Improved ielisa for strangles

Abstract

The present disclosure relates to a polypeptide fragment selected from the group consisting of a fragment of Eq48, and a fragment of Eq39. Eq48 and Eq39 are two surface proteins encoded by Streptococcus equi subspecies equi. The polypeptide fragments are useful for detection of exposure of a subject to Streptococcus equi 5 subspecies equi. Thus, the present disclosure furthermore relates to methods of use of such polypeptides.

Description

[0001] IMPROVED IELISA FOR STRANGLES

[0002] Technical field

[0003] The present invention relates to polypeptides useful for detection of exposure of a subject to Streptococcus equi subspecies equi, and to methods of use of such polypeptides.

[0004] Background

[0005] Strangles, caused by Streptococcus equi subspecies equi (5. equi), is one of the most frequently diagnosed infectious disease in horses worldwide. 5. equi invades the lymph nodes of the head and neck of horses within a few hours of exposure. Here the bacterium multiplies, forming abscesses that rupture into the guttural pouches or through the skin. Abscesses can grow to such a size that they restrict the airway, hence the naming of this disease.

[0006] If biosecurity is poor, an outbreak may result in clinical disease in every horse on a farm (i.e. 100% morbidity), and if exposure to 5. equi is high, then 1 in 10 horses may die from strangles, or complications thereof (i.e. 10% mortality). Clinical signs in each individual horse can take several weeks to resolve and some outbreaks can take more than 6 months to resolve. Therefore, outbreaks of strangles lead to significant health and economic cost (Boyle, A. G. et al., J Vet Intern Med 32, 633- 647, 2018).

[0007] Purulent material from abscesses in the retropharyngeal lymph nodes that ruptured into the guttural pouches drains via the nostrils leading to the typical clinical sign of mucopurulent nasal discharge associated with strangles cases. However, over time the incomplete drainage of abscess material from horses that have recovered from clinical disease leads to the formation of dried balls of pus in the guttural pouch, known as chondroids. 5. equi can persist in chondroids for up to several years and can intermittently shed from these so-called 'carrier' horses into the environment where the bacterium can go on to infect new horses and thereby trigger new outbreaks of disease. Thus, the identification and treatment of carrier horses is important to the control of outbreaks of strangles.

[0008] Persistently infected carrier horses can be identified and treated by the physical removal of residual purulent material by guttural pouch endoscopy and lavage. Therefore, the identification of horses exposed to 5. equi during an outbreak is important in order to facilitate the screening of exposed and recovered horses so that carriers can be identified and treated. However, screening of populations of horses using guttural pouch endoscopy and lavage, whilst recognized as the gold standard in terms of sensitivity and specificity, is not practical in the majority of outbreaks due to the invasiveness of this procedure and its associated financial cost. A much more practical and cost-effective approach to identify which horses have been exposed to 5. equi during an outbreak of strangles is to collect a blood sample and, following clotting of the blood, to test the serum for antibody responses towards fragments of two sortase-processed proteins (SEQ_2190, also known as antigen A; and SEQ_2017, also known as M-protein, mpepl and antigen C) that are produced on the surface of 5. equi. This assay was reported to have high levels of sensitivity and specificity for horses exposed to 5. equi (Robinson, C. et al., Vet J 197, 188-191; 2013). However, its ability to specifically identify persistently infected horses has been questioned in recent years (Pringle, J., et al., J Vet Intern Med 34, 2751-2757, 2020; Durham, A. E. & Kemp-Symonds, J., Equine Vet J 53, 38-43, 2021).

[0009] One explanation for the observed decrease in specificity is cross-reaction of the antigen fragments with antibodies towards the endemic opportunistic pathogen Streptococcus zooepidemicus, which is the most frequently recovered streptococcal species of horses. Such identification of 'false-positive' horses can lead to significant welfare and economic cost as a result of the need to further determine infection status of false-positive seropositive horses by guttural pouch endoscopy and lavage. There remains a need in the art to enable detection of horses that have been exposed to 5. equi. There remains a need in the art to enable detection of horses that have been exposed to 5. equi with high specificity. There remains a need in the art to enable detection of horses that have been exposed to 5. equi with high selectivity. As such, there remains a need in the art for means of detection of exposure to 5. equi.

[0010] Disclosure of the invention

[0011] It is an object of the present invention to at least partly reduce or overcome challenges in the prior art, and provide means that enable detection of horses exposed to 5. equi, such as Streptococcus equi subspecies equi. It is further an object to provide means that enable detection of horses exposed to 5. equi with higher specificity than what is provided by the prior art. It is further an object to provide means that enable detection of horses exposed to 5. equi with high sensitivity. It is further an object to provide means that enable detection of horses exposed to 5. equi with higher sensitivity than what is provided by the prior art.

[0012] These and other objects, which will be apparent to a skilled person from the present disclosure, are achieved by the different aspects of the disclosure as defined in the appended claims and as generally disclosed herein.

[0013] In a first aspect of the disclosure, there is provided a polypeptide fragment selected from the group consisting of a polypeptide A fragment of Eq48, and a polypeptide C fragment of Eq39. Eq48 and Eq39 are two surface proteins encoded by 5. equi.

[0014] In particular, there is provided a polypeptide fragment selected from the group consisting of a polypeptide A fragment of Eq48, and a polypeptide C fragment of Eq39; wherein said polypeptide A fragment comprises an amino acid sequence as shown in SEQ ID NO:2; said polypeptide A fragment has a maximum length of about 200 amino acids and has a maximum C-terminal extension of about 60 amino acids in relation to SEQ ID NO:3, and wherein said polypeptide A fragment has at least 85% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment; and wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:6, and wherein said polypeptide C fragment has at least 85% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment.

[0015] As used herein, the "term over the length of" in reference to the sequence identity of a polypeptide fragment of a longer (or full length) polypeptide sequence is to be understood to refer to the sequence of the full length polypeptide corresponding to the fragment. For example, if the fragment corresponds to amino acid residues 10- 50 of a full length polypeptide, the sequence identity of the 40 amino acid residues long fragment is to be calculated compared to the amino acid residues 10-50 of said full length polypeptide.

[0016] The term "% identity", as used throughout the specification, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity. The polypeptide A fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:2; said polypeptide A fragment has a maximum length of about 200 amino acids and has a maximum C-terminal extension of about 60 amino acids in relation to SEQ ID NO:3, and wherein said polypeptide A fragment has at least 85% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment.

[0017] The polypeptide A fragment has a maximum length of about 200 amino acids. In addition, said polypeptide A fragment has a maximum C-terminal extension of about 60 amino acids in relation to SEQ ID NO:3. In other words, polypeptide A fragment has a maximum C-terminal extension of about 129 amino acids.

[0018] Without being bound by any theory, it is believed that too long extension at the C- terminal may cause cross-reactivity with 5. zooepidemicus, which may be disadvantageous. According to the present disclosure, a too long extension may be a C-terminal extension of more than about 60 amino acids in relation to SEQ ID NO:3, such as about 70 amino acids or more. In other words, a too long extension may be a C-terminal extension of more than about 129 amino acids, such as about 139 amino acids or more.

[0019] That the polypeptide A fragment has a maximum C-terminal extension of about 60 amino acids in relation to SEQ ID NO:3 is to be understood as that said polypeptide A fragment comprises SEQ ID NO: 2 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2, and that fragment A corresponds with at least 85% sequence identity to position 1-71 in SEQ ID NO: 3. In addition, said polypeptide A fragment may comprise further amino acids on its C-terminal part such that the resulting fragment can be aligned with SEQ ID NO: 3 over the length of the polypeptide A fragment and wherein said alignment is an at least 85% sequence identity match. In such alignment, the extension starts at the position corresponding to position 72 in SEQ ID NO:3 and continues according to the sequence SEQ ID NO: 3, with the proviso of that the resulting alignment is at least 85% sequence identity match. For example, the polypeptide A fragment may correspond with at least 85% sequence identity to position 1-72 in SEQ ID NO: 3; or 1-73, or 1-74, or 1-75, or 1-80, or 1-85, or 1-90, or 1-95, or 1-100, or 1-105, or 1-110, or 1-115, or 1-120, or 1-125, or 1-130, or 1-135, or 1-140, or 1-145, or 1-150, or 1-155, or 1-160, or 1-165, or 1- 170, or 1-175, or 1-180, or 1-185, or 1-190, or 1-195, or the polypeptide A fragment may correspond with at least 85% sequence identity to position 1-200 in SEQ ID NO: 3.

[0020] The polypeptide C fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:6, and wherein said polypeptide C fragment has at least 85% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment. The present inventors have surprisingly found that the novel polypeptide fragments (also referred to herein as "antigens") of Eq48 and Eq39 according to the present disclosure are surprisingly beneficial for the identification of horses exposed to 5. equi, such as Streptococcus equi subspecies equi.

[0021] In one embodiment, the polypeptide fragment is the polypeptide A fragment. The present inventors show in the examples below that the new assay utilizing antigen A2 showed higher specificity than prior art assay utilizing antigen A.

[0022] In one embodiment, the polypeptide fragment is the polypeptide C fragment. The present inventors show in the examples below that the new assay utilizing antigen C3 showed higher sensitivity when compared to prior art assay utilizing antigen C.

[0023] In one embodiment, the polypeptide C fragment has a maximum length of about 250 amino acids. In one embodiment, the polypeptide C fragment has a maximum length of about 245 amino acids, such as a maximum length of about 240 amino acids, such as a maximum length of about 235 amino acids, such as a maximum length of about 230 amino acids, such as a maximum length of about 225 amino acids, such as a maximum length of about 220 amino acids, such as a maximum length of about 215 amino acids, such as a maximum length of about 210 amino acids, such as a maximum length of about 205 amino acids, such as a maximum length of about 200 amino acids, such as a maximum length of about 190 amino acids, such as about 185 amino acids; or such as a maximum length of about 180 amino acids, such as a maximum length of about 175 amino acids, such as a maximum length of about 170 amino acids, such as a maximum length of about 165 amino acids, such as a maximum length of about 160 amino acids, such as a maximum length of about 155 amino acids, such as a maximum length of about 150 amino acids, such as a maximum length of about 145 amino acids, such as a maximum length of about 140 amino acids, such as a maximum length of about 135 amino acids, such as a maximum length of about 130 amino acids, such as a maximum length of about 125 amino acids, such as a maximum length of about 120 amino acids, such as a maximum length of about 115 amino acids, such as a maximum length of about 110 amino acids, such as about 106 amino acids, or such as a maximum length of about 100 amino acids, such as a maximum length of about 95 amino acids, such as a maximum length of about 90 amino acids, such as a maximum length of about 85 amino acids. In one embodiment, the polypeptide C fragment is from about 70 to about 250 amino acids long, such as from about 75 to about 240 amino acids long, such as from about 80 to about 230 amino acids long, such as from about 85 to about 220 amino acids long, such as from about 90 to about 210 amino acids long, such as from about 95 to about 200 amino acids long, such as from about 100 to about 190 amino acids long, such as from about 106 to about 185 amino acids long, such as about 106 amino acids long or such as about 185 amino acids long.

[0024] In one embodiment, Eq48 comprises or consists of an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:1 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:1, such as wherein said Eq48 comprises or consists of an amino acid sequence as shown in SEQ ID NO:1. In one embodiment, Eq48 has at least 87% sequence identity to SEQ ID NO:1, such as at least 90% sequence identity, at least 92% sequence identity, at least 97% sequence identity, or at least 100% sequence identity to SEQ ID NO:1.

[0025] In one embodiment, Eq39 comprises or consists of an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:4 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:4, such as wherein said Eq39 comprises or consists of an amino acid sequence as shown in SEQ ID NO:4. In one embodiment, Eq39 has at least 87% sequence identity to SEQ ID NO:4, such as at least 90% sequence identity, at least 92% sequence identity, at least 97% sequence identity, or at least 100% sequence identity to SEQ ID NO:4.

[0026] In one embodiment, the polypeptide A fragment has a maximum length of about 195 amino acids, such as about 190 amino acids, such as about 185 amino acids, such as about 180 amino acids, such as about 175 amino acids, such as about 170 amino acids, such as about 165 amino acids, such as about 160 amino acids, such as about 155 amino acids, such as about 150 amino acids, such as about 145 amino acids, such as about 142 amino acids, such as about 140 amino acids, such as about 135 amino acids, such as about 130 amino acids, such as about 125 amino acids, such as about 120 amino acids, such as about 115 amino acids, such as about 110 amino acids, such as about 105 amino acids, such as about 100 amino acids, such as about 95 amino acids, such as about 90 amino acids, such as about 85 amino acids, such as about 80 amino acids, such as about 75 amino acids, such as about 71 amino acids.

[0027] In one embodiment, the polypeptide A fragment has a minimum length of about 50 amino acids, such as about 55 amino acids, such as about 60 amino acids, such as about 65 amino acids, such as a minimum length of about 70 amino acids.

[0028] In one embodiment, the polypeptide A fragment from about 40 amino acids to about 200 amino acids, such as rom about 45 amino acids to about 190 amino acids, such as from about 50 amino acids to about 180 amino acids, such as from about 55 amino acids to about 170 amino acids, such as from about 60 amino acids to about

[0029] 160 amino acids, such as from about 65 amino acids to about 150 amino acids, such as from about 70 amino acids to about 145 amino acids.

[0030] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity, such as having 100% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment.

[0031] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:2 and 3 and any amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:2 and 3.

[0032] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NO:2 and 3, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity, such as having 100% sequence identity to any one of SEQ ID NO:2 and 3. In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:3 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:3.

[0033] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:2 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:2.

[0034] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:3, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity, such as having 100% sequence identity to SEQ ID NO:3.

[0035] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:2, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity, such as having 100% sequence identity to SEQ ID NO:2.

[0036] In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:2 and 3. In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:2. In one embodiment, the polypeptide A fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:3.

[0037] In one embodiment, the polypeptide A fragment does not comprise an amino acid sequence from amino acid position 252 to amino acid position 673, such as from amino acid position 250 to amino acid position 673, such as from amino acid position 245 to amino acid position 673, such as from amino acid position 240 to amino acid position amino acid position 673, such as from amino acid position 235 to amino acid position 673, such as from amino acid position 230 to amino acid position 673, such as from amino acid position 225 to amino acid position 673, such as from amino acid position 220 to amino acid position 673, such as from amino acid position 215 to amino acid position 673, such as from amino acid position 210 to amino acid position 673, such as from amino acid position 205 to amino acid position 673, such as from amino acid position 200 to amino acid position 673, such as from amino acid position 195 to amino acid position 673, such as from amino acid position 190 to amino acid position 673, such as from amino acid position 189 to amino acid position 673, of SEQ ID NO:1, or fragments thereof.

[0038] In one embodiment, the polypeptide A fragment does not comprise or consist of an amino acid sequence as shown in SEQ ID NO:7.

[0039] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:6 , such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:6 , or such as having 100% sequence identity to SEQ ID NO:6. In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:5, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:5 , or such as having 100% sequence identity to SEQ ID NO:5.

[0040] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:5 and 6 and any amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:5 and 6.

[0041] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NO:5 and 6 , such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to any one of SEQ ID NO:5 and 6, or the fragment has 100% sequence identity to any one of SEQ ID NO:5 and 6.

[0042] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:5 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:5. In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:6 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:6. In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:5, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:5, or the fragment has 100% sequence identity to SEQ ID NO:5.

[0043] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence having at least 86% sequence identity to SEQ ID NO:6, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:6, or the fragment has 100% sequence identity to SEQ ID NO:6.

[0044] In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:5 and 6. In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:6. In one embodiment, the polypeptide C fragment comprises or consists of an amino acid sequence as shown in SEQ ID NO:5.

[0045] In one embodiment, the polypeptide fragment is linked at the N-terminal thereof to an amino acid sequence, wherein said amino acid sequence comprises or consists of about 1 to about 60, such as about 1 to about 55, such as about 1 to about 50, such as about 1 to about 45, such as about 1 to about 40, such as about 1 to about 35, such as about 1 to about 30, such as about 1 to about 25, such as about 1 to about 20, such as about 1 to about 15, such us about 1 to about 10, such as about 1 to about 5 amino acids. Such amino acid sequence may for example in some embodiments be a linker, a tag such as an affinity tag, a cloning artefact. In one embodiment, the polypeptide fragment is linked at the C-terminal thereof to an amino acid sequence, wherein said amino acid sequence comprises or consists of about 1 to about 60, such as about 1 to about 55, such as about 1 to about 50, such as about 1 to about 45, such as about 1 to about 40, such as about 1 to about 35, such as about 1 to about 30, such as about 1 to about 25, such as about 1 to about 20, such as about 1 to about 15, such us about 1 to about 10, such as about 1 to about 5 amino acids. Such amino acid sequence may for example in some embodiments be a linker, a tag such as an affinity tag, a cloning artefact.

[0046] The skilled person is aware of different kinds of linkers and tag with different properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers as well as various tags used for example for purification, such as affinity tags, His-tags, Myc-tags and more. Linkers and / or tags are known to have been used to for example increase stability or improve folding of polypeptides, to increase expression, improve biological activity, improve purification and other purposes.

[0047] In one embodiment, the polypeptide A fragment further comprises or consists of the amino acid sequence from amino acid position 1 to amino acid position 46 of SEQ ID NO:1 , or a fragment thereof. A fragment of the amino acid sequence from the amino acid position 1 to amino acid position 46 of SEQ ID NO:1, may be a fragment of from about 1 to about 45 amino acids, such as from about 1 to about 40 amino acids, such as from about 1 to about 35 amino acids, such as from about 1 to about 30 amino acids, such as from about 1 to about 25 amino acids, such as from about 1 to about 20 amino acids, such as from about 1 to about 15 amino acids, such as from about 1 to about 10 amino acids, such as a fragment from about 1 to about 5 amino acids long.

[0048] In one embodiment, the polypeptide C fragment further comprises or consists of the amino acid sequence from amino acid position 1 to amino acid position 37 of SEQ ID NO:4, or a fragment thereof. A fragment of the amino acid sequence from the amino acid position 1 to amino acid position 37 of SEQ ID NO:1 may be a fragment of from about 1 to about 37 amino acids, such as from about 1 to about 35 amino acids, such as from about 1 to about 30 amino acids, such as from about 1 to about 25 amino acids, such as from about 1 to about 20 amino acids, such as from about 1 to about 15 amino acids, such as from about 1 to about 10 amino acids, such as a fragment from about 1 to about 5 amino acids long.

[0049] In one embodiment, the polypeptide C fragment has a maximum length of about 185 amino acids, such as about 180 amino acids, such as about 175 amino acids, such as about 170 amino acids, such as about 165 amino acids, such as about 160 amino acids, such as about 155 amino acids, such as about 150 amino acids, such as about 145 amino acids, such as about 142 amino acids, such as about 140 amino acids, such as about 135 amino acids, such as about 130 amino acids, such as about 125 amino acids, such as about 120 amino acids, such as about 115 amino acids, such as about 110 amino acids, such as about 106 amino acids.

[0050] In one embodiment, The polypeptide fragment or a combination of polypeptide fragments is in a form of an isolated fragment or a fragment of a fusion protein. In one embodiment, the fusion protein comprises or consists of both the polypeptide A fragment and the polypeptide C fragment. As discussed elsewhere in the present disclosure, using both the polypeptide A fragment and the polypeptide C fragment may increase the sensitivity of the assay. Having both proteins as a fusion, may be advantageous, since only a single protein has to be prepared, and / or since only a single protein has to be brought into contact with a sample while testing a sample for both polypeptide fragments, as apparent to a person of skill in the art. In one embodiment, the fragment is in a form of an isolated fragment.

[0051] In one embodiment, the polypeptide fragment is an antigen.

[0052] In second aspect of the present disclosure, there is provided a combination of polypeptide fragments, which combination comprises a polypeptide A fragment as defined herein and a polypeptide C fragment as defined herein. The skilled person will appreciate that the embodiments recited in connection in the first aspect as disclosed herein are equally relevant for the second aspect and are merely for the sake of brevity not repeated herein.

[0053] The skilled person appreciates that the polypeptide fragment according to the first aspect and / or combination according to the second aspect is / are useful for the detection of antibodies, which antibodies are indicative of exposure of a subject to Streptococcus equi subsp. equi; and / or which antibodies are indicative of an infection caused by Streptococcus equi subsp. equi in a subject. The exposure may be previous and / or present exposure to Streptococcus equi subsp. equi.

[0054] The skilled person appreciates that the subject may have developed antibodies in response to exposure to Streptococcus equi subsp. equi, without the subject developing an infection. Such subject may be a carrier of Streptococcus equi subsp. equi and may thus spread the infection to one or more further subjects. As such, it may be of importance to identify a subject having antibodies indicative of exposure in order to prevent further spread of the disease.

[0055] Thus, in a third aspect of the present disclosure there is provided a use of the polypeptide fragment as defined herein, or of the combination of polypeptide fragments as defined herein, for the detection of antibodies indicative of exposure of a subject to Streptococcus equi subsp. equi; and / or indicative of an infection caused by Streptococcus equi subsp. equi in a subject.

[0056] In a fourth aspect, there is provided a method for determining the presence or the absence of one or more antibody / antibodies in a sample. The antibody is capable of binding to a polypeptide fragment as defined in the first aspect. Thus, the antibody is capable of binding to a polypeptide fragment A or a polypeptide fragment C. The method comprises the steps of: (i) bringing said sample into contact with said polypeptide fragment under conditions permissive of binding of said antibody to said polypeptide fragment; and

[0057] (ii) detecting binding of said antibody to said polypeptide fragment, wherein said binding is indicative of the presence of said antibody in said sample. No binding in this step is indicative of the absence of said antibody in said sample, as apparent to a person of skill in the art.

[0058] In one embodiment, the method detects the presence of an antibody which is capable of binding to a polypeptide fragment A and the presence of an antibody which is capable of binding to a polypeptide fragment C. In one embodiment, the method detects the presence of an antibody which is capable of binding to a polypeptide fragment A or a polypeptide fragment C. In one embodiment, the method detects the presence of an antibody which is capable of binding to a polypeptide fragment A. In one embodiment, the method detects the presence of an antibody which is capable of binding to a polypeptide fragment C.

[0059] To clarify, the method may detect an antibody which is capable of binding to a polypeptide fragment A or a polypeptide fragment C. Alternatively, the method may simultaneously detect one or more antibodies capable of binding to a polypeptide fragment A and one or more antibodies capable of binding to a polypeptide fragment C. Thus, in one embodiment, said method comprises in step (i) bringing said sample into contact with the combination of polypeptide fragments as defined herein under conditions permissive of binding of said one or more antibody / antibodies to at least one of said polypeptide fragments, such as both polypeptide fragments.

[0060] In one embodiment, the antibody is, or the antibodies are, produced in response to exposure to Streptococcus equi subsp. equi in a subject from which said sample is obtained. In one embodiment, the antibody is, or the antibodies are, produced in response to an active Streptococcus equi subsp. equi infection. In one embodiment, the antibody is, or the antibodies are, produced in response a dormant Streptococcus equi subsp. equi infection.

[0061] A subject with a dormant infection may be referred to as a carrier subject. As used herein, the term "carrier subject" refers to a subject who does not exhibit clinical symptoms and / or clinical signs of infection, however is infected with the infectious disease agent. A carrier subject may serve as a potential source of infection. The carrier state may occur in an subject with an infection that is inapparent throughout its course (known as an asymptomatic carrier) or the carrier state may exist only during the incubation period, convalescence, and postconvalescence of an subject with a clinically recognizable disease (also referred to as incubatory carrier subject or convalescent carrier subject). The carrier subject status may be of short or long duration (temporary or transient carrier or chronic carrier). As shown in the appended examples, the present invention is useful in method for determining the presence or the absence of one or more antibody / antibodies in samples obtained from potential carriers subjects, whereby carrier subjects may be identified and isolated to prevent the spread of disease in a population.

[0062] In one embodiment, the sample in step (i) is selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample, a fecal sample and a tissue sample (for example a biopsy), such as a sample selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample and a fecal sample. In one particular embodiment, said sample is a blood sample or a serum sample.

[0063] In one embodiment, the method further comprises a step of quantifying the amount of said antibody or antibodies in said sample. In one embodiment, the step of quantifying is an absolute quantification. In one embodiment, the step of quantifying is a relative quantification. In one embodiment, the presence or the absence of said antibody is determined relative to a control sample, such as a control sample which does not comprise any antibody capable of binding to said polypeptide fragment. In one embodiment, the presence or the absence of said antibodies is determined relative to a control sample, such as a control sample which does not comprise any antibody capable of binding to said polypeptide fragments.

[0064] Methods of quantification are apparent to the skilled person and the skilled person is familiar with selecting a suitable method for quantification, such as various enzyme-linked immunosorbent assays (ELISA).

[0065] In one embodiment, the method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. In one embodiment, the method is direct ELISA. In one embodiment, the method is indirect ELISA. In one embodiment, the method is sandwich ELISA. In one embodiment, the method is competitive ELISA.

[0066] In one embodiment, the method comprises direct coupling of said polypeptide fragment to a solid support. In one embodiment, the method comprises indirect coupling of said polypeptide fragment to a solid support. In one embodiment, the method comprises direct coupling of polypeptide A fragment to a solid support and indirect coupling of polypeptide C fragment to a solid support. In one embodiment, the method comprises direct coupling of polypeptide C fragment to a solid support and indirect coupling of polypeptide A fragment to a solid support.

[0067] The skilled person knows about solid supports that are suitable for use in the present method as well as is familiar with various methods for coupling polypeptides to different solid support materials. Possible supports may be solid supports selected from the group consisting of a microtiter plate, a vial, a collection tube, a bottle, a bead, a pre-coated filter paper, a blood tube, a Whatman paper, a DBS collection device, a dried plasma spot device, a dried serum spot device and a culturing plate. In one embodiment, the solid support is selected from the group consisting of microtiter plate, a vial, a collection tube, a bottle, and a culturing plate. In a preferred embodiment, the solid support is a microtiter plate. The microtiter plate may be a 6-, 12-, 24-, 48-, 96-, 384- or 1536-well plate, such as a 96-well plate. In one embodiment, the direct or indirect coupling is performed prior to step (i). According to the present disclosure, the solid support is as defined in the seventh aspect.

[0068] In one embodiment, the concentration of said polypeptide fragment at said direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0069] The skilled person is aware of common types of detection methods that may be employed in ELISA assays. Non-limiting examples of such methods include chromogenic assays, for example calorimetric assays using horseradish peroxidase (HRP-) or alkaline phosphatase (AP-) conjugated antibodies are used in combination with a chromogenic substrate; chemiluminescent assays, for example using peroxidase-conjugated detection antibodies (ie, AP and HRP) in a chemiluminescent reaction using a luminol-based enhancer solution added with a substrate; or fluorescent assay, for example using peroxidase-conjugated detection antibodies (ie, AP and HRP) with fluorescent substrates. Thus, in one embodiment said binding is detected using a method selected from the group consisting of a colorimetric method, a chemiluminescent method and a fluorescence method.

[0070] In one embodiment, the binding is detected using a colorimetric method or a fluorescence method. In one embodiment, the presence or the absence of said antibody is determined based on an optical density of the sample. In one embodiment, the optical density is measured at 450 nm. In one embodiment, an optical density value equal to or higher than 0.5 for said sample is indicative of the presence of said antibody in the sample. As discussed above, the identification of horses exposed to 5. equi during an outbreak is important in order to facilitate the screening of exposed and recovered horses so that carriers can be identified and treated. Thus, a method that is able to identify animals that have been exposed to Streptococcus equi subsp. equi, and have developed an antibody response against the antigens used in the assay, but are not necessarily infected with Streptococcus equi subsp. equi, may be beneficial from the point of managing an outbreak.

[0071] Thus, in a fifth aspect, the is provided a method for detecting exposure to Streptococcus equi subsp. equi in a subject. The method comprises the steps of

[0072] (i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in the first aspect, and

[0073] (ii) determining the presence or the absence of one or more antibodies in the sample. The one or more antibodies are capable of binding to said at least one polypeptide fragment.

[0074] As apparent to a person of skill in the art, step (i) and / or step (ii) is performed under conditions permissive of binding of the one or more antibodies to the at least one polypeptide fragment. The presence of the one or more antibodies in the sample is indicative of present or previous exposure to Streptococcus equi subsp. equi. As explained in the context of the fourth aspect, the method may thus be used for the identification of carrier subjects, who do not exhibit clinical symptoms / clinical signs of infection, however are infected with Streptococcus equi subsp. equi. A carrier subject may serve as a potential source of infection. The carrier subject may be an asymptomatic carrier, incubatory carrier subject or convalescent carrier subject. The carrier subject status may be temporary or transient or chronic, for example having a chronic dormant infection. As shown in the appended examples, the present invention is useful in method for detecting exposure to Streptococcus equi subsp. equi of a subject when the subject does not exhibit clinical symptoms and / or clinical signs of infection. This way carrier subjects may be identified and isolated to prevent the spread of disease in a population. In one embodiment, the exposure to Streptococcus equi subsp. equi results in a transient, an active or a dormant infection. In one embodiment, the infection is a transient, an active or a dormant infection.

[0075] In one embodiment, said sample is selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample, a fecal sample and a tissue sample (for example a biopsy), such as a sample selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample and a fecal sample. In one particular embodiment, said sample is a blood sample or a serum sample.

[0076] The skilled person will appreciate that the details discussed in connection with the embodiments as well as the embodiments per se of the fourth aspect of the present disclosure are equally relevant for the present fifth aspect and are not repeated here merely for the sake of brevity.

[0077] In one embodiment, the method in step (i) comprises bringing a sample obtained from the subject into contact with the polypeptide A fragment as defined in the first aspect, and the polypeptide C fragment as defined in the first aspect. As demonstrated in the Examples herein, using both the polypeptide A fragment and the polypeptide C fragment may increase the sensitivity of the assay, as when used in a dual antigen iELISA.

[0078] In one embodiment, the sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment simultaneously. In one embodiment, the sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment subsequently.

[0079] In one embodiment, the sample is brought into contact with the polypeptide A fragment and the polypeptide C fragment in the same single assay.

[0080] In one embodiment, the sample is brought into contact with the polypeptide A fragment and the polypeptide C fragment in two separate assays. If two separate assays are used, the results thereof may be combined and a conclusion based on the two assays can be determined. It will be understood that the order of bringing the sample into contact with said polypeptide A fragment and said polypeptide C fragment does not matter. Thus, said sample may be brought in contact with polypeptide A fragment first followed by contact with said polypeptide C fragment. Alternatively, said sample may be brought in contact with polypeptide C fragment first followed by contact with said polypeptide A fragment. Thus, the two assays may be performed in any order.

[0081] In one embodiment, step (ii) comprises detecting the binding of the one or more antibodies to said at least one polypeptide fragment. The skilled person appreciates different methods and ways of the detection. Thus, in one embodiment said binding is detected using method selected from the group consisting of a colorimetric method, a chemiluminescent method and a fluorescence method. In one embodiment, the binding is detected using a colorimetric method or a fluorescence method. In one embodiment, the binding is detected using a colorimetric method. In one embodiment, the binding is detected using a fluorescence method. In one embodiment, the binding is detected using a colorimetric method and a fluorescence method. Suitable methods in the present context may be various forms of ELISA. Thus in one embodiment, the method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. In one embodiment, the method is direct ELISA. In one embodiment, the method is indirect ELISA. In one embodiment, the method is sandwich ELISA. In one embodiment, the method is competitive ELISA. In a preferred embodiment, the method is indirect ELISA method demonstrated by the examples herein.

[0082] In one embodiment, the method further comprises direct or indirect coupling of said at least one polypeptide fragment to a solid support. In one embodiment, the direct or indirect coupling is performed prior to step (i). In one embodiment, the concentration of the at least one polypeptide fragment at the direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL. In one embodiment, the concentration of each of the at least one polypeptide fragment at the direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0083] In one embodiment, the at least one polypeptide fragment in step (i) is directly or indirectly coupled to a solid support. In one embodiment, the at least one polypeptide fragment in step (i) is directly coupled to a solid support. In one embodiment, the at least one polypeptide fragment in step (i) is indirectly coupled to a solid support. According to the present disclosure, the solid support is as defined in the fourth and / or seventh aspect.

[0084] As discussed in more detail in the context of aspect four, quantification of the amount of said one or more antibodies in a sample is possible. In one embodiment, the method further comprises a step of (iii); quantifying the amount of the one or more antibodies in the sample. In one embodiment, the step of quantifying is an absolute quantification or a relative quantification.

[0085] In one embodiment, the presence or the absence of said antibody is determined relative to a control sample. For example, the presence or the absence of said antibody may be determined relative to a control sample which does not comprise any antibody capable of binding to said at least one polypeptide fragment.

[0086] In one embodiment, the presence or the absence of said one or more antibodies is determined based on an optical density of the sample. In one embodiment, the optical density is measured at 450 nm. In one embodiment, an optical density value equal to or higher than 0.5 for the sample is indicative of the presence of the one or more antibodies in the sample. Thus, in this embodiment, 0.5 is the threshold value, and performing the method of the present aspect results in an value of 0.5 or more, it may be determined that the subject from which the sample is taken has been exposed to Streptococcus equi subsp. equi. Moreover, it may be determined that the subject has developed an antibody response against the antigens used in the assay. It should be noted that the subject may not necessarily be infected with Streptococcus equi subsp. equi at the time point at which the sample was taken.

[0087] In a sixth aspect there is provided a method for diagnosis of an infection caused by Streptococcus equi subsp. equi in a subject, said method comprising the steps of

[0088] (i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in the first aspect, and

[0089] (ii) determining the presence or the absence of one or more antibodies in the sample. The one or more antibodies are capable of binding to the at least one polypeptide fragment.

[0090] The skilled person will appreciate that the details discussed in connection with the embodiments as well as the embodiments per se of the fourth and fifth aspects of the present disclosure are equally relevant for the present sixth aspect and are not repeated here merely for the sake of brevity.

[0091] As apparent to a person of skill in the art, step (i) and / or step (ii) is performed under conditions permissive of binding of the one or more antibodies to the at least one polypeptide fragment. The presence of the one or more antibodies in the sample is indicative of present or previous infection to Streptococcus equi subsp. equi. It will be appreciated that the method for diagnosis as disclosed herein may thus be used for the identification of subjects infected with Streptococcus equi subsp. equi. who do exhibit various severity of clinical symptoms and / or signs of infection or who are asymptomatic. As used herein, the term "clinical sign(s)" refers to any objective evidence of a disease as observed by for example a clinician or other person. As used herein, the term "clinical symptom(s)" refers correspond to individual experience by the infected animal.

[0092] In one embodiment, said sample is selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample, a fecal sample and a tissue sample (for example a biopsy), such as a sample selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample and a fecal sample, such as a sample selected from the group consisting of a blood sample, a serum sample, a mucus sample, a urine sample and a fecal sample. In one particular embodiment, said sample is a blood sample or a serum sample.

[0093] The skilled person appreciates that all embodiments of aspect five are within the scope and disclosure of aspect six and are not repeated herein for the sake of brevity nor discussed extensively if repeated below.

[0094] In one embodiment, the method in step (i) comprises bringing a sample obtained from the subject into contact with the polypeptide A fragment as defined in the first aspect, and the polypeptide C fragment as defined in the first aspect. As demonstrated in the Examples herein, using both the polypeptide A fragment and the polypeptide C fragment may increase the sensitivity of the assay, as when used in a dual antigen iELISA.

[0095] In one embodiment, the sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment simultaneously. In one embodiment, the sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment subsequently.

[0096] In one embodiment, the sample is brought into contact with the polypeptide A fragment and the polypeptide C fragment in the same single assay.

[0097] In one embodiment, the sample is brought into contact with the polypeptide A fragment and the polypeptide C fragment in two separate assays. If two separate 1 assays are used, the results thereof may be combined and a conclusion based on the two assays can be determined.

[0098] In one embodiment, step (ii) comprises detecting the binding of the one or more antibodies to the at least one polypeptide fragment. The skilled person appreciates different methods and ways of the detection. Thus, in one embodiment said binding is detected using method selected from the group consisting of a colorimetric method, a chemiluminescent method and a fluorescence method. In one embodiment, the binding is detected using a colorimetric method or a fluorescence method. In one embodiment, the binding is detected using a colorimetric method. In one embodiment, the binding is detected using a fluorescence method. In one embodiment, the binding is detected using a colorimetric method and a fluorescence method. Suitable methods in the present context may be various forms of ELISA. Thus, in one embodiment, the method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA (i ELISA), sandwich ELISA and competitive ELISA. In one embodiment, the method is direct ELISA. In one embodiment, the method is indirect ELISA. In one embodiment, the method is sandwich ELISA. In one embodiment, the method is competitive ELISA. In a preferred embodiment, the method is indirect ELISA method demonstrated by the Examples herein.

[0099] In one embodiment, the method further comprises direct or indirect coupling of said at least one polypeptide fragment to a solid support. In one embodiment, the direct or indirect coupling is performed prior to step (i).

[0100] In one embodiment, the concentration of the at least one polypeptide fragment at the direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL. In one embodiment, the concentration of each of the at least one polypeptide fragment at the direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0101] In one embodiment, the at least one polypeptide fragment in step (i) is directly or indirectly coupled to a solid support. In one embodiment, the at least one polypeptide fragment in step (i) is directly coupled to a solid support. In one embodiment, the at least one polypeptide fragment in step (i) is indirectly coupled to a solid support. According to the present disclosure, the solid support is as defined in the fourth and / or seventh aspect.

[0102] As discussed in more detail in the context of aspect four, quantification of the amount of said one or more antibodies in a sample is possible. In one embodiment, the method further comprises a step of (iii); quantifying the amount of the one or more antibodies in the sample.

[0103] In one embodiment, the step of quantifying is an absolute quantification or a relative quantification.

[0104] In one embodiment, the presence or the absence of said antibody is determined relative to a control sample. For example, the presence or the absence of said antibody may be determined relative to a control sample which does not comprise any antibody capable of binding to said at least one polypeptide fragment.

[0105] In one embodiment, the presence or the absence of said one or more antibodies is determined based on an optical density of the sample. In one embodiment, the optical density is measured at 450 nm.

[0106] In one embodiment, an optical density value equal to or higher than 0.5 for the sample is indicative of the presence of the one or more antibodies in the sample. Thus, in this embodiment, 0.5 is the threshold value, and performing the method of the present aspect results in an value of 0.5 or more, it may be determined that subject from which the sample is taken has been exposed to Streptococcus equi subsp. equi. Moreover, it may be determined that the subject have developed an antibody response against the antigens used in the assay. It should be noted that the subject may not necessarily be exhibiting clinical symptoms and / or clinical signs of infection with Streptococcus equi subsp. equi at the time point at which the sample was taken.

[0107] In a seventh aspect there is provided a solid support comprising at least one polypeptide fragment as defined in any embodiment of the first aspect.

[0108] The skilled person knows about solid supports that are suitable for comprising a polypeptide fragment according to first aspect. The skilled person is also familiar with various methods for coupling polypeptides to different solid support materials. Possible supports may be solid supports selected from the group consisting of a microtiter plate, a vial, a collection tube, a bottle, a bead, a pre-coated filter paper, a blood tube, a Whatman paper, a DBS collection device, a dried plasma spot device, a dried serum spot device and a culturing plate. In one embodiment, the solid support is selected from the group consisting of microtiter plate, a vial, a collection tube, a bottle, and a culturing plate. In one embodiment, the solid support is a microtiter plate. The microtiter plate may be a 6-, 12-, 24-, 48-, 96-, 384- or 1536-well plate, such as a 96-well plate.

[0109] In one embodiment, the at least one polypeptide fragment is directly or indirectly coupled to the solid support. In one embodiment, the at least one polypeptide fragment is directly coupled to the solid support. In one embodiment, the at least one polypeptide fragment is indirectly coupled to the solid support.

[0110] In one embodiment, the solid support comprises said polypeptide A fragment as defined in the first aspect, and said polypeptide C fragment as defined in the first aspect. Such set up enables a dual antigen i ELISA. As discussed elsewhere the present inventors envision that a dual antigen i ELISA may provide high sensitivity, such as higher sensitivity compared to an individual iELISA.

[0111] In one embodiment, the polypeptide A fragment and said polypeptide C fragment are directly or indirectly coupled to the solid support.

[0112] In an eight aspect of the present disclosure, there is provided a kit for carrying out the method according to the fourth, fifth or sixth aspect of the present disclosure. The kit comprises one or more polypeptide fragment according to the first aspect and instructions for carrying out the method of the fourth, fifth or sixth aspect. In one embodiment, the kit comprises the solid support as defined in the seventh aspect.

[0113] In one embodiment, the kit comprises said polypeptide A fragment as defined in the first aspect, and said polypeptide C fragment as defined in the first aspect. Thus, in this embodiment the kit provides for a dual iELISA. Advantages of dual iELISA are discussed elsewhere in the present disclosure and demonstrated in the appended examples

[0114] In one embodiment, the polypeptide A fragment and said polypeptide C fragment are directly or indirectly coupled to two separate solid supports.

[0115] In one embodiment, the kit further comprises one or more reagents required for a conventional ELISA method. The skilled person is well aware of reagents required for in a conventional ELISA method, and examples thereof are presented in Example 2 herein. A conventional ELISA method may be for example direct ELISA, indirect ELISA, sandwich ELISA and / or competitive ELISA.

[0116] In a nineth aspect, there is provided a use of a solid support as defined in the seventh aspect or kit as defined in the eight aspect, in a method according to any one of the fourth, fifth or the sixth aspect as disclosed herein. In a tenth aspect of the present disclosure, there is provided a fusion polypeptide comprising a polypeptide A fragment as defined herein and a polypeptide C fragment as defined herein.

[0117] In a eleventh aspect, there is provided a polynucleotide encoding a polypeptide fragment according to any embodiment of the first aspect.

[0118] In one embodiment, the polynucleotide encodes a fusion protein comprising said polypeptide A fragment and said polypeptide C fragment according to the tenth aspect as defined herein.

[0119] In an twelfth aspect, there is provided an expression vector comprising a polynucleotide according to the eleventh aspect.

[0120] In a thirteenth h aspect, there is provided a cloning vector comprising a polynucleotide according to the eleventh aspect.

[0121] In a fourteenth aspect, there is provided a host cell comprising an expression vector according to according to the twelfth aspect.

[0122] In a fifteenth aspect, there is provided a host cell comprising a cloning vector according to according to the fourteenth aspect.

[0123] In a sixteenth aspect, there is provided a method of producing a polypeptide fragment according to any embodiment of the first aspect. The method comprises: culturing a host cell according to the fourteenth aspect under conditions permissive of expression of the polypeptide from the expression vector, and isolating said polypeptide.

[0124] In a seventeenth aspect, there is provided a composition comprising at least one polypeptide fragment according to any embodiment of the first aspect, and optionally an excipient and / or adjuvant.

[0125] In a eighteenth aspect, there is provided a composition according to the seventeenth aspect, for use in a method according to any one of the fourth, fifth or the sixth aspect.

[0126] In a nineteenth aspect, there is provided a method for direct or indirect coupling of a polypeptide fragment according to any embodiment of the first aspect. The method comprising the steps of

[0127] (i) bringing a solid support as defined in the fifth aspect into contact with the polypeptide fragment or a combination of polypeptide fragments under conditions permissive of binding of the polypeptide fragment or a combination of polypeptide fragments to the solid support, thereby obtaining said polypeptide fragment coupled to said solid support.

[0128] In a twentieth aspect, there is provided a method of treating Streptococcus equi infection, such as a Streptococcus equi subsp. equi infection. The method comprises

[0129] (i) identifying a subject suspected to carry an antibody, which antibody is capable of binding to a polypeptide fragment according to any embodiment of the first aspect;

[0130] (ii) providing a sample from the subject;

[0131] (iii) testing the sample in a method according to any one of the fourth, fifth or the sixth aspect;

[0132] (iii) determining that the subject carries said antibody; and

[0133] (iv) administrating a therapeutic agent suitable for treatment of said Streptococcus equi infection, such as a Streptococcus equi subsp. equi infection.

[0134] In one embodiment, said therapeutic agent is a prophylactic agent, such as a vaccine. In one embodiment, the therapeutic agent comprises recombinant proteins derived from Streptococcus equi subsp. equi.

[0135] In one embodiment, said therapeutic agent comprises a polypeptide comprising at least two, three, four or all five, polypeptides selected from the group consisting of SEQ ID NO:12, 13, 14, 15 and 16. In one embodiment, the therapeutic agent comprises a polypeptide according to SEQ ID NO:9 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0136] In one embodiment, said therapeutic agent comprises at least one of both polypeptide according at SEQ ID NO:17 and 18. In one embodiment, the therapeutic agent comprises a polypeptide according to SEQ ID NQ:10 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0137] In one embodiment, the therapeutic agent comprises a polypeptide according to SEQ ID NO:11 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0138] In one embodiment, the therapeutic agent comprises at least one, such as at least two or all three, polypeptide(s) selected from the group consisting of a polypeptide comprising the amino acid sequence according to SEQ ID NO:9 starting from position 12, a polypeptide comprising the amino acid sequence according to SEQ ID NQ:10 starting from position 12 and a polypeptide comprising the amino acid sequence according to SEQ ID NO:11 starting from position 12. Such composition is described in WO 2011 / 149419 and reference is made thereto. In one embodiment, the therapeutic agent is Strangvac®.

[0139] In one embodiment, said therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding at least two, three, four or all five polypeptides selected form the group consisting of SEQ ID NO:12, 13, 14, 15 and 16. In one embodiment, the therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding a polypeptide according to SEQ ID NO:9 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0140] In one embodiment, said therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding at least one or both polypeptides SEQ ID NO:17 and 18. In one embodiment, the therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding a polypeptide according to SEQ ID NQ:10 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0141] In one embodiment, the therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding a polypeptide according to SEQ ID NO:11 starting from position 12 or any polypeptide exhibiting at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0142] In one embodiment, said therapeutic agent comprises nucleic acid(s), such as DNA or mRNA, encoding at least one, such as encoding at least two or all three, polypeptide(s) selected from the group consisting of a polypeptide comprising the amino acid sequence according to SEQ ID NO:9 starting from position 12, a polypeptide comprising the amino acid sequence according to SEQ ID NQ:10 starting from position 12 and a polypeptide comprising the amino acid sequence according to SEQ ID NO:11 starting from position 12.

[0143] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the invention. Therefore, it is intended that the invention is not limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims. The invention will be further illustrated by the following non-limiting Examples.

[0144] Brief description of the figure

[0145] Figure 1 shows a summary table listing the polypeptides (SEQ ID NO:l-8) of the present disclosure and polypeptide A fragments SEQ ID NO:2 and 3 and polypeptide C fragments 5 and 6 of the present invention.

[0146] Examples

[0147] Summary

[0148] In the following non-limiting examples, the inventors demonstrate that novel polypeptide fragments (also referred to herein as "antigens") of Eq48 and Eq39 according to the present disclosure are surprisingly beneficial for the identification of horses exposed to 5. equi. As discussed earlier, Eq48 (SEQ ID NO:1) and Eq39 (SEQ ID NO:4) are two surface proteins encoded by 5. equi and the novel polypeptide fragments are predicted by the inventors to interact with antibodies specific to 5. equi.

[0149] The new antigens of the present disclosure and known antigens from the prior art were produced as described in Example 1. Amino acid sequences of the novel antigens together with the known control antigens are listed in Fig. 1 with the sequence identifiers SEQ ID NO:l-8. The Examples further describe the use of the novel antigens in the detection of 5. equi infected horses using indirect ELISA (iELISA) assays, as described in Example 2, against various selection criteria and sample groups in comparison to known antigens in the art (Examples 3-8). Specificity and sensitivity of the assays were evaluated. In particular, testing of specificity was performed using serum samples from horses that were known negative of strangles ("negative samples" from "Icelandic" horses, as described in Example 3). Sensitivity was tested using samples known positive of strangles (serum samples from "Rossdales" and "Dartmoor" horses, Examples 4-8). The latter sample groups included sera from persistently infected horses ("carrier samples") and sera from horses in an acute disease state, i.e. in early stages of the disease before they had sufficient time to develop a strong antibody response ("acute disease samples"). The sample groups are further described in Example 2.

[0150] Example 1: Protein expression and purification of polypeptide fragments of Eq48 and Eq39

[0151] The inventors herein demonstrate advantageous characteristics of novel polypeptide fragments (also referred to herein as "new" and "novel" antigens) according to the present disclosure for improved iELISA assays that are useful for identifying horses exposed to 5. equi with high specificity and / or sensitivity. Among these, the polypeptide A2 fragment (also referred to herein is "antigen A2" or "A2 antigen") is a fragment of the N-terminal region of Eq48 (SEQ ID NO:1), as shown in SEQ ID NO:3. The polypeptide C3 fragment (also referred to herein is "antigen C3" or "C3 antigen") is a fragment of the N-terminal region of Eq39 (SEQ ID NO:4), as shown in SEQ ID NO:5.

[0152] The polypeptide C4 fragment (also referred to herein is "antigen C4" or "C4 antigen") is a fragment of Eq39 (SEQ ID NO:4), as shown in SEQ ID NO:6.

[0153] These novel polypeptide fragments were tested in comparison to two known polypeptide fragments (also referred to herein as "known", "old" and "previous" antigens) of Eq48 and Eq39 (referred to herein as "antigen A" and "antigen C", respectively) which have been used in the prior art for testing serum samples from horses for the detection of 5. equi infection. The amino acid sequences of antigen A and antigen C are shown in SEQ ID NO:7 and 8, respectively. In this example, materials and methods for the expression and purification of these new and old antigens are described in detail.

[0154] Materials and methods

[0155] All DNA constructs were ordered from GenScript (Leiden, Netherlands). The DNA constructs were delivered as dried DNA (inserted in the IPTG-inducible expression plasmid pGEX-6P-l) encoding the protein of interest and an ampicillin resistance gene for plasmid selection. The DNA was dissolved according to manufacturer's instructions. A small part of the resulting DNA solution was transformed into E. coli BL21 by electroporation using 2 mm electroporation cuvettes and an electric pulse generator (Gene Pulser, BIO-RAD), the settings were 2.5 kV and 25 pFD. Following the electric discharge, 1 mL of room tempered Luria Broth (LB) was added to the cuvette and the cell suspension transferred to a 1.5 mL micro-centrifuge tube that was incubated for one hour at room temperature before plating. Successfully transformed bacterial clones were confirmed by plating approximately 100 pL of the cell suspension onto LB plates with agar and ampicillin (50 pg / mL) with incubation overnight at +37°C.

[0156] A single clone was transferred to 20 mL of LB with ampicillin in a 250 mL E-flask as a starter culture for expression of the target protein. The culture flask was incubated overnight at +37°C with shaking at 150 rpm. The starter culture was subsequently used to inoculate a larger expression culture, for example, 1000 mL LB with ampicillin (50 pg / mL) in a 5000 mL E-flask at 37°C with shaking at 150 rpm. The expression of the target protein was induced by the addition of 50 pM of IPTG at ODeoonm = 0.6. The temperature was subsequently lowered to 25°C and the culture was allowed to express protein overnight with shaking set to 150 rpm.

[0157] Bacterial cells were harvested by centrifugation in 1000 mL centrifuge flasks for 30 minutes at 4000 rpm in a Beckman centrifuge. The supernatant was removed and the bacterial cell pellet was resuspended in 150-160 ml of lysis buffer (100 mM NaCI, 20 mM Tris-HCL, 0.05% Tween 20) containing lysozyme at a concentration of 50 pg / mL. The bacterial suspension was divided into six 50 mL tubes that were freeze- thawed two to three times. The resulting bacterial cell lysate was sonicated gently using one-second pulses for 20-30 seconds, with the amplitude set to 30% of maximum until the first viscous lysate was clear and less viscous, the lysate was kept on ice to avoid heating and if needed, the sonication step was repeated. The sonicated lysate was centrifuged at 3800 rpm for 20 minutes and the supernatant sterile filtered (0.45 pm).

[0158] The sterile filtered sonicated lysate was loaded onto 2 mL of washed Glutathione Sepharose beads in a tube according to the manufacturer's description for batch purification (Cytiva), where the expressed target protein was allowed 30 minutes to bind to the beads via the GST-tag included in the N-terminal end of the target protein. After washing away the contaminating E. coli protein from the beads with the bound GST-tagged target protein, the target protein was released by adding PreScission Protease (Cytiva) which cleaves the target protein from the GST-tag. After confirmation that the cleavage process had reached completion (by running samples from a time series on a protein gel), the supernatant containing cleaved target protein was dialyzed in a dialysis buffer (150 mM NaCI, 50 mM Tris, pH 7.4) with several buffer changes to remove impurities. The final protein concentration was measured using a Qubit Fluorometer with a Qubit protein assay kit (ThermoFisher Scientific). Purified proteins were stored at -20 °C until needed.

[0159] Results and conclusions

[0160] Antigens A, A2, C and C3 were expressed well regarding both protein quantity and solubility. The antigens were utilized in i ELI SA assays as described in the following Examples. For the sake of clarity, assays using the novel antigens are also referred to herein as "new" and "novel" assays, and assays using the old antigens are also referred to herein as "old", "known" and "previous" assays. Example 2

[0161] Sample groups and i ELISA assay

[0162] This Example describes details of the tested sample groups and the general protocol of the i ELI SA assays used in the following examples.

[0163] Samples

[0164] Three groups of serum samples were selected for demonstrating an improved i ELI SA against strangles according to the present disclosure. The first group of serum samples (a total number of 200 serum samples) were from Icelandic horses negative of strangles, received from Vilhjalmur Svansson, Institute for Experimental Pathology, University of Iceland, Reykjavik, Iceland. 5. equi is not present in Iceland and all 200 serum samples from Icelandic horses should test negative in an i ELI SA that is specific to 5. equi. However, the closely related pathogen, 5. zooepidemicus, does infect horses in Iceland and this may trigger false positive results. Therefore, the use of sera from Icelandic horses provides an overview of the specificity of a serology assay for 5. equi. In Example 3, the antigens were tested against the Icelandic samples.

[0165] The second group of serum samples (referred to herein as "acute disease samples") were from horses with early stages of strangles from two horse populations: Rossdales and Dartmoor horses. The third group of serum samples (referred to herein as "carrier samples") were from persistently infected horses from the two horse populations. In particular, a total number of 81 Rossdales samples were from UK, provided by Liz Medcalf, Rossdales Laboratories. Rossdales samples were selected for testing, based on the availability of clinical information on disease status from the attending veterinarian and / or matched testing for 5. equi bacteria and / or 5. equi DNA to confirm infection with 5. equi. 35 of the 81 samples were paired samples from 17 different horses. Out of 81 samples, 30 samples were acute disease samples and 51 samples were carrier samples. A total number of 48 Dartmoor samples were collected from UK, provided by Nic de Brauwere from Dartmoor, wherein 24 of the samples were acute disease samples and 24 of the samples were carrier samples. In Examples 4, 5 and 6, overall assessment of sensitivity for testing Rossdales and Dartmoor samples is shown. In Examples 7 and 8, assessment of sensitivity for acute disease and carrier samples, respectively, is presented.

[0166] Iceland serum sample number 70 (shown in Example 3) was included as a negative control in all the assays performed. Absorbance (at 450 nm) of the negative control sample was measured between 0.00 and 0.03 for the antigen A iE LISA, 0.00 for the antigen C i ELI SA, 0.00 and 0.03 for the antigen A2 i ELI SA and 0.01 and 0.03 for the antigen C3 iELISA. A pooled serum sample, pooled from several horses with strangles, was used as a positive control in all the assays performed. Absorbance (at 450 nm) of the positive control sample was measured between 2.03 and 3.72 (average 2.91) for the antigen A iELISA, 1.61 and 2.25 (average 2.00) for the antigen C iELISA, 0.88 and 1.41 (average 1.18) for the antigen A2 iELISA and 2.16 to 3.01 (average 2.64) for the antigen C3 iELISA. iELISA

[0167] This test has been designed for determining the presence or the absence of 5. equi antigen-specific antibodies present in equine sera using indirect ELISA (iELISA, enzyme-linked immunosorbent assay). The reaction took place in the wells of microplates (or strips) that have been pre-coated with new and old antigens of 5. equi as described in Example 1. Each tested antigen was coupled to a separate microplate. Serum samples were then added to the antigen-coated wells under conditions that enable antibodies specific for these antigens to recognise and bind to the immobilised antigens, if said antibodies are present in the sample. These bound, specific antibodies were subsequently detected using horseradish peroxidase (HRP)-conjugated protein G, which has high binding affinity for equine IgG. The visualisation of binding was done by adding tetramethylbenzidine (TMB), a substrate for the enzyme HRP, that results in a coloured product. The coloured product was subsequently measured with a spectrophotometer at an optical density of 450 nm (A450). While the inventors herein utilized this particular variant of ELISA in the context of the present disclosure, it will be appreciated by those skilled in the art that other ELISA variants are equally suitable for the methods as disclosed herein.

[0168] Coating of iELISA plates

[0169] 1) The content of one capsule of carbonate-bicarbonate coating buffer (C-3041, Sigma-Aldrich, Merck) was dissolved in 100 mL ultra-pure water.

[0170] 2) The stock of each antigen (A2 and C3) was diluted separately in carbonate buffer to a final concentration of 0.8 pg / mL for antigen A2 and 0.5 pg / mL for antigen C3, allowing 10 mL of each diluted antigen per plate. For comparison, antigen A and antigen C were also diluted separately in carbonate buffer but to the final concentration of 0.8 pg / mL for antigen A and 1.2 pg / mL for antigen C.

[0171] 3) The diluted antigens were dispensed to separate Maxisorp plates (Nunc, ThermoFisher Scientific), 100 pL of corresponding antigen to each individual well and the plates were covered with Microwell plate lids (Nunc, Thermo Scientific).

[0172] 4) The plates with antigen in carbonate buffer were incubated overnight at +5 °C with a piece of flat Styrofoam placed on top of each plate to avoid any condensation in the plate lid.

[0173] Performing the iELISA

[0174] 1) One tablet of phosphate buffered saline (PBS; Millipore) was dissolved in 1 L of ultra-pure water. To one litre of PBS, 500 pL of Tween 20 (Sigma-Aldrich, Merck) was added and mixed to produce PBST.

[0175] 2) PBST was used as a plate washing solution in an automatic plate washer (Wellwash; ThermoFisher Scientific) programmed to do a wash procedure comprising of four washing cycles with 400 pL per well. The washed plates were tapped, wells down, onto dry paper tissue to ensure that any remaining droplets were removed.

[0176] 3) After completion of the first wash procedure, blocking was done for 1 h at room temperature (222C ±2) using 200 pL per well of PBST with the addition of 1 % (w / v) of non-fat skimmed milk powder (PBSTN; AppliChem / ITW, Avantor / VWR). The plates were covered with Microwell plate lids during the incubation time.

[0177] 4) During the blocking incubation, the test samples, either equine serum or plasma, were diluted 1 / 800 in PBSTN. Dilution was made in two steps, first by taking 10 pL of sample to 90 pL of PBSTN followed by vortex-mixing (dilution 1 / 10), second by taking 10 pL of the first dilution to 790 pL of PBSTN in a new tube and again vortex-mixing (dilution 1 / 80).

[0178] 5) After completion of the blocking incubation, another four-cycle wash procedure was performed.

[0179] 6) To the antigen-containing plates, triplicate wells with 100 pL per well of PBSTN (as blank), negative control (from a naive horse, sample no. 70 from the Icelandic samples), positive control (pooled serum samples from several horses with strangles) and test samples were added in identical order for all the antigen-coated plates. Incubation was done for 1 h at room temperature (222C ±2) with the plates covered.

[0180] 7) After completion of the sample incubation, another four-cycle wash procedure was performed.

[0181] 8) Directly before use, the protein G-HRP conjugate was diluted 1 / 600 in PBSTN from a stock solution of 1 mg / mL in PBS, pH 7.4 (P21041, HRP-Protein G, 1 mg, Invitrogen). For two full plates, 40 pL of stock solution was added to 24 mL of PBSTN and mixed carefully and then 100 pL of the diluted conjugate was added to each well. Incubation was done for 1 h at room temperature (222C ±2) with the plates covered. 9) After completion of the protein G-HRP incubation, another four-cycle wash procedure was performed.

[0182] 10) Approximately 10 mL per plate, of the peroxidase substrate solution (TMB, 500 mL, 00-2023 / 10445723, Novex / Life Technologies Ltd / ThermoFisher Scientific), was brought to room temperature (protected from light) before use. To each well, a 100 pL of TMB was added and allowed to react for 15 minutes covered with a plate lid and protected from light.

[0183] 11) After allowing for 15 minutes of the TMB substrate conversion reaction, the reaction was stopped and the i ELI SA developed by adding 100 pL of stop solution (0.18 M sulphuric acid, Avantor / VWR) to each well, causing a pH- dependent colour shift from blue to yellow.

[0184] 12) The stopped / developed plates (without the plate lid) were read at A450 (450 nm) using an ELISA plate reader (Multiscan FC, Thermo Scientific) and the raw data was exported to Excel for further data handling.

[0185] Data handling

[0186] The raw data from the spectrophotometer readings were imported to Excel where a script calculated the average of the triplicate values and from each average value the average blank value (PBSTN) was subtracted. Occasionally, some absorbance values deviated within a triplicate set of wells. These outlier values were manually removed and instead the values of duplicate wells were used for calculating the final absorbance values. The average absorbance values for each plate were normalized based on the absorbance value of the positive control sera that was obtained on that plate relative to the average across the assays (2.91 for antigen A, 2.00 for antigen C, 1.18 for antigen A2 and 2.64 for antigen C3). The cut-off values for a positive readout of the i ELI SA were set to A450 > 0.5 for one (or both) of the antigens in any of the dual antigen iE LISA test (Robinson, C. et al. Combining two serological assays optimizes sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure. Vet J 197, 188-191 (2013)). In general, sensitivity of the herein described assays was measured as the number of positives detected compared to the total number of true positives in the given sample group. Accordingly, a lower number of false negatives indicated higher sensitivity.

[0187] Specificity of the herein described assays was measured as the number of negative samples detected compared to the total number of true negatives in the given sample group. Accordingly, a lower number of false positives indicated a higher specificity.

[0188] Example 3: Assessment of specificity of iELISA assays using antigens A2 and C3 compared to iELISA assays using antigens A and C against negative Icelandic samples This Example illustrates assessment of specificity of iELISA assays using either antigen A2 (SEQ ID NO:3) or C3 (SEQ ID NO:5) and serum samples from Icelandic horses. For comparison, the old antigens, antigen A (SEQ ID NO:7) and C (SEQ ID NO:8) were also assessed. Assay results for the new and old individual antigens were also analyzed in combination, referred to herein as combined A2 and C3 dual antigen iELISA or combined A and C dual antigen iELISA, respectively. For the sake of clarity, these combined iELISA assays refer to testing of the samples in separate assays using the individual antigens and the analysis of the results in combination from both assays.

[0189] As discussed above, samples from Icelandic horses should test negative in an iELISA that is specific to 5. equi and the use of sera from Icelandic horses provides an overview of the specificity of a serology assay for 5. equi. Specifically, an increasing amount of false positives detected in the assay reflects a decreased specificity of the assay. Hence, the lower the number of false positives detected, the higher the specificity. Materials and methods

[0190] The coating of the iELISA plates with the antigens tested (either antigen A, C, A2 or C3), assay procedures and data handling were performed as described in Example 2.

[0191] Results and conclusions

[0192] Table 1 illustrates the individual absorbance values (A450) measured for the 200 Icelandic samples using iELISA plates coated with either antigen A, A2, C or C3. An absorbance value at or above the cut-off value 0.5 was defined as a sample detected as being positive for strangles (shown with highlight in light gray), i.e. the sample would be deemed to contain antibodies specific for S.equi based on the assay result. As explained above, strangles is not present in Iceland, and samples from Icelandic horses should test negative in an iELISA which is specific for 5. equi. For the sake of clarity, Icelandic horses neither classify as being exposed to 5. equi nor as being carriers - based on criteria for the clinical assessment of horses infected with 5. equi. Accordingly, Icelandic samples were considered as true negatives, and any positive test result was considered as a false positive.

[0193] Table 1. Normalised absorbance values (A450) measured for 200 Icelandic samples in iELISA assays using plates coated with any of the four antigens tested (antigen A2, C3, A or C). The gray fillings refer to samples identified as positive tests.

[0194]

[0195] The results are summarized in Table 2 below. The known antigen A assay identified

[0196] 26 (13.0%) negative Icelandic samples as being positive, resulting in a specificity of 87.0% for this assay. The known antigen C assay identified 1 (0.5%) negative Icelandic sample as being positive, resulting in a specificity of 99.5% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen i ELISA identified 26 (13.0%) Icelandic serum samples as being positive, resulting in a specificity of 87.0% for the dual antigen assay known in the prior art.

[0197] The new antigen A2 assay identified 5 (2.5%) negative Icelandic samples as being positive, resulting in a specificity of 97.5% for this assay. The new antigen C3 assay identified 7 (3.5%) negative Icelandic samples as being positive, resulting in a specificity of 96.5% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen i ELISA identified 11 (5.5%) negative Icelandic samples as being positive, resulting in a specificity of 94.5% for this new dual antigen assay.

[0198] Table 2. A summary table of the number of false positives identified by the iELISA assays for Icelandic samples and the corresponding specificities.

[0199] In conclusion, the antigen A2 assay showed higher specificity than the antigen A assay (P = 0.0001, Fisher's exact 2-tailed test), whereas the antigen C3 assay showed similar specificity to that of the assay using antigen C. When combining the data for the new A2 and C3 dual antigen iELISA, the specificity was significantly better than that is for the previous A and C dual antigen iELISA (P = 0.015, Fisher's exact 2-tailed test). Example 4: Assessment of sensitivity of iE LISA assays using antigens A2 and C3 compared to iELISA assays using antigens A and C against the Rossdales samples This Example illustrates assessment of sensitivity of iELISA assays using either antigen A2 or C3 and a total of 81 Rossdales samples from horses with strangles, including both carrier samples and acute disease samples. Out of 81 samples, 30 samples were acute disease samples and 51 samples were carrier samples. For comparison, the old antigens, antigen A and C were also assessed. Assay results for the new and old individual antigens were also analyzed in combination, referred to herein as combined A2 and C3 dual antigen iELISA or combined A and C dual antigen iELISA, respectively. For the sake of clarity, these combined iELISA assays refer to testing of the samples in separate assays using the individual antigens and the analysis of the results in combination from both assays.

[0200] In general, an increasing amount of false negative samples detected reflects a decreased sensitivity of the assay. Hence, the lower the number of false negative samples detected, the higher the sensitivity of the assay.

[0201] Materials and methods

[0202] The coating of the iELISA plates with the antigens tested (either antigen A, C, A2 or C3), assay procedures and data handling were performed as described in Example 2.

[0203] Results and conclusions

[0204] Table 3 illustrates the individual absorbance values (A450) measured for the 81 Rossdales samples using iELISA plates coated with either antigen A, A2, C or C3. An absorbance value at or above the cut-off value 0.5 is defined as a sample detected as being positive for strangles (shown with highlight in light gray), i.e. the sample would be deemed to contain antibodies specific for 5. equi based on the assay result. As explained above, each Rossdales sample was obtained from horses that have been exposed to 5. equi, and based on the clinical assessment of these animals, the samples were obtained from horses which were either in the early stages of disease (acute disease state) or which were with confirmed persistent infection (carriers). Accordingly, any negative test result was considered as a false negative. Table 3. Normalised absorbance values (A450) measured for 81 Rossdales samples in iELISA assays using plates coated with any of the four antigens tested (antigen A2, C3, A or C).

[0205]

[0206]

[0207]

[0208] The results are summarized in Table 4 below. The known antigen A assay identified 57 (70.4%) Rossdales samples as being positive, resulting in a sensitivity of 70.4% for this assay. The known antigen C assay identified 52 (64.2%) Rossdales samples as being positive, resulting in a sensitivity of 64.2% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen iELISA identified 61 (75.3%) Rossdales samples as being positive, resulting in a sensitivity of 75.3% for the dual antigen assay known in the prior art.

[0209] The new antigen A2 assay identified 48 (59.3%) Rossdales samples as being positive, resulting in a sensitivity of 59.3% for this assay. The new antigen C3 assay identified 56 (69.1%) Rossdales samples as being positive, resulting in a sensitivity of 69.1% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 63 (77.8%) Rossdales samples as being positive, resulting in a sensitivity of 77.8% for this new dual antigen assay.

[0210] Table 4. A summary table of the number of positives identified by the iELISA assays for Rossdales samples and the corresponding sensitivities. In conclusion, the i ELISA assay using antigen C3 showed higher sensitivity than the assay using antigen C (P = 0.62, Fisher's exact 2-tailed test). Furthermore, the new A2 and C3 dual antigen i ELI SA had better sensitivity for the identification of horses from the Rossdales samples with strangles compared to the previous A and C dual antigen i ELISA (P = 0.85, Fisher's exact 2-tailed test).

[0211] Example 5: Assessment of sensitivity of iE LISA assays using antigens A2 and C3 compared to i ELISA assays using antigens A and C against the Dartmoor samples This Example illustrates assessment of sensitivity of iE LISA assays using either antigen A2 or C3 and a total of 48 Dartmoor samples from horses with strangles, including both carrier samples and acute disease samples. Out of 48 samples, 24 samples were acute disease samples and 24 samples were carrier samples. For comparison, the old antigens, antigen A and C were also assessed. Assay results for the new and old individual antigens were also analyzed in combination, referred to herein as combined A2 and C3 dual antigen i ELISA or combined A and C dual antigen iE LISA, respectively. For the sake of clarity, these combined i ELI SA assays refer to testing of the samples in separate assays using the individual antigens and the analysis of the results in combination from both assays.

[0212] As mentioned in Example 4, an increasing amount of false negative samples detected reflects a decreased sensitivity of the assay. Hence, the lower the number of false negative samples detected, the higher the sensitivity of the assay.

[0213] Materials and methods

[0214] The coating of the iELISA plates with the antigens tested (either antigen A, C, A2 or C3), assay procedures and data handling were performed as described in Example 2. Results and conclusions

[0215] Table 5 illustrates the individual absorbance values (A450) measured for the 48 Dartmoor samples using i ELISA plates coated with either antigen A, A2, C or C3. An absorbance value at or above the cut-off value 0.5 is defined as a sample detected as being positive for strangles (shown with highlight in light gray), i.e. the sample would be deemed to contain antibodies specific for 5. equi based on the assay result. As explained above, each Dartmoor sample was obtained from horses that have been exposed to 5. equi, and based on the clinical assessment of these animals, the samples were obtained from horses which were either in the early stages of disease (acute disease state) or which were with confirmed persistent infection (carriers). Accordingly, any negative test result was considered as a false negative.

[0216] Table 5. Normalised absorbance values (A450) measured for 48 Dartmoor samples in iE LISA assays using plates coated with any of the four antigens tested (antigen A2, C3, A or C).

[0217]

[0218] The results are summarized in Table 6 below. The known antigen A assay identified 44 (91.7%) Dartmoor samples as being positive, resulting in a sensitivity of 91.7% for this assay. The known antigen C assay identified 40 (83.3%) Dartmoor samples as being positive, resulting in a sensitivity of 83.3% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen i ELISA identified 45 (93.8%) Dartmoor samples as being positive, resulting in a sensitivity of 93.8% for the dual antigen assay known in the prior art.

[0219] The new antigen A2 assay identified 40 (83.3%) Dartmoor samples as being positive, resulting in a sensitivity of 83.3% for this assay. The new antigen C3 assay identified 41 (85.4%) Dartmoor samples as being positive, resulting in a sensitivity of 85.4% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 45 (93.8%) Dartmoor samples as being positive, resulting in a sensitivity of 93.8% for this new dual antigen assay. Table 6. A summary table of the number of positives identified by the iELISA assays for Dartmoor samples and the corresponding sensitivities.

[0220] In conclusion, the iELISA assay using antigen C3 showed a similar sensitivity to that of the assay using antigen C. The new A2 and C3 dual antigen iELISA had the same sensitivity for the identification of horses from the Dartmoor samples with strangles compared to the previous A and C dual antigen iELISA assay. Example 6: Overall assessment of sensitivity of iE LISA assays using antigens A2 and C3 compared to i ELISA assays using antigens A and C against Rossdales and Dartmoor samples

[0221] This Example illustrates the overall assessment of sensitivity when assay results for all horses which has been exposed to 5. equi have been analyzed in combination. This includes results for samples from all horses, both in acute disease state and with persistent infection, and concerning both horse populations (Rossdales and Dartmoor), a total of 129 samples.

[0222] Materials and methods, as well as assay results are as explained in Examples 4 and 5, and as shown in Table 3 and Table 5.

[0223] Results and conclusions

[0224] The summary of analysis for all horses exposed to 5. equi is shown in Table 7. The known antigen A assay identified 101 (78.3%) samples as being positive, resulting in a sensitivity of 78.3% for this assay. The known antigen C assay identified 92 (71.3%) samples as being positive, resulting in a sensitivity of 71.3% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen i ELISA identified 106 (82.2%) samples as being positive, resulting in a sensitivity of 82.2% for the dual antigen assay known in the prior art.

[0225] The new antigen A2 assay identified 88 (68.2%) samples as being positive, resulting in a sensitivity of 68.2% for this assay. The new antigen C3 assay identified 97 (75.2%) samples as being positive, resulting in a sensitivity of 75.2% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 108 (83.7%) samples as being positive, resulting in a sensitivity of 83.7% for this new dual antigen assay.

[0226] Table 7. A summary table of the number of positives identified by the iELISA assays for Rossdales and Dartmoor samples and the corresponding sensitivities.

[0227] In conclusion, the iELISA assay using antigen C3 showed higher sensitivity for the overall horse population exposed to 5. equi than the assay using antigen C (P = 0.57, Fisher's exact 2-tailed test). Furthermore, the new A2 and C3 dual antigen iELISA showed a trend towards improved sensitivity for the identification of horses from the samples with strangles compared to the previous A and C dual antigen iELISA (P = 0.87, Fisher's exact 2-tailed test).

[0228] Example 7: Assessment of sensitivity of iELISA assays using antigens A2 and C3 compared to iELISA assays using antigens A and C against acute disease samples

[0229] This Example illustrates the assessment of sensitivity when assay results for horses which has been exposed to 5. equi and were in an acute disease state were analyzed. This includes results for acute disease samples from both horse populations (Rossdales and Dartmoor), a total of 54 samples. The analysis results are shown for the Rossdales and Dartmoor acute disease samples separately (30 and 24 samples, respectively) as well as in combination. Materials and methods, as well as assay results are as explained in Examples 4 and 5, and as shown in Table 3 and Table 5.

[0230] Results and conclusions

[0231] The results of the acute disease Rossdales samples are shown in Table 3, wherein the column for clinical results for the acute disease samples are given the value 0. The summary of analysis for this horse population is presented in Table 8 below. The known antigen A assay identified 22 (73.3%) acute disease samples as being positive, resulting in a sensitivity of 73.3% for this assay. The known antigen C assay identified 20 (66.7%) acute disease samples as being positive, resulting in a sensitivity of 66.7% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen iELISA identified 23 (76.7%) acute disease samples as being positive, resulting in a sensitivity of 76.7% for the dual antigen assay known in the prior art.

[0232] The new antigen A2 assay identified 18 (60%) acute disease samples as being positive, resulting in a sensitivity of 60% for this assay. The new antigen C3 assay identified 21 (70.0%) acute disease samples as being positive, resulting in a sensitivity of 70.0% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 24 (80%) acute disease samples as being positive, resulting in a sensitivity of 80% for this new dual antigen assay. Table 8. A summary table of the number of positives identified by the iELISA assays for acute disease Rossdales samples and the corresponding sensitities.

[0233] In conclusion, the iELISA assay using antigen C3 showed a higher sensitivity for the Rossdales horse population in the acute disease state compared to the assay using antigen C (P = 0.84, Fisher's exact 2-tailed test). Furthermore, the new A2 and C3 dual antigen iELISA showed a trend for improved sensitivity for the identification of acute disease Rossdales samples compared to the previous A and C dual antigen iELISA assay (P = 1.0, Fisher's exact 2-tailed test).

[0234] The results of the acute disease Dartmoor samples are shown in Table 5, wherein the column for clinical results for the acute disease samples are given the value 0. The summary of analysis for this horse population is presented in Table 9 below. The known antigen A assay identified 23 (95.8%) acute disease samples as being positive, resulting in a sensitivity of 95.8% for this assay. The known antigen C assay identified 20 (83.3%) acute disease samples as being positive, resulting in a sensitivity of 83.3% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen iELISA identified 23 (95.8%) acute disease samples as being positive, resulting in a sensitivity of 95.8% for the dual antigen assay known in the prior art.

[0235] The new antigen A2 assay identified 21 (87.5%) acute disease samples as being positive, resulting in a sensitivity of 87.5% for this assay. The new antigen C3 assay identified 21 (87.5%) acute disease samples as being positive, resulting in a sensitivity of 87.5% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 23 (95.8%) acute disease samples as being positive, resulting in a sensitivity of 95.8% for this new dual antigen assay.

[0236] Table 9. A summary table of the number of positives identified by the iELISA assays for acute disease Dartmoor samples and the corresponding sensitivities.

[0237] In conclusion, the iELISA assay using antigen C3 showed a higher sensitivity for the Dartmoor horse population in the acute disease state compared to the assay using antigen C (P = 1.0, Fisher's exact 2-tailed test). The new A2 and C3 dual antigen iELISA showed a similar sensitivity for the identification of acute disease Dartmoor samples compared to the previous A and C dual antigen iELISA assay (P = 1.0, Fisher's exact 2-tailed test).

[0238] The summary of analysis for all horses in the acute disease state is shown in Table 10. The known antigen A assay identified 45 (83.3%) acute disease samples as being positive, resulting in a sensitivity of 83.3% for this assay. The known antigen C assay identified 40 (74.1%) acute disease samples as being positive, resulting in a sensitivity of 74.1% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen i ELISA identified 46 (85.2%) acute disease samples as being positive, resulting in a sensitivity of 85.2% for the dual antigen assay known in the prior art.

[0239] The new antigen A2 assay identified 39 (72.2%) acute disease samples as being positive, resulting in a sensitivity of 72.2% for this assay. The new antigen C3 assay identified 42 (77.8%) acute disease samples as being positive, resulting in a sensitivity of 77.8% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen i ELISA identified 47 (87.0%) acute disease samples as being positive, resulting in a sensitivity of 87.0% for this new dual antigen assay.

[0240] Table 10. A summary table of the number of positives identified by the iELISA assays for acute disease Rossdales and Dartmoor samples and the corresponding sensitivities.

[0241] In conclusion, the iELISA assay using antigen C3 showed a higher sensitivity for the overall horse population in the acute disease state compared to the assay using antigen C (P = 0.82, Fisher's exact 2-tailed test). The new A2 and C3 dual antigen iELISA showed a trend towards better sensitivity for the identification of acute disease samples compared to the previous A and C dual antigen i ELISA assay (P = 1.0, Fisher's exact 2-tailed test).

[0242] Example 8: Assessment of sensitivity of i ELI SA assays using antigens Al and C3 compared to iELISA assays using antigens A and C against carrier samples

[0243] This Example illustrates the assessment of sensitivity when assay results for horses which has been exposed to 5. equi and were classified with persistent infection based on clinical assessment (i.e. carriers). This includes results for carrier samples from both horse populations (Rossdales and Dartmoor), a total of 75 samples. The analysis results are shown for the Rossdales and Dartmoor carrier samples separately (51 and 24 samples, respectively) as well as in combination.

[0244] Materials and methods, as well as assay results are as explained in Examples 4 and 5, and as shown in Table 3 and Table 5.

[0245] Results and conclusions

[0246] The results of the carrier Rossdales samples are shown in Table 3, wherein the column for clinical results for the carrier samples are given the value 1. The summary of analysis for this horse population is presented in Table 11 below. The known antigen A assay identified 35 (68.6%) carrier samples as being positive, resulting in a sensitivity of 68.6% for this assay. The known antigen C assay identified 32 (62.7%) carrier samples as being positive, resulting in a sensitivity of 62.7% for this assay.

[0247] The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen iELISA identified 38 (74.5%) carrier samples as being positive, resulting in a sensitivity of 74.5% for the dual antigen assay known in the prior art.

[0248] The new antigen A2 assay identified 30 (58.8%) carrier samples as being positive, resulting in a sensitivity of 58.8% for this assay. The new antigen C3 assay identified 35 (68.6%) carrier samples as being positive, resulting in a sensitivity of 68.6% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 39 (76.5%) acute disease samples as being positive, resulting in a sensitivity of 76.5% for this new dual antigen assay.

[0249] Table 11. A summary table of the number of positives identified by the iELISA assays for carrier Rossdales samples and the corresponding sensitivities.

[0250] In conclusion, the iELISA assay using antigen C3 showed higher sensitivity for the carrier Rossdales horse population compared to the assay using antigen C (P = 0.68, Fisher's exact 2-tailed test). Furthermore, the new A2 and C3 dual antigen iELISA showed a trend towards better sensitivity for the identification of carrier Rossdales samples compared to the previous A and C dual antigen iELISA assay (P = 1.0, Fisher's exact 2-tailed test).

[0251] The results of the carrier Dartmoor samples are shown in Table 5, wherein the column for clinical results for the carrier samples are given the value 1. The summary of analysis for this horse population is presented in Table 12 below. The known antigen A assay identified 21 (87.5%) carrier samples as being positive, resulting in a sensitivity of 87.5% for this assay. The known antigen C assay identified 20 (83.3%) carrier samples as being positive, resulting in a sensitivity of 83.3% for this assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen i ELISA identified 22 (91.7%) carrier samples as being positive, resulting in a sensitivity of 91.7% the dual antigen assay known in the prior art.

[0252] The new antigen A2 assay identified 19 (79.2%) carrier samples as being positive, resulting in a sensitivity of 79.2% for this assay. The new antigen C3 assay identified 20 (83.3%) carrier samples as being positive, resulting in a sensitivity of 83.3% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 22 (91.7%) carrier samples as being positive, resulting in a sensitivity of 91.7% for this new dual antigen assay.

[0253] Table 12. A summary table of the number of positives identified by the iELISA assays for carrier Dartmoor samples and the corresponding sensitivities.

[0254] In conclusion, the iELISA assay using antigen C3 showed a similar sensitivity for the carrier Dartmoor horse population compared to the assay using antigen C. Similarly, the new A2 and C3 dual antigen iELISA showed a similar sensitivity for the identification of carrier Dartmoor samples compared to the previous A and C dual antigen i ELISA assay (P=1.0, Fisher's exact 2-tailed test).

[0255] The summary of analysis for all carrier horses is shown in Table 13. The known antigen A assay identified 56 (74.7%) carrier samples as being positive, resulting in a sensitivity of 74.7% for this previous dual antigen assay. The known antigen C assay identified 52 (69.3%) carrier samples as being positive, resulting in a sensitivity of 69.3% for this previous dual antigen assay. The individual assay results were also analysed in combination for the two old antigens. Overall, the combined A and C dual antigen iELISA identified 60 (80.0%) carrier samples as being positive, resulting in a sensitivity of 80.0% the dual antigen assay known in the prior art.

[0256] The new antigen A2 assay identified 49 (65.3%) carrier samples as being positive, resulting in a sensitivity of 65.3% for this assay. The new antigen C3 assay identified 55 (73.3%) carrier samples as being positive, resulting in a sensitivity of 73.3% for this assay. The individual assay results were also analysed in combination for the two new antigens. Overall, the new combined A2 and C3 dual antigen iELISA identified 61 (81.3%) carrier samples as being positive, resulting in a sensitivity of 81.3% for this new dual antigen assay.

[0257] Table 13. A summary table of the number of positives identified by the iELISA assays for carrier Rossdales and Dartmoor samples and the corresponding sensitivities. In conclusion, the i ELISA assay using antigen C3 showed a higher sensitivity for the overall carrier horse population compared to the assay using antigen C (P = 0.65, Fisher's exact 2-tailed test). The new A2 and C3 dual antigen iELISA showed a trend towards higher sensitivity for the identification of carrier samples compared to the previous A and C dual antigen iELISA assay (P=0.83, Fisher's exact 2-tailed test).

[0258] Conclusive remarks for Examples 1-8

[0259] Maintaining very high levels of specificity of a serological assay is critical for screening horses for exposure to 5. equi in order to minimize the number of false positive horses that need to go under expensive follow-up investigations. As demonstrated in Example 3, the iELISA assay using the novel A2 antigen showed a higher specificity (97.5%) than the assay using the old antigen from Eq48, antigen A (87.0%), which was a highly significant improvement (P = 0.0001, Fisher's exact 2- tailed test). Thus, the A2 antigen for determining the presence or absence for antibodies specific for 5. equi in a serum sample is surprisingly beneficial for minimizing the detection of false positives in a sample cohort. The inventors has also found that the novel C3 antigen is useful in an iELISA with a similar specificity to an assay utilizing the old antigen, antigen C. In an overall conclusion, the new dual antigen A2 and C3 iELISA had a significantly better specificity of 94.5% than the previous A and C dual antigen iELISA (87.0%) (P = 0.015). These results indicate that serological assays using the novel antigens, and especially a dual assay using both of these, are particularly useful for determining with high specificity if a serum sample contains antibodies specific to 5. equi. It is of particular importance, as the closely related pathogen, 5. zooepidemicus, also infects horses and this may trigger false positive results in an assay with suboptimal specificity.

[0260] Beside specificity, it is also critical for a serological assay for outbreak prevention to identify horses for exposure to 5. equi with high sensitivity, i.e. that low levels of antibodies are also detected, for example at a very early stage of infection or at a dormant stage. It may be particularly challenging in certain horse populations (e.g. as observed for the Rossdales horses). As demonstrated in Examples 4-8, the novel C3 antigen appears to be especially advantageous to minimize the number of false negative results in all sample cohorts. Except of the case of Dartmoor carrier samples, i ELISA assays using the C3 antigen showed a trend towards improved sensitivity in all sample cohorts in comparison to the assay utilizing the old antigen C. In case of the Dartmoor carrier samples, the sensitivity of these two assays was comparable (Table 14). Moreover, the combined sensitivity of the two assays for acute cases was slightly improved in the new dual antigen A2 and C3 i ELI SA 87.0% cf. 85.2% (P = 1.0, Fisher's exact 2-tailed test)). The sensitivity for the identification of persistently infected carriers was also slightly improved in the new dual antigen A2 and C3 i ELISA (81.3%) compared with the previous A and C dual antigen iELISA (80.0%, P = 1.0). Notably, assays using the novel C3 antigen, and in particular the new dual antigen A2 and C3 iELISA, were surprisingly beneficial in sample cohorts of the challenging Rossdales samples and acute disease samples from horses in the early stages of disease before they had sufficient time to develop a strong antibody response.

[0261] Table 14. Summarizing comparison of iELISA sensitivity (%) in different sample cohorts - using antigen C vs. C3 or dual antigens C and A vs. C3 and A2. Taken together, the novel C3 antigen, and especially in combination with the novel A2 antigen, is particularly useful for determining with high sensitivity if a serum sample contains antibodies specific to 5. equi. This is of high importance, as false negative assay results may hinder efficient outbreak prevention. In particular, the antigen A2 and C3 dual antigen iELISA offers the ability to detect horses that have been exposed to, or are persistently infected with 5. equi, with high sensitivity whilst identifying significantly fewer false positive horses, providing a significant advantage in the management of this endemic and important disease of horses.

[0262] Hypothetical example 9

[0263] Analysis according to Examples 1-8 will be performed for antigen C4 (SEQ ID NO: 6), using the same methods as described above. Results are expected to be similar as to the C3 fragment in all of Examples 1-8.

[0264] Itemized list of embodiments

[0265] 1. A polypeptide fragment selected from the group consisting of a polypeptide A fragment of Eq48, and a polypeptide C fragment of Eq39; wherein said polypeptide A fragment comprises an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2; said polypeptide A fragment has a maximum length of about 200 amino acids and has a maximum C-terminal extension of aboutl29 amino acids , and wherein said polypeptide A fragment has at least 85% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment; and wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:6, and wherein said polypeptide C fragment has at least 85% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment.

[0266] 2. The polypeptide fragment according to item 1, wherein said polypeptide fragment is the polypeptide A fragment.

[0267] 3. The polypeptide fragment according to item 1, wherein said polypeptide fragment is the polypeptide C fragment.

[0268] 4. The polypeptide fragment according to item 1 or 3, wherein said polypeptide C fragment has a maximum length of about 250 amino acids.

[0269] 5. The polypeptide fragment according to any one of items 1-4, wherein said Eq48 comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:1 (Eq48) and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:1 (Eq48), such as wherein said Eq48 comprises an amino acid sequence as shown in SEQ ID NO:1 (Eq48).

[0270] 6. The polypeptide fragment according to any one of items 1-5, wherein said Eq39 comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:4 (Eq39) and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:4 (Eq39), such as wherein said Eq39 comprises an amino acid sequence as shown in SEQ ID NO:4 (Eq39).

[0271] 7. The polypeptide fragment according to any one of items 1, 2 and 5-6, wherein said polypeptide A fragment has a maximum length of about 195 amino acids, such as about 190 amino acids, such as about 185 amino acids, such as about 180 amino acids, such as about 175 amino acids, such as about 170 amino acids, such as about 165 amino acids, such as about 160 amino acids, such as about 155 amino acids, such as about 150 amino acids, such as about 145 amino acids, such as about 142 amino acids, such as about 140 amino acids, such as about 135 amino acids, such as about 130 amino acids, such as about 125 amino acids, such as about 120 amino acids, such as about 115 amino acids, such as about 110 amino acids, such as about 105 amino acids, such as about 100 amino acids, such as about 95 amino acids, such as about 90 amino acids, such as about 85 amino acids, such as about 80 amino acids, such as about 75 amino acids, such as about 71 amino acids.

[0272] 8. The polypeptide fragment according to any one of items 1, 2 and 5-7, wherein said polypeptide A fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:lover the length of said polypeptide A fragment.

[0273] 9. The polypeptide fragment according to any one of items 1, 2 and 5-8, wherein said polypeptide A fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:2 and 3 and any amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:2 and 3. 10. The polypeptide fragment according to item 9, wherein said polypeptide A fragment comprises an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NO:2 and 3, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to any one of SEQ ID NO:2 and 3.

[0274] 11. The polypeptide fragment according to any one of items 1, 2 and 5-10, wherein said polypeptide A fragment comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:3 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:3; or wherein polypeptide A fragment comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:2 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:2.

[0275] 12. The polypeptide fragment according to item 11, wherein said polypeptide A fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:3, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:3; or wherein said polypeptide A fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:2, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:2.

[0276] 13. The polypeptide fragment according to any one of items 1, 2 and 5-12, wherein said polypeptide A fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:2 and 3.

[0277] 14. The polypeptide fragment according to any one of items 1, 2 and 5-13, wherein said polypeptide A fragment comprises an amino acid sequence as shown in SEQ ID NO:2.

[0278] 15. The polypeptide fragment according to any one of items 1, 2 and 5-14, wherein said polypeptide A fragment comprises an amino acid sequence as shown in SEQ ID NO:3.

[0279] 16. The polypeptide fragment according to any one of items 1, 2 and 5-15, wherein said polypeptide A fragment does not comprise an amino acid sequence from amino acid position 252 to amino acid position 673, such as from amino acid position 250 to amino acid position 673, such as from amino acid position 245 to amino acid position 673, such as from amino acid position 240 to amino acid position amino acid position 673, such as from amino acid position 235 to amino acid position 673, such as from amino acid position 230 to amino acid position 673, such as from amino acid position 225 to amino acid position 673, such as from amino acid position 220 to amino acid position 673, such as from amino acid position 215 to amino acid position 673, such as from amino acid position 210 to amino acid position 673, such as from amino acid position 205 to amino acid position 673, such as from amino acid position 200 to amino acid position 673, such as from amino acid position 195 to amino acid position 673, such as from amino acid position 190 to amino acid position 673, such as from amino acid position 189 to amino acid position 673, of SEQ ID NO:1, or fragments thereof.

[0280] 17. The polypeptide fragment according to any one of items 1, 2 and 5-16, wherein said polypeptide A fragment does not comprise an amino acid sequence as shown in SEQ ID NO:7. 18. The polypeptide fragment according to any one of items 1 and 3-6, wherein polypeptide C fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment.

[0281] 19. The polypeptide fragment according to any one of items 1, 3-6 and 18, wherein said polypeptide C fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:5 and 6 and any amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:5 and 6.

[0282] 20. The polypeptide fragment according to item 19, wherein said polypeptide C fragment comprises an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NO:5 and 6, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to any one of SEQ ID NO:5 and 6.

[0283] 21. The polypeptide fragment according to any one of items 1, 3-6 and 18-20, wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:5 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:5; or wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:6 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:6. 22. The polypeptide fragment according to item 21, wherein said polypeptide C fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:5 (C3), such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:5 (C3); or wherein said polypeptide C fragment comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:6, such as 87% sequence identity, such as 88% sequence identity, such as 89% sequence identity, such as 90% sequence identity, such as 91% sequence identity, such as 92% sequence identity, such as 93% sequence identity, such as 94% sequence identity, such as 95% sequence identity, such as 96% sequence identity, such as 97% sequence identity, such as 98% sequence identity, such as 99% sequence identity to SEQ ID NO:6.

[0284] 23. The polypeptide fragment according to any one of items 1, 3-6 and 18-22, wherein said polypeptide C fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:5 and 6.

[0285] 24. The polypeptide fragment according to any one of items 1, 3-6 and 18-23, wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:6.

[0286] 25. The polypeptide fragment according to any one of items 1, 3-6 and 18-24, wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:5.

[0287] 26. The polypeptide fragment according to any one of items 1-25, wherein said polypeptide fragment is linked at the N-terminal thereof to an amino acid sequence, wherein said amino acid sequence comprises or consists of about 1 to about 60, such as about 1 to about 55, such as about 1 to about 50, such as about 1 to about 45, such as about 1 to about 40, such as about 1 to about 35, such as about 1 to about 30, such as about 1 to about 25, such as about 1 to about 20, such as about 1 to about 15, such us about 1 to about 10, such as about 1 to about 5 amino acids.

[0288] 27. The polypeptide fragment according to any one of items 1-25, wherein said polypeptide fragment is linked at the C-terminal thereof to an amino acid sequence, wherein said amino acid sequence comprises or consists of about 1 to about 60, such as about 1 to about 55, such as about 1 to about 50, such as about 1 to about 45, such as about 1 to about 40, such as about 1 to about 35, such as about 1 to about 30, such as about 1 to about 25, such as about 1 to about 20, such as about 1 to about 15, such us about 1 to about 10, such as about 1 to about 5 amino acids.

[0289] 28. The polypeptide fragment according to any one of items 1, 2, 5-17, 26 and 27, wherein said polypeptide A fragment further comprises the amino acid sequence from amino acid position 1 to amino acid position 46 of SEQ ID NO:1, or a fragment thereof.

[0290] 29. The polypeptide fragment according to any one of items 1, 3-6 and 18-27, wherein said polypeptide C fragment further comprises the amino acid sequence from amino acid position 1 to amino acid position 37 of SEQ ID NO:4 , or a fragment thereof.

[0291] 30. The polypeptide fragment according to any one of items 1, 3-6, 18-27 and 29, wherein said polypeptide C fragment has a maximum length of about 185 amino acids, such as about 180 amino acids, such as about 175 amino acids, such as about 170 amino acids, such as about 165 amino acids, such as about 160 amino acids, such as about 155 amino acids, such as about 150 amino acids, such as about 145 amino acids, such as about 142 amino acids, such as about 140 amino acids, such as about 135 amino acids, such as about 130 amino acids, such as about 125 amino acids, such as about 120 amino acids, such as about 115 amino acids, such as about 110 amino acids, such as about 106 amino acids.

[0292] 31. The polypeptide fragment according to any one of items 1-30, wherein said polypeptide fragment is in a form of an isolated fragment or a fragment of a fusion protein. 32. The polypeptide fragment according to item 31, wherein said fusion protein comprises both the polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26 and 27-28, and the polypeptide C fragment as defined in any one of itemsl, 3-6, 18-27 and 29-30.

[0293] 33. The polypeptide fragment according to item 31, wherein said polypeptide fragment is in a form of an isolated fragment.

[0294] 34. The polypeptide fragment according to any one of items 1-33, wherein said polypeptide fragment is an antigen.

[0295] 35. A combination of polypeptide fragments comprising a polypeptide A fragment and a polypeptide C fragment, wherein said polypeptide A fragment is as defined in any one of items 1, 2, 5-17, 26-28 and 31-34, and said polypeptide C fragment is as defined in any one of items 1, 3-6, 18-24, 26, 27 and 29-34.

[0296] 36. Use of the polypeptide fragment as defined in any one of items 1-34 or of the combination of polypeptide fragments as defined in item 35 for the detection of antibodies indicative of exposure of a subject to Streptococcus equi subsp. equi; and / or indicative of an infection caused by Streptococcus equi subsp. equi in a subject.

[0297] 37. A method for determining the presence or the absence of an antibody in a sample, which antibody is capable of binding to a polypeptide fragment as defined in any one of items 1-34, said method comprising the steps of:

[0298] (i) bringing said sample into contact with said polypeptide fragment under conditions permissive of binding of said antibody to said polypeptide fragment, such as bringing said sample into contact with a polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26-28 and 31-34, and / or a polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34; and

[0299] (ii) detecting binding of said antibody to said polypeptide fragment, wherein said binding is indicative of the presence of said antibody in said sample. 38. The method for determining according to item 37, wherein said antibody is produced in response to exposure to Streptococcus equi subsp. equi in a subject from which said sample is obtained, or wherein said antibody is produced in response to an active or a dormant Streptococcus equi subsp. equi infection.

[0300] 39. The method for determining according to item 37 or 38, wherein said method further comprises a step of quantifying the amount of said antibody in said sample.

[0301] 40. The method for determining according to item 39, wherein said step of quantifying is an absolute quantification or a relative quantification.

[0302] 41. The method for determining according to any one of items 37-40, wherein the presence or the absence of said antibody is determined relative to a control sample, such as a control sample which does not comprise any antibody capable of binding to said polypeptide fragment.

[0303] 42. The method for determining according to any one of items 37-41, wherein said method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA.

[0304] 43. The method for determining according to any one of items 37-42, wherein said method comprises direct or indirect coupling of said polypeptide fragment to a solid support.

[0305] 44. The method for determining according to item 43, wherein said direct or indirect coupling is performed prior to step (i).

[0306] 45. The method for determining according to item 43 or 44, wherein the concentration of said polypeptide fragment at said direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0307] 46. The method for determining according to any one of items 37-45, wherein said binding is detected using a colorimetric method or a fluorescence method. 47. The method for determining according to any one of items 37-46, wherein the presence or the absence of said antibody is determined based on an optical density of the sample.

[0308] 48. The method for determining according to item 47, wherein said optical density is measured at 450 nm.

[0309] 49. The method for determining according to item 47 or 48, wherein an optical density value equal to or higher than 0.5 for said sample is indicative of the presence of said antibody in the sample.

[0310] 50. A method for detecting exposure to Streptococcus equi subsp. equi in a subject, said method comprising the steps of:

[0311] (i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in any one of items 1-34,

[0312] (ii) determining the presence or the absence of one or more antibodies in said sample, wherein said one or more antibodies are capable of binding to said at least one polypeptide fragment; wherein said step (i) and / or step (ii) is performed under conditions permissive of binding of said one or more antibodies to said at least one polypeptide fragment; and wherein the presence of said one or more antibodies in the sample is indicative of previous or present exposure to said Streptococcus equi subsp. equi.

[0313] 51. The method for detecting exposure according to item 50, wherein said method in step (i) comprises bringing a sample obtained from said subject into contact with said polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26-28 and 31- 34, and said polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34.

[0314] 52. The method for detecting exposure according to item 51, wherein said sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment simultaneously or subsequently. 53. The method for detecting exposure according to item 51 or 52, wherein said sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment in the same single assay or in two separate assays.

[0315] 54. The method for detecting exposure according to any one of items 50-53, wherein said exposure to 5. equi is a transient, an active or a dormant infection.

[0316] 55. The method for detecting exposure according to any one of items 50-54, wherein step (ii) comprises detecting the binding of said one or more antibodies to said at least one polypeptide fragment.

[0317] 56. The method for detecting exposure according to item 55, wherein said binding is detected using a colorimetric method or a fluorescence method.

[0318] 57. The method for detecting exposure according to any one of items 50-56, wherein said method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA.

[0319] 58. The method for detecting exposure according to any one of items 50-57, wherein said method further comprises direct or indirect coupling of said at least one polypeptide fragment to a solid support.

[0320] 59. The method for detecting exposure according to item 58, wherein said direct or indirect coupling is performed prior to step (i).

[0321] 60. The method according to item 58 or 59, wherein the concentration of said at least one polypeptide fragment at said direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0322] 61. The method for detecting exposure according to any one of items 50-60, wherein said at least one polypeptide fragment in step (i) is directly or indirectly coupled to a solid support, such as wherein both said polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26-28 and 31-34, and said polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34 are directly or indirectly coupled to a solid support.

[0323] 62. The method for detecting exposure according to any one of items 50-61, further comprising a step of (iii) quantifying the amount of said one or more antibodies in said sample.

[0324] 63. The method for detecting exposure according to item 62, wherein said step of quantifying is an absolute quantification or a relative quantification.

[0325] 64. The method for detecting exposure according to any one of items 50-63, wherein the presence or the absence of said antibody is determined relative to a control sample, such as a control sample which does not comprise any antibody capable of binding to said at least one polypeptide fragment.

[0326] 65. The method for detecting exposure according to any one of items 50-64, wherein the presence or the absence of said one or more antibodies is determined based on an optical density of the sample.

[0327] 66. The method for detecting exposure according to item 65, wherein said optical density is measured at 450 nm.

[0328] 67. The method for detecting exposure according to item 65 or 66, wherein an optical density value equal to or higher than 0.5 for said sample is indicative of the presence of said one or more antibodies in the sample.

[0329] 68. A method for diagnosis of an infection caused by Streptococcus equi subsp. equi in a subject, said method comprising the steps of

[0330] (i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in any one of items 1-34,

[0331] (ii) determining the presence or the absence of one or more antibodies in said sample, wherein said one or more antibodies are capable of binding to said at least one polypeptide fragment; wherein said step (i) and / or step (ii) is performed under conditions permissive of binding of said one or more antibodies to said at least one polypeptide fragment; and wherein the presence of said one or more antibodies in the sample is indicative of said infection.

[0332] 69. The method for diagnosis according to item 68, wherein said method in step (i) comprises bringing a sample obtained from said subject into contact with said polypeptide A fragment as defined in any one of items 1, 3, 5-15, 24, 26-28 and 31- 34, and said polypeptide C fragment as defined in any one of items 1, 2, 4, 16-23, 25- 27 and 29-34.

[0333] 70. The method for diagnosis according to item 69, wherein said sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment simultaneously or subsequently.

[0334] 71. The method for diagnosis according to item 69 or 70, wherein said sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment in the same single assay or in two separate assays.

[0335] 72. The method for diagnosis according to any one of items 68-71, wherein said infection is a transient, an active or a dormant infection.

[0336] 73. The method for diagnosis according to any one of items 68-72, wherein step (ii) comprises detecting the binding of said one or more antibodies to said at least one polypeptide fragment.

[0337] 74. The method for diagnosis according to item 73, wherein said binding is detected using a colorimetric method or a fluorescence method.

[0338] 75. The method for diagnosis according to any one of items 68-74, wherein said method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. 76. The method for diagnosis according to any one of items 68-75, wherein said method further comprises direct or indirect coupling of said at least one polypeptide fragment to a solid support.

[0339] 77. The method for diagnosis according to item 76, wherein said direct or indirect coupling is performed prior to step (i).

[0340] 78. The method for diagnosis according to item 76 or 77, wherein the concentration of said at least one polypeptide fragment at said direct or indirect coupling is from about 0.1 pg / mL to about 1 pg / mL, such as from about 0.2 pg / mL to about 1 pg / mL, such as from about 0.3 pg / mL to about 1 pg / mL, such as from about 0.4 pg / mL to about 0.9 pg / mL, such as from about 0.5 pg / mL to about 0.8 pg / mL.

[0341] 79. The method for diagnosis according to any one of items 68-78, wherein said at least one polypeptide fragment in step (i) is directly or indirectly coupled to a solid support.

[0342] 80. The method for diagnosis according to any one of items 68-79, further comprising a step of (iii) quantifying the amount of said one or more antibodies in said sample.

[0343] 81. The method for diagnosis according to item 80, wherein said step of quantifying is an absolute quantification or a relative quantification.

[0344] 82. The method for diagnosis according to any one of items 68-81, wherein the presence or the absence of said antibody is determined relative to a control sample, such as a control sample which does not comprise any antibody capable of binding to said at least one polypeptide fragment.

[0345] 83. The method for diagnosis according to any one of items 68-82, wherein the presence or the absence of said one or more antibodies is determined based on an optical density of the sample.

[0346] 84. The method for diagnosis according to item 83, wherein said optical density is measured at 450 nm. 85. The method for diagnosis according to item 83 or 84, wherein an optical density value equal to or higher than 0.5 for said sample is indicative of the presence of said one or more antibodies in the sample.

[0347] 86. A solid support comprising at least one polypeptide fragment as defined in any one of the items 1-34 or a combination of fragments as defined in item 35..

[0348] 87. The solid support according to item 86, wherein said solid support is a microtiter plate.

[0349] 88. The solid support according to item 86 or 87, wherein said at least one polypeptide fragment is directly or indirectly coupled to the solid support.

[0350] 89. The solid support according to any one of items 86-88, wherein the solid support comprises said polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26- 28 and 31-34, and said polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34.

[0351] 90. The solid support according to item 89, wherein said polypeptide A fragment and said polypeptide C fragment are directly or indirectly coupled to the solid support.

[0352] 91. A kit for carrying out a method for determining as defined in any one of items 36-49 and / or in a method detecting exposure as defined in any one of items 50-67, and / or in a method for diagnosis as defined in any one of items 68-85; comprising a polypeptide fragment as defined in any one of the items 1-34 or a combination as defined in item 35 and instructions for carrying out the method.

[0353] 92. The kit according to item 91, wherein said kit comprises the solid support as defined in any one of items 86-90.

[0354] 93. The kit according to item 91 or 92, wherein said kit comprises said polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26-28 and 31-34, and said polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34.

[0355] 94. The kit according to item 93, wherein said polypeptide A fragment and said polypeptide C fragment are directly or indirectly coupled to two separate solid supports. 95. The kit according to any one of items 91 to 94, wherein said kit further comprises one or more reagents required for a conventional ELISA method.

[0356] 96. Use of a solid support as defined in any one of items 86-90 or a kit as defined in any one of items 91-95 in a method for determining as defined in any one of items 37-49 and / or in a method detecting exposure as defined in any one of items 50-67, and / or in a method for diagnosis as defined in any one of items 68-85.

[0357] 97. A fusion polypeptide comprising a polypeptide A fragment as defined in any one of items 1, 2, 5-17, 26-28 and 31-34, and a polypeptide C fragment as defined in any one of items 1, 3-6, 18-27 and 29-34.

[0358] 98. A polynucleotide encoding a polypeptide fragment according to any one of the items 1-34 or a fusion polypeptide according to item 97.

[0359] 99. The polynucleotide according to item 98, wherein said polynucleotide encodes a fusion protein comprising said polypeptide A fragment and said polypeptide C fragment as defined in item 32.

[0360] 100. An expression vector comprising a polynucleotide according to any one of items 97 or 99.

[0361] 101. A cloning vector comprising a polynucleotide according to to any one of items 97 or 99.

[0362] 102. A host cell comprising an expression vector according to item 100.

[0363] 103. A host cell comprising a cloning vector according to according to item 101.

[0364] 104. A method of producing a polypeptide fragment according to any one of items 1- 34 comprising culturing a host cell according to item 102 under conditions permissive of expression of said polypeptide from said expression vector, and isolating said polypeptide.

[0365] 105. A composition comprising at least one polypeptide fragment according to any one of items 1-34 and optionally an excipient and / or adjuvant.

[0366] 106. A composition according item 105 for use in a method for determining as defined in any one of items 37-49 and / or in a method detecting exposure as defined in any one of items 50-67, and / or in a method for diagnosis as defined in any one of items 68-85.

[0367] 107. A method for direct or indirect coupling of a polypeptide fragment as defined in any one of items 1-34, said method comprising the steps of

[0368] (i) bringing a solid support as defined in any one of items 86-90 into contact with said polypeptide fragment under conditions permissive of binding of said polypeptide fragment to said solid support, thereby obtaining said polypeptide fragment coupled to said solid support.

[0369] 108. A method of treating a Streptococcus equi infection, such as a Streptococcus equi subsp. equi infection, the method comprising

[0370] (i) identifying a subject suspected to carry an antibody, which antibody is capable of binding to a polypeptide fragment as defined in any one of items 1-34;

[0371] (ii) providing a sample from said subject;

[0372] (iii) testing said sample in a method according to any one of items 37-85;

[0373] (iii) determining that said subject carries said antibody; and

[0374] (iv) administrating a therapeutic agent suitable for treatment of said Streptococcus equi infection.

[0375] 109. The method according to item 108, wherein said therapeutic agent is a prophylactic agent, such as a vaccine.

[0376] 110. The method according to item 108 or 109, wherein said therapeutic agent comprises at least one, such as at least two or all three, polypeptide(s) selected from the group consisting of a polypeptide comprising the amino acid sequence according to SEQ ID NO:9 starting from position 12, a polypeptide comprising the amino acid sequence according to SEQ ID NO:10 starting from position 12 and a polypeptide comprising the amino acid sequence according to SEQ ID NO:11 starting from position 12.

[0377] 111. The method according to any one of items 108-110, wherein said therapeutic agent is Strangvac®.

Claims

93Claims1. A polypeptide fragment selected from the group consisting of a polypeptide A fragment of Eq48, and a polypeptide C fragment of Eq39; wherein said polypeptide A fragment comprises an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2; said polypeptide A fragment has a maximum length of about 200 amino acids and has a maximum C-terminal extension of about 129 amino acids , and wherein said polypeptide A fragment has at least 85% sequence identity to SEQ ID NO:1 over the length of said polypeptide A fragment; and wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:6, and wherein said polypeptide C fragment has at least 85% sequence identity to SEQ ID NO:4 over the length of said polypeptide C fragment.

2. The polypeptide fragment according to claim 1, wherein said polypeptide fragment is the polypeptide A fragment.

3. The polypeptide fragment according to claim 1, wherein said polypeptide fragment is the polypeptide C fragment.

4. The polypeptide fragment according to claim 1 or 3, wherein said polypeptide C fragment has a maximum length of about 250 amino acids.

5. The polypeptide fragment according to any one of claims 1 and 2, wherein said polypeptide A fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:2 and 3 and any amino94 acid sequence having at least 85% sequence identity to any one of SEQ ID NO:2 and 3.

6. The polypeptide fragment according to any one of claims 1, 2 and 5, wherein said polypeptide A fragment comprises an amino acid sequence selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:3 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:3.

7. The polypeptide fragment according to any one of claims 1 and 3-4, wherein said polypeptide C fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences as shown in SEQ ID NO:5 and 6 and any amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO:5 and6.

8. The polypeptide fragment according to any one of claims 1, 3-4 and 7 wherein said polypeptide C fragment comprises an amino acid sequence as shown in SEQ ID NO:5 and any amino acid sequence having at least 85% sequence identity to SEQ ID NO:5.

9. A combination of polypeptide fragments comprising a polypeptide A fragment and a polypeptide C fragment, wherein said polypeptide A fragment is as defined in any one claims 1, 2 and 5-6, and said polypeptide C fragment is as defined in any one of claims 1, 3-4 and 7-8.

10. A method for determining the presence or the absence of an antibody in a sample, which antibody is capable of binding to a polypeptide fragment as defined in any one of claims 1-8, said method comprising the steps of:(i) bringing said sample into contact with said polypeptide fragment under conditions permissive of binding of said antibody to said polypeptide fragment;95 and(ii) detecting binding of said antibody to said polypeptide fragment, wherein said binding is indicative of the presence of said antibody in said sample.

11. A method for detecting exposure to Streptococcus equi subsp. equi in a subject, said method comprising the steps of:(i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in any one of claims 1-8,(ii) determining the presence or the absence of one or more antibodies in said sample, wherein said one or more antibodies are capable of binding to said at least one polypeptide fragment; wherein said step (i) and / or step (ii) is performed under conditions permissive of binding of said one or more antibodies to said at least one polypeptide fragment; and wherein the presence of said one or more antibodies in the sample is indicative of previous or present exposure to said Streptococcus equi subsp. equi.

12. A method for diagnosis of an infection caused by Streptococcus equi subsp. equi in a subject, said method comprising the steps of(i) bringing a sample obtained from said subject into contact with at least one polypeptide fragment as defined in any one of claims 1-8,(ii) determining the presence or the absence of one or more antibodies in said sample, wherein said one or more antibodies are capable of binding to said at least one polypeptide fragment; wherein said step (i) and / or step (ii) is performed under conditions permissive of binding of said one or more antibodies to said at least one polypeptide fragment; and96 wherein the presence of said one or more antibodies in the sample is indicative of said infection.

13. The method for detecting exposure to Streptococcus equi subsp. equi according to claim 11 or the method for diagnosis of an infection caused by Streptococcus equi subsp. equi according to claim 12, wherein said sample is brought into contact with said polypeptide A fragment and said polypeptide C fragment simultaneously or subsequently.

14. The method for determining presence or absence of an antibody in a sample according to claim 10; the method for detecting exposure to Streptococcus equi subsp. equi according to claim 11 or 13; or the method for diagnosis of an infection caused by Streptococcus equi subsp. equi according to claim 12 or 13, wherein said method is an ELISA method, such as an ELISA method selected from the group consisting of direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA.

15. A solid support comprising at least one polypeptide fragment as defined in any one of the claims 1-8 or a combination of fragments as defined in claim 9.

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