 76.01 (0-177.3) 52.2 (1.3-103.1) 7.4 (0-49.7)

Comparison with existing serological tests.

To compare the multichannel SBA assay with existing commercially available methodology, ASO and ADB assays were performed in a commercial laboratory in the serum of 20 participants for whom sufficient sample volumes were available. ASO was measured using the widely used turbidimetric method, and exact values were obtained in international units (IU/mL). In contrast, ADB titers were measured using an enzyme inhibition assay that produces titer ranges (100, 200, 300, 400, 600, 800, 1200, and ∼1600). As shown in FIG. 4A, there was an excellent correlation between the IgG ASO concentration determined by the CBA assay and the commercially available turbidimetric method (R2=0.968). As shown in FIG. 4B, a good correlation also occurred between the IgG ADB concentration determined by the CBA assay and the commercially available enzyme inhibition assay (R2=0.934).

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However, the drawing also illustrates the lack of accuracy of the ADB assay based on enzyme inhibition. In the enzyme inhibition assay, three samples were classified as

1200, but the anti-DNase B IgG concentration measured in our CBA assay was 1508.

1070 and 914 μg/ml, respectively (data points in box).

Discussion.

This case study presents the preparation of a multichannel bead-based GAS serology assay and the application of this assay to determine antibody concentrations in a range of samples from healthy and ARF subjects. This example successfully demonstrated that three Streptococcus pyogenes antigens could be used in a single multichannel assay in combination to detect the presence of different populations of Streptococcus pyogenes-specific antibodies without significant cross-reactivity using very small sample volumes (1 μl or less ).

The multichannel assay presented here can be used quantitatively for each of the antigen:antibody complexes, unlike, in particular, existing DNase B assays. In addition, improved sensitivity/specificity is expected, in part due to the inclusion of SpnA, which is more characterized low background values in healthy subjects, and partly due to improved sensitivity, which in particular is not negatively affected by the multi-channel embodiment.

Example 2.

This case study describes the development of a multichannel bead-based assay using the Luminex platform to determine the presence and quantity of GAS-specific antibodies in biological samples.

Ways.

Subjects under study.

In this study, human serum was obtained as described above in Example 1. Similarly, all participants provided written informed consent and appropriate ethics review board approval was obtained.

Obtaining serum.

Serum samples were collected and diluted 1:15,000 for analysis. The IVIG standard was diluted 1:60,000 before use.

Luminex bead preparation and analysis.

Each of the antigens was coupled to xMAP microspheres using an amine-carboxyl cross-linker according to the manufacturer's instructions (Luminex Corporation). Three different ratios of antigen beads were tested for conjugation, and beads conjugated with 12.5 μg of antigen were selected for further analysis.

Three well-separable granule positions were chosen: granules with DNase B at position 030; granules with SLO at position 072; and SpnA beads at position 078. Conjugated beads were incubated with anti-human IgG secondary antibody diluted 1:30 for multichannel reactions.

The Luminex assay was performed on a Magpix™ system (Merck) according to the manufacturer's instructions.

Results.

Single-channel and multi-channel analyses.

To evaluate the feasibility of simultaneously measuring titers of three antigens, the results of single-channel assays were compared with multichannel assays. Single-channel assays were performed in which each bead was incubated with participant sera, and then the same sera were tested in multichannel assays in which three antigen beads were mixed in equal parts and incubated with the test serum in one assay well. The MFI in these multichannel assays showed an extremely strong correlation with the single-channel MFI for each antigen, as shown in Fig. 9; R ² values were: SLO=0.999 (Fig. 9A); DNase B=0.998 (Fig. 9B); and SpnA=0.998 (Fig. 9C). This indicates no interference or IgG cross-reactivity between beads and demonstrates the feasibility of a multichannel Luminex-based assay incorporating three streptococcal antigens.

The coefficient of variation (CV) for the multichannel Luminex assay was estimated using the same 10 sera as in the single/multichannel assay comparison above. IgG concentrations in these 10 sera were measured in an assay that included IgG standard curves; and the mean intra-assay and inter-assay CV values for each antigen were <2% and <11%, respectively (Table 3A).

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Table FOR

π = 10 Coefficient SLO you are variations DNase B SpnA CV within analysis (%) 1.8 1.5 1.8 CV between assays (%) 9.3 10.4 10.4

These CVs demonstrate good accuracy and precision for Luminex-based multichannel analysis. The reproducibility of both the standard curve and the assay results means that these reagents can also be used to test the coupling efficiency and integrity of future batches of antigen-coupled beads.

Comparison with existing serological tests.

To compare the Luminex assay with existing commercially available methodology, ASO and ADB assays were performed in the serum of 61 participants for whom sufficient sample volumes were available. ASO was measured as described above in Example 1 using the widely used turbidimetric method, and accurate values were obtained in international units (IU/ml). ADB titers were measured (in a similar manner as described above in Example 1) using an enzyme inhibition assay that provides titer ranges (100, 200, 300, 400, 600, 800, 1200 and ~1600). As shown in FIG. 10A, there was a good correlation between the IgG ASO concentration determined by a commercially available turbidimetric method and the anti-SLO antibody titer determined by Luminex analysis (R ² =0.933). As shown in FIG. 10B, a good correlation also occurred between the IgG ADB concentration determined by a commercially available enzyme inhibition assay and the anti-DNase B antibody titer determined by the Luminex assay (R2=0.942).

Immunokinetics in ARF.

Serum from patients diagnosed with ARF (RFRF study) was stratified by days after hospitalization. As can be seen in FIG. 11, levels of anti-SpnA IgG were significantly reduced in sera collected more than 20 days after hospitalization (n=17) compared with levels in sera collected less than 20 days (n=19), indicating that they were more shorter half-life than anti-SLO antibodies or anti-DNase B antibodies.

This, in turn, indicates that SpnA has favorable immunokinetics for serological streptococci, and supports the possibility of its use in diagnostic assays, especially in multi-channel assays, such as those described and exemplified herein. It should be appreciated that the assays performed using the invention described herein allow rapid identification and treatment of patients recently exposed to Streptococcus pyogenes for whom long-term antibiotic treatment, often lasting for many years with associated costs and risks, is unnecessary and can be avoided.

Example 3.

This case study describes further evaluation of a multichannel bead-based assay using the Luminex platform to determine the presence, quantity, and immunokinetics of GAS-specific antibodies in biological samples.

Ways.

Subjects under study.

In this study, human serum was obtained as described above in Example 1.

Likewise, all participants provided written informed consent and appropriate ethics board approval was obtained.

Obtaining serum.

Serum samples from patients with acute rheumatic fever were grouped according to the day the blood samples were obtained: less than 20 days after the date of hospital admission; 20 days or more after hospitalization. Serum samples were diluted for analysis at a ratio of 1:15,000. The IVIG standard was diluted 1:60,000 before use.

Luminex bead preparation and analysis.

Each of the antigen:xMAP microspheres was prepared for use in the assay as described in Example 2. The Luminex assay was performed on a Magpix™ system (Merck) according to the manufacturer's instructions.

Comparisons of serum antibody titers for SLO, DNase B, and SpnA were determined using a three-channel Luminex assay. Statistical analysis was performed using GraphPad Prism 7.0 software.

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Results.

Immunokinetics in ARF.

Serum from patients diagnosed with ARF (RFRF study) was stratified by days after hospitalization. The results of this analysis are shown in the table below. 4 and fig. 12.

Table 4

Immunokinetics

Antigen < 20 days (n = 48) 20+days (n = 37) p value SLO average(95% CI) 834.9 (598.3 -1072) 695.2 (552.1-838.4) median value (95% CI) 558.1 (430.9 - 740.6) 592.0(465-727.8) n/s (p = 0.8983) DNase B average(95% CI) 341.2(267.7-414.6) 288.1(220-356.2) median value (95% CI) 279.6(238.2-355.7) 223.3 (184.2 - 293) ns (p = 0.3248) SpnA average(95% CI) 103.4 (79.4 -127.5) 70.1 (51.7-88.5) median value (95% CI) 75.1(61.2-91.6) 59.3 (43.0 - 69.8) p = 0.0389

There were no significant differences in antibody titers against SLO and DNase B between these groups. However, as can be seen in Fig. 12, anti-SpnA IgG levels were significantly reduced in sera collected more than 20 days after hospitalization (n=37) compared with levels in sera collected less than 20 days after hospitalization (n=48), which is consistent with results presented above in Example 2. This supports the conclusion that anti-SpnA antibodies have a shorter half-life than anti-SLO antibodies or anti-DNase B antibodies.

This, in turn, indicates that SpnA has favorable immunokinetics for serological streptococci, and supports its utility in diagnostic assays (especially multichannel assays, such as those described and exemplified herein), therapeutic evaluations, and treatment regimens.

Example 4.

This example describes a study of the thermal stability of SpnA as part of an assessment of suitability for use in diagnostic assays, such as multichannel bead-based assays such as the Luminex platform, or test strip/indicator strip-based assays (particularly where storage using a cold chain is not feasible or available).

Ways.

The stability of SpnA was determined using thermal melts according to published protocols (Moreau, M. J. J., Morin, I. & Schaeffer, P. M. Mol. BioSyst. 6, 1285-1292 (2010)). Briefly, the thermostability of the original SpnA construct (AK 28-877) was compared with the truncated construct (AK 28-854) by incubating the proteins in identical buffers for 5 min at the temperatures shown in FIG. 13 A.

This was followed by a cooling and centrifugation step to remove protein aggregates. The percentage of protein with a characteristic three-dimensional structure was determined using SDS-PAGE, and Tagg values (the temperature at which 50% of the protein molecules aggregate) were calculated from thermal aggregation profiles using GraphPad Prism 7.0 software.

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Results.

The percentage of protein with a characteristic three-dimensional structure at each temperature, as assessed by SDS-PAGE, is shown in the chromatogram in FIG. 13A. A Tagg plot (the temperature at which 50% of the protein molecules aggregate) was plotted for each protein at each temperature (Figure 13B), which clearly shows the differences in the melt curves of the two proteins. The mean Tagg for the truncated construct, 51.0 ±0.6°C across triplicate experiments, was significantly higher than the Tagg for the original construct, which was 47.5 ±0.9°C (p<0.05), which can be clearly seen in Fig. 13C.

This clearly demonstrates the increased thermal stability of the truncated SpnA polypeptide, supporting its potential for use in diagnostic assays, therapeutic evaluations, and treatment regimens, especially under conditions of long-term reagent storage or storage under conditions that are suboptimal for typical protein-based formulations, such as in the absence of a cold chain.

Publications.

1. Johnson DR, Kurlan R, Leckman J, Kaplan EL. The human immune response to streptococcal extracellular antigens: clinical, diagnostic, and potential pathogenetic implications. Clin. Infect. Dis. 2010;50:481-90.

2. Chang A, Khemlani A, Kang H, ProftT. Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor. Molecular Microbiology. 2011;79:1629-42.

3. Atatoa-Carr P, Lennon D, Wilson N, New Zealand Rheumatic Fever Guidelines Writing Group.

Rheumatic fever diagnosis, management, and secondary prevention: a New Zealand guideline. N Z Med J 2008;121:59-69.

4. Raynes JM, Frost HRC, Williamson DA, Young PG, Baker EN, Steemson JD, et al. Serological Evidence of Immune Priming by Group A Streptococci in Patients with Acute Rheumatic Fever. Front Microbiol. 2016;7:1119.

5. Dabitao D, Margolick JB, Lopez J, Bream JH. Multiplex measurement of proinflammatory cytokines in human serum: comparison of the Meso Scale Discovery electrochemiluminescence assay and the Cytometric Bead Array. J Immunol Methods. 2011;372:71-7.

6. Steer AC, Vidmar S, Ritika R, Kado J, Batzloff M, Jenney AWJ, et al. Normal ranges of streptococcal antibody titers are similar whether streptococci are endemic to the setting or not. Clin. Vaccine Immunol. 2009;16:172-5.

7. Moreau, M. J. J., Morin, I. & Schaeffer, P. M. Mol. BioSyst. 6, 1285-1292 (2010).

The complete descriptions of all applications, patents and publications mentioned above and below (if any) are incorporated herein by reference.

Any reference to prior art in this description is not and should not be taken as an endorsement or any form of indication that said prior art is part of the general general knowledge of the field in any country in the world.

Furthermore, the invention may be said to be, in a broad sense, composed of the parts, elements and features mentioned or indicated in the specification of the application, individually or together, in any possible combination of two or more of the said parts, elements or features.

Where integers or components having known equivalents are mentioned in the foregoing description, those integers are incorporated herein as if set forth individually.

It should be noted that various changes and modifications to the preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit of the invention and without diminishing its attendant advantages. Therefore, it is intended that such changes and modifications be included in the present invention.

Furthermore, the invention may be said to be, in a broad sense, composed of the parts, elements and features mentioned or indicated in the specification of the application, individually or together, in any possible combination of two or more of the said parts, elements or features.

Aspects of the present invention are described by way of example only, and it should be understood that modifications and additions may be made thereto without departing from the spirit thereof.

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Claims (15)

CLAIM 1. A method for detecting recent exposure to Streptococcus pyogenes in a subject, the method comprising: i) obtaining a biological sample previously obtained from the subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes antigens; ii) contacting said biological sample with one or more antigens from Streptococcus pyogenes nuclease A (SpnA), wherein said one or more SpnA antigens are capable of binding SpnA antigen-specific antibodies present in the biological sample to form one or more SpnA antigen complexes :SpnA antigen-specific antibody, if SpnA antigen-specific antibodies are present in the biological sample; and iii) detecting the presence or absence of specified SpnA antigen:SpnA antigen-specific antibody complexes; and iv) assessing one or more other diagnostic criteria for the presence of Streptococcus pyogenes or rheumatism or post-streptococcal glomerulonephritis in the subject, wherein detecting the presence of one or more SpnA antigen:antigen complexes SpnA-specific antibody or detection of an amount of one or more SpnA antigen:SpnA-specific antibody complexes above the threshold indicates recent exposure to Streptococcus pyogenes in the subject and the presence of one or more other diagnostic criteria for rheumatism or post-streptococcal glomerulonephritis in the absence of SpnA antigen:SpnA-specific antigen complexes antibody or numbers of SpnA antigen:SpnA antigen-specific antibody complexes below the threshold indicate recent exposure to Streptococcus pyogenes in the subject. 2. The method according to claim 1, including: i) providing a biological sample previously obtained from the subject that may contain or is suspected of containing one or more antibodies specific for SpnA; ii) contacting said biological sample with one or more SpnA antigens, wherein said one or more SpnA antigens are capable of binding antigen-specific antibodies present in the biological sample to form one or more SpnA antigen:SpnA antigen-specific antibody complexes, if the biological sample contains antigen SpnA-specific antibodies; and iii) detecting the presence or absence of said SpnA antigen:SpnA antigen-specific antibody complexes, wherein detection of an amount of one or more SpnA antigen:SpnA antigen-specific antibody complexes above a threshold value indicates recent exposure to Streptococcus pyogenes in the subject. 3. The method of claim 1 or 2, comprising assessing one or more other diagnostic criteria for the presence of Streptococcus pyogenes or rheumatism or post-streptococcal glomerulonephritis (PSGN) in the subject, wherein: i) detection of the presence of SpnA antigen:SpnA-specific antibody antigen complexes or a number of SpnA antigen:SpnA-specific antibody antigen complexes above a threshold value indicates recent exposure to Streptococcus pyogenes in the subject and ii) the presence of one or more other diagnostic criteria for rheumatic fever or PSGN in the absence SpnA antigen:SpnA antigen-specific antibody complexes or the number of Streptococcus pyogenes antigen SpnA-specific complexes below the threshold indicates recent exposure to Streptococcus pyogenes in the subject. 4. A method of detecting or diagnosing rheumatic fever or post-streptococcal glomerulonephritis (PSGN), including acute post-streptococcal glomerulonephritis (APSGN) in a subject, or determining the increased likelihood of developing rheumatic fever, PGSN or APSGN in a subject, the method comprising: i) providing a biological sample previously obtained from the subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes; ii) contacting said biological sample with one or more antigens from Streptococcus pyogenes nuclease A (SpnA), wherein said one or more SpnA antigens are capable of binding SpnA antigen-specific antibodies present in the biological sample to form one or more SpnA antigen complexes :SpnA antigen-specific antibody, if SpnA antigen-specific antibodies are present in the biological sample; and iii) detecting the presence or absence of SpnA antigen:SpnA antigen-specific antibody complexes; and iv) assessing one or more other diagnostic criteria for the presence of Streptococcus pyogenes or rheumatic fever or PSGN in the subject; - 37 044128 wherein the presence of one or more other diagnostic criteria for rheumatism, PGSN or APSGN together with the presence of one or more SpnA antigen:SpnA complexes. antigen-specific antibody or detection of the number of SpnA complexes. antigen:SpnA. an antigen-specific antibody above the threshold indicates an increased likelihood of developing rheumatic fever, PGSN or APSGN or indicates recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever, PGSN or APSGN in the subject; and wherein the presence of one or more other diagnostic criteria for rheumatic fever, PGSN or APSGN in the absence of one or more SpnA complexes. antigen:SpnA. antigen-specific antibody or when an amount of one or more SpnA complexes is detected. antigen:SpnA. an antigen-specific antibody below the threshold indicates the presence of rheumatic fever or PSGN in the subject, recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever or PGSN in the subject, such as an increased risk of subsequent Streptococcus pyogenes infection. 5. The method according to claim 4, characterized in that said method includes: i) providing a biological sample previously obtained from the subject that may contain or is suspected of containing one or more antibodies specific for Streptococcus pyogenes SpnA; ii) bringing said biological sample into contact with one or more Streptococcus pyogenes SpnA antigens, wherein said one or more Streptococcus pyogenes SpnA antigens are capable of binding antigen-specific antibodies present in the biological sample to form one or more SpnA antigen:SpnA antigen complexes -specific antibody, if the biological sample contains the antigen SpnA-specific antibodies; and iii) detecting said complexes, wherein detection of an amount of one or more SpnA antigen:SpnA antigen-specific antibody complexes above a threshold value indicates an increased likelihood of developing rheumatic fever, PGSN or APSGN, or indicates recent exposure to Streptococcus pyogenes in the subject as one of the criteria for the presence of rheumatic fever. , PGSN or APSGN in the subject; and iv) assessing one or more diagnostic criteria for the presence of rheumatism or post-streptococcal glomerulonephritis in the subject; and wherein the detection of an amount of one or more SpnA antigen:SpnA antigen-specific antibody complexes above a threshold value in combination with one or more other diagnostic criteria for rheumatism or acute post-streptococcal glomerulonephritis indicates the presence of rheumatism or acute post-streptococcal glomerulonephritis in the subject. 6. The method according to any one of claims 1 to 5, characterized in that said threshold value represents a control level of antigen established for each study population. 7. Method according to any one of claims 1 to 6, characterized in that said one or more diagnostic criteria is the presence or absence of one or more clinical symptoms associated with rheumatism, PSGN or APSGN. 8. The method according to claim 7, characterized in that said one or more of the clinical symptoms are selected from migratory polyarthritis, carditis, hematuria, erythema rheumatica, subcutaneous nodules, Sydenham's chorea or pyoderma. 9. The method according to any one of claims 1 to 8, characterized in that the assessment of one or more of the other diagnostic criteria includes the following steps: i) contacting the biological sample with one or more antigens from Streptococcus pyogenes DNase B and/or one or more antigens from Streptococcus pyogenes SLO, wherein said one or more Streptococcus pyogenes antigens can bind antigen-specific antibodies present in the biological sample to the formation of one or more antigen:antigen-specific antibody complexes in the presence of antigen-specific antibodies in a biological sample; and ii) detecting said complexes, wherein: a) the presence of Streptococcus pyogenes SpnA-specific complexes, or a determination of the number of Streptococcus pyogenes SpnA-specific complexes above a threshold value, indicates recent exposure to Streptococcus pyogenes in the subject; And b) the presence of Streptococcus pyogenes DNase B-specific complexes and/or Streptococcus pyogenes SLO-specific complexes and the absence of Streptococcus pyogenes SpnA-specific complexes, or detection of the number of Streptococcus SpnA-specific complexes pyogenes, below the threshold indicates the subject's past exposure to Streptococcus pyogenes. 10. Method according to any one of claims 1 to 9, characterized in that one or more of the Streptococcus pyogenes antigens is selected from the group consisting of: i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 8, or iv) deoxyribonuclease B (DNase B) or - 38 044128 v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, or vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic fragment an SLO comprising, essentially consisting of, or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, or x) any combination of two or more of i)-ix) above. 11. Method according to any one of claims 1 to 10, characterized in that the biological sample is brought into contact with each of the following Streptococcus pyogenes antigens: i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 8, and iv) deoxyribonuclease B (DNase B) or v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, and vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or ix) an antigenic fragment SLO containing, essentially consisting of, or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. 12. Method according to any one of claims 1 to 11, characterized in that the biological sample is brought into contact with each of the following Streptococcus pyogenes antigens: i) Streptococcus pyogenes nuclease A (SpnA) or an antigenic fragment of SpnA containing, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 8; and ii) deoxyribonuclease B (DNase B) or a DNase B antigenic fragment containing, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 5, and iii) streptolysin-O (SLO) or an SLO antigenic fragment containing essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 13. The method according to any one of claims 1 to 12, characterized in that the detection of antigen:antibody complexes involves exposing the complexes to a specific binding partner bearing a detectable label, and detecting the label signal in the presence of antigen-specific antibodies in the biological sample. 14. Method according to any one of claims 1 to 13, characterized in that one or more of the Streptococcus pyogenes antigens are labeled with a detectable label. 15. A kit for detecting or diagnosing rheumatism or PSGN in a subject for detecting the presence of Streptococcus pyogenes infection in the subject or for detecting antigen-specific antibodies to Streptococcus pyogenes in a biological sample, wherein said kit contains a composition containing at least one of Streptococcus pyogenes antigens, selected from the group consisting of: i) nuclease A (SpnA) of Streptococcus pyogenes or ii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 8, or iii) an antigenic fragment of SpnA containing, essentially consisting of or consisting of at least of at least 10 contiguous amino acids from the amino acid sequence of SEQ ID NO: 8, and at least one of the Streptococcus pyogenes antigens is optionally selected from the group consisting of: iv) deoxyribonuclease B (DNase B) or v) a DNase B antigenic fragment containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 5, or vi) a DNase B antigenic fragment containing, essentially consisting of or consisting of at least 10 contiguous amino acids from the amino acid sequence SEQ ID NO: 5, or vii) streptolysin-O (SLO) or viii) an antigenic fragment of SLO containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or -