Molecular probes for the diagnosis of Lyme borreliosis

Abstract

Molecular probes for the diagnosis of Lyme borreliosis The invention relates to an aptamer which binds to the CspZ protein present on the surface of bacteria of the genus Borrelia and their use in an in vitro method for diagnosing an infection by a bacterium of the genus Borrelia in a subject.

Description

Title of the invention: Molecular probes for the diagnosis of Lyme borreliosis. Technical field

[0001] The invention relates to an aptamer that binds to the CspZ protein present on the surface of bacteria of the genus Borrelia and their use in an in vitro method for diagnosing an infection by a bacterium of the genus Borrelia in a subject. Previous technique

[0002] Lyme disease, commonly known as Lyme borreliosis (LB), was first discovered in 1975. The most common tick-borne disease in France, Europe, and the world, with approximately 700,000 estimated annual cases (USA / Europe), BL is transmitted by ticks of the genus Ixodes. The causative agent of BL is a group of bacteria belonging to the genus Borrelia (B.) and collectively referred to as Borrelia burgdorferi sensu lato (Bbsl) (Stanek et al. 2012; Stanek and Strie 2018).

[0003] The genus Borrelia, belonging to the phylum Spirochaetes, is characterized by three distinct groups (Trevisan et al. 2021a), including the Lyme Borrelia (LBB) group. Lyme disease is characterized by three stages of progression: (i) early localized, (ii) early disseminated, and (iii) late disseminated, with evolving symptoms. In the first stage of Lyme disease, erythema migrans (EM), near the tick bite site, is a pathognomonic cutaneous manifestation that allows for the clinical diagnosis of the disease. It is present in the majority of cases (Stanek and Strie 2018). Such a symptom necessitates the specific and rapid diagnosis of Lyme disease, and therefore the initiation of antibiotic therapy, which resolves 80 to 90% of Borrelia infection cases (Cabello et al. 2022). However, ME is not systematically observed / detected and, in the absence of appropriate antibiotic treatment (Trevisan et al.2022b), non-characteristic symptoms related to stages 2 and 3 may be expressed, including cases of neuroborreliosis, carditis or arthritis).

[0004] Currently, the two-part serological test, whether standard or modified, is the official recommendation for the diagnosis of BL (Miraglia 2016). Generally speaking, serology is based on the detection of immunoglobulins produced by the host's immune response following an infection. However, this approach has many limitations. First, due to the latency period of the immune response after a bacterial or viral infection, the early detection of BL, particularly during its acute phase, proves to be of very limited effectiveness, as the diagnosis rarely exceeds a sensitivity of 50% (Marques 2015). Furthermore, after treatment of an active infection, immunoglobulins can still be detected in the serum for months or even years after the infection (Feder et al. 1992). This last point makes serology unsuitable for monitoring the response to antibiotic treatment or for identifying new infections.

[0005] Since 1994, CDC (Centers for Disease Control and Prevention) guidelines and recommendations have included standard two-tiered testing (STTT) to maintain high sensitivity and optimize specificity. First, an ELISA test is performed. If the result is borderline or positive, then a confirmatory Western blot test is recommended (Centers for Disease Control and Prevention (CDC) 1995).

[0006] However, sensitivity and specificity remain major problems for these techniques (Hoeve-Bakker et al. 2022; Kobayashi and Auwaerter 2022). Indeed, in addition to the risk of false negative results, these techniques generate numerous false positives (Grqzlewska and Holec-Gqsior 2023). A recent study showed that false positives lead to inappropriate treatment and therapeutic uncertainty (Wojciechowska-Koszko et al. 2022).

[0007] The diagnostic challenge is particularly hampered by the complexity of the Borrelia genus and the BGL group. Indeed, these species exhibit numerous disparities in clinical manifestations, dissemination within the human body, the expression of their genome(s) / antigens, and their geographic distribution (Trevisan et al. 2021b; Trevisan et al. 2022a). Their great diversity worldwide thus leads to regional disparities in diagnosis (Steere et al. 2016). Even though the 910-kilobase (kb) linear chromosome has been shown to be highly conserved in the BLG genome, the entire plasmid set (9 linear and 12 circular plasmids) (Casjens et al. 2000) shows a high degree of variation (lyer et al. 2013; Schüler et al. 2015). These variations, which are the source of great complexity within Borrelia species (Hanincova et al. 2013; Cerar et al. 2016; Pearson et al.2022), also highlight the need to improve the diagnostic arsenal capable of rapidly detecting all pathogens responsible for BL and any co-infections that may be present in order to avoid any therapeutic errors.

[0008] Many innovations and improvements in diagnostics have been proposed recently but have not yet proven to be totally satisfactory.

[0009] It is in this context that the inventors developed aptamers that bind to the CspZ protein, also called Borrelia burgdorferi Complement regulator-acquiring surface protein-2 (BbCRASP-2), which can be used in the diagnosis of BL, particularly at an early stage of BL. The CspZ protein is a protein present on the surface of Borrelia strains and which is expressed particularly at an early stage of the disease. Summary of the invention

[0010] According to a first aspect, the invention relates to an aptamer that binds to CspZ, said aptamer comprises a nucleotide sequence SEQ ID NO: 1 of formula (I) or a nucleotide sequence having at least 90% identity with the nucleotide sequence of formula (I): 5'-TGGCAATGGN1GGGGTGGTN2GGAGGGGGN3N4N5N6N7N8N9GGGTTN1oN11-3' (I) in which Ni, Nio and Nu are independently chosen from A, T, G, C or a deletion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently chosen from A, T, G or C.

[0011] According to a second aspect, the invention relates to an aptamer (hereinafter "competitor aptamer") which competes with an aptamer according to the invention as described above (hereinafter "reference aptamer") for binding to CspZ.

[0012] According to a third aspect, the invention relates to a support on which an aptamer according to the invention is immobilized.

[0013] According to a fourth aspect, the invention relates to the use of an aptamer according to the invention or a solid support according to the invention to detect the CspZ protein in a biological sample.

[0014] According to a fifth aspect, the invention relates to the use of an aptamer according to the invention or a solid support according to the invention to detect a bacterium of the genus Borrelia in a biological sample.

[0015] According to a sixth aspect, the invention relates to an in vitro method for detecting the CspZ protein in a biological sample, said method comprises the following steps: a) bringing the biological sample into contact with an aptamer according to the invention or a solid support according to the invention in order to form a non-covalent complex between the CspZ protein and said aptamer; b) analyze the biological sample to detect the presence of the non-covalent complex formed in step a).

[0016] According to a seventh aspect, the invention relates to an in vitro method for detecting a bacterium of the genus Borrelia in a biological sample, said method comprises the following steps: a) bringing the biological sample into contact with an aptamer according to the invention or a solid support according to the invention in order to form a non-covalent complex between the bacterium of the genus Borrelia and said aptamer; b) analyze the biological sample to detect the presence of the non-covalent complex formed in step a).

[0017] According to an eighth aspect, the invention relates to an in vitro method for diagnosing infection with a bacterium of the genus Borrelia in a subject, said method comprising (i) the detection of the CspZ protein in a biological sample of said subject by implementing the method according to the sixth aspect of the invention, or (ii) the detection of a bacterium of the genus Borrelia in a biological sample of said subject by implementing the method according to the seventh aspect of the invention. Detailed description

[0018] Definitions

[0019] The term “aptamer” refers to synthetic single-stranded nucleotide (or oligonucleotide) sequences (DNA or RNA), generally ranging in length from 20 to 100 nucleotides (nt), which are capable of adopting unique and complex three-dimensional (3D) conformations, enabling them to bind specifically and with high affinity to a wide variety of targets. The recognition and binding of aptamers to their respective targets are non-covalent and rely on a range of intermolecular interactions, such as hydrophobic and electrostatic interactions, hydrogen bonds, van der Waals forces, shape complementarity, and base stacking.

[0020] The term “aptatope”, by analogy with “epitope” for antibodies, designates the binding site of an aptamer at the level of its target.

[0021] The term “nucleotide sequence” (or oligonucleotide) refers to a sequence of nucleotides.

[0022] The term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide, or a modified form thereof, as well as their analogues. Nucleotides include species that include purines (e.g., adenine, hypoxanthine, guanine and their derivatives and analogues) as well as pyrimidines (e.g., cytosine, uracil, thymine and their derivatives and analogues).

[0023] In the context of the present invention, the nucleotide sequence is preferably an unmodified ribonucleotide sequence (i.e., DNA sequence).

[0024] For the purposes of the present invention, "identity" is calculated by comparing two sequences aligned in a comparison window. Aligning the sequences allows us to determine the number of positions (nucleotides or amino acids) in common for the two sequences in the comparison window. The number of common positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of identity. The percentages of identity referred to in the present invention are determined on the basis of a global alignment of the sequences to be compared, that is, on an alignment of sequences taken in their entirety along their full length, using any algorithm well known to those skilled in the art, for example, the Needleman and Wunsch algorithm (1970). This sequence comparison can be performed using any software well known to those skilled in the art, for example, using the Needle software with the "Gap open" parameter set to 10.0, the "Gap Extend" parameter set to 0.5, and a "BLOSUM 62" matrix. The Needle software is, for example, available worldwide from ebi.ac.uk under the name "Align". Other methods for determining the percentage of identity can also be used, for example, those described in the following reference: Computational Molecular Biology, Lesk AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and genome Projects, Smith DW, ed.Academy Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin AM, and Griffin HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje G., Academy Press, 1987; and Sequence Analysis Primer, Gribskov M. and Devereux J., eds., M. Stockton Press, New York, 1991; and Carillo H. and Lipman D., SIAM J. Applied Math., 48:1073 (1998). The methods for determining identity are designed to provide the greatest possible match between the tested sequences. Furthermore, these methods are codified in publicly available computer programs. Computer program methods for determining the identity between two sequences include, but are not limited to, the GCG software (Devereux J. et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and PASTA (Altschul SF et al., J. Molec. Biol. 215: 403-410 (1990)).The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul S. et al., NCBI NLM NUH Bethesda, MD 20894; Altschul S. et al., J. Mol Biol. 215: 403-410 (1990)).

[0025] When a sequence of an aptamer according to the invention has a nucleotide sequence that is not 100% identical to one of those described in the list of sequences (reference sequences) but has at least 90% identity with such a reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence.

[0026] For the purposes of the invention, "at least 90% identity" means at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99%. Thus, in particular embodiments of the invention, the term "at least 90% identity" corresponds to at least 90% identity, at least 91% identity, at least 92% of identity, at least 93% of identity, at least 94% of identity, at least 95% of identity, at least 96% of identity, at least 97% of identity, at least 98% of identity, or at least 99%.

[0027] When a sequence has at least 90% identity with a reference sequence, it may have, for example, at least one insertion, at least one deletion, and / or at least one substitution with respect to the reference sequence. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, substitutions, and / or deletions with respect to the reference sequence. Thus, in particular embodiments of the invention, the term "at least 90% identity" corresponds to at least one insertion, at least one deletion, and / or at least one substitution with respect to the reference sequence. For example, the term "at least 90% identity" may correspond to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, substitutions, and / or deletions with respect to the reference sequence. For example, a sequence that has at least 90% identity with a 100 nt reference sequence may have 1 to 10 insertions, deletions and / or substitutions compared to the reference sequence.For example, a sequence that has at least 90% identity with a 75 nt reference sequence may have 7 insertions, deletions, and / or substitutions compared to the reference sequence. For example, a sequence that has at least 90% identity with a 39 or 40 nt reference sequence may have 4 insertions, deletions, and / or substitutions compared to the reference sequence. It is understood that an aptamer according to the invention whose sequence has "at least 90% identity" with a reference sequence does not lose its ability to bind to CspZ. Preferably, such an aptamer does not lose its ability to bind the CspZ protein expressed on the outer membrane of Borrelia bacteria.

[0028] A person skilled in the art uses their general knowledge to determine the number of modifications that can be made and their location in order to preserve the function of the aptamer. To determine the ability of an aptamer variant to bind specifically to its target (e.g., CspZ), several appropriate methods, well known to a person skilled in the art and described in the prior art, can be used. Aptamers can therefore be tested by binding methods, such as ELISA, affinity chromatography, SPR, etc.

[0029] The term "CspZ" refers to a lipoprotein anchored to the outer leaflet of the outer membrane of bacteria of the genus Borrelia. CspZ is largely universal among Borrelia species responsible for LBD and is highly conserved both within and between species (>85% protein identity between Borrelia burdorferi sensu stricto (BZ?ss) isolates and 98% between Borrelia burgdorferi sensu lato (Bèsl) strains) (Rogers and Marconi 2007; Marcinkiewicz et al. 2023). The CspZ protein is also called Complement Regulator-Acquiring Surface Protein 2 (BbCRASP-2) or BBH06. It is described in the literature that CspZ (which is expressed, for example, at the surface of Borrelia burgdorferi) binds to FH via domains 5 to 7, while FhbA (which is expressed on the surface of Borrelia hermsii) can bind to FH via domains 5 to 7 or 19 to 20 (Hovis et al. 2004; Kraiczy et al. 2008a; Meri et al. 2013; Kogan et al. 2022).

[0030] The term “solid support” refers to any support having a surface on which aptamers can be immobilized, directly or indirectly, by covalent or non-covalent bonds.

[0031] The term “affinity” refers to the strength of all non-covalent interactions between a molecule, for example an aptamer, and its target, for example the CspZ protein. Affinity is generally represented by the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD) can be measured by well-known methods, for example by Surface Plasmon Resonance (SPR). The implementation of an SPR method (BIACORE™) for measuring the affinity of an aptamer according to the invention is described in Example 4.

[0032] The term “biological sample” refers to any biological material capable of containing a bacterium of the genus Borrelia (e.g., Borrelia burgdorferi, Borrelia garinii, and / or Borrelia afzelii), including any liquid or fluid sample or any solid material, in particular a sample from a biological source such as a subject, for example, a patient. This may include biological fluids from a tissue sample taken from a subject, preferably a human being, for example, by surgical resection or biopsy. In the context of the present invention, the sample is preferably selected from whole blood, blood serum, blood plasma, saliva, urine, cerebrospinal fluid, synovial fluid, and genital secretions.

[0033] The term "subject" refers to a human or non-human mammal (such as a cat, a dog, a horse or a primate). Preferably, the subject is a human being, male or female.

[0034] Aptamers according to the invention

[0035] The inventors have made considerable efforts to develop aptamers that bind remarkably well to the CspZ protein, including when the CspZ protein is naturally expressed on the surface of Borrelia bacteria. Such aptamers have proven particularly interesting for diagnostic use in detecting Borrelia infections in subjects, especially Borrelia bacteria that cause Lyme borreliosis.

[0036] According to a first aspect, the invention relates to an aptamer that binds to CspZ, said aptamer comprises a nucleotide sequence SEQ ID NO: 1 of formula (I) or a nucleotide sequence having at least 90% identity with the nucleotide sequence of formula (I): 5'- TGGCAATGGN1GGGGTGGTN2GGAGGGGN3N4N5N6N7N8N9GGGTTN1oN11-3' (I) in which Ni, Nio and Nu are independently chosen from A, T, G, C or a deletion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently chosen from A, T, G or C.

[0037] In particular embodiments, Ni is chosen from A or a deletion; and / or N2 is chosen from T or C; and / or N3 is chosen from G or T; and / or N4 is chosen from A or T; and / or N5 is chosen from A or T; and / or N6 is chosen from T or A; and / or N7 is chosen from G or C; and / or N8 is chosen from T or G; and / or N9 is chosen from G or T; and / or Ni0 is chosen from a deletion or C; and / or Nu is chosen from a deletion or G.

[0038] In particularly preferred embodiments, said nucleotide sequence of formula (I) is selected from: (i) the sequence 5'-TGGCAATGGAGGGGTGGTTGGAGGGGGGAATGTGGGGTT-3' (SEQ ID NO: 2) or a sequence having at least 90% identity with SEQ ID NO: 2; or (ii) the sequence 5'-TGGCAATGGGGGGTGGTCGGAGGGGGTTTACGTGGGTTCG-3' (SEQ ID NO: 3) or a sequence having at least 90% identity with SEQ ID NO: 3.

[0039] In particular, the aptamer according to the invention may consist of: (i) the sequence 5'-TGGCAATGGAGGGGTGGTTGGAGGGGGGAATGTGGGGTT-3' (SEQ ID NO: 2) or a sequence having at least 90% identity with SEQ ID NO: 2; or (ii) the sequence 5'-TGGCAATGGGGGGTGGTCGGAGGGGGTTTACGTGGGTTCG-3' (SEQ ID NO: 3) or a sequence having at least 90% identity with SEQ ID NO: 3.

[0040] In particular embodiments, the aptamer according to the invention comprises a nucleotide sequence SEQ ID NO: 4 of formula (II) or a nucleotide sequence having at least 90% identity with the nucleotide sequence of formula (II): 5' - ATACCAGCTTATTCAATTTGGCAATGGNi GGGGTGGTN2GGAGGGGN3N4N5N6N7N8N9 GGGTTN10NnAGATAGTAAGTGCAATCT-3' (II). in which Ni, Nio and Nu are independently chosen from A, T, G, C or a deletion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently chosen from A, T, G or C.

[0041] Preferably, said nucleotide sequence of formula (II) is chosen from: (i) the sequence 5 '-ATACCAGCTTATTC AATTTGGC AATGGAGGG GTGGTTGGAGGGGGGAATGTGGGGTTAGATAG TAAGTGCAATCT-3' (SEQ ID NO: 5) or a sequence having at least 90% identity with SEQ ID NO: 5; or (ii) the sequence 5' - ATACCAGCTTATTC AATTTGGC AATGGGGGGTG GTCGGAGGGGGTTTACGTGGGTTCGAGATAGTAA GTGCAATCT-3' (SEQ ID NO: 6) or a sequence having at least 90% identity with SEQ ID NO: 6.

[0042] In particular, the aptamer according to the invention may consist of: (i) the sequence 5' - ATACCAGCTTATTC AATTTGGC AATGGAGGGGTGG TTGGAGGGGGGAATGTGGGGTTAGATAGTAAGTGCA ATCT-3' (SEQ ID NO: 5) or a sequence having at least 90% identity with SEQ ID NO: 5; or (ii) the sequence 5 ' - ATACCAGCTTATTC AATTTGGC AATGGGGGGTGGTC GGAGGGGGTTTACGTGGGTTCGAGATAGTAAGTGCAA TCT-3' (SEQ ID NO: 6) or a sequence having at least 90% identity with SEQ ID NO: 6.

[0043] The affinity of the aptamer according to the invention for CspZ must be sufficient for diagnostic use. Preferably, the aptamer according to the invention binds to CspZ with a Kd of less than 1 pM measured by SPR (Surface Plasmon Resonance), preferably a Kd of less than 500 nM measured by SPR.

[0044] According to a second aspect, the invention relates to an aptamer (hereinafter "competitor aptamer") which competes with an aptamer according to the invention as described above (hereinafter "reference aptamer") for binding to CspZ.

[0045] The ability of an aptamer to compete with the reference aptamer for binding to CspZ can be tested by a competitive method. A "competitive method" consists of testing an aptamer for its ability to block binding between a reference aptamer and a target or to compete with a reference aptamer for binding to the target. In other words, an aptamer that competes with the reference aptamer binds to the same aptatope as the reference aptamer or to an aptatope that is sufficiently close to the aptatope recognized by the reference aptamer to prevent binding of the reference aptamer due to steric hindrance.

[0046] Many types of competitive methods can be used to determine whether an aptamer competes with a reference aptamer, for example by a competitive ELISA, by direct or indirect solid-phase radioimmunoassay (RIA), by direct or indirect solid-phase enzyme immunoassay (EIA), by surface plasmon resonance technology (e.g., BIACORE), by flow cytometry, by fluorescence polarization (e.g., between a fluorescent protein and the aptamer to be tested), by BLI (BioLayer Interferometry), etc. For example, the competitive ELISA method involves the use of purified CspZ bound to a solid surface or to Borrelia bacteria, the aptamer to be tested which binds to unlabeled CspZ, and a labeled reference aptamer.Typically, the reference aptamer is present at a non-saturating concentration (relative to its dissociation constant KD for CspZ), and the signal is measured at increasing concentrations of the aptamer under test. When an aptamer is present in excess, it can block, displace, or inhibit (e.g., reduce) the specific binding of a reference aptamer to CspZ by at least 40–45%, 45–50%, 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or 75% or more. In some cases, binding is inhibited by at least 80–85%, 85–90%, 90–95%, 95–97%, or 97% or more.

[0047] In particular embodiments, a competing aptamer according to the invention comprises: - a nucleotide sequence SEQ ID NO: 8 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 8; - a nucleotide sequence SEQ ID NO: 9 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 9; - a nucleotide sequence SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 10; - a nucleotide sequence SEQ ID NO: 11 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 11; - a nucleotide sequence SEQ ID NO: 12 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 12; or - a nucleotide sequence SEQ ID NO: 13 or a nucleotide sequence having at least 90% identity with the nucleotide sequence SEQ ID NO: 13.

[0048] The aptamers according to the invention (reference aptamers and competitor aptamers) as defined above are hereinafter referred to collectively as "aptamer according to the invention".

[0049] The aptamer according to the invention can bind to CspZ in an isolated form and / or in its biological environment, namely CspZ expressed on the outer membrane of bacteria of the genus Bb si, such as Bb ss, Borrelia garinii and / or Borrelia afzelii.

[0050] Preferably, the CspZ protein to which the aptamer according to the invention binds has the peptide sequence SEQ ID NO: 7, that is to say the peptide sequence of the CspZ protein of the species Borrelia burgdorferi sensu stricto.

[0051] The aptamer according to the invention preferably comprises 120 nucleotides or less, for example 100 nucleotides or less. For example, the aptamer according to the invention may comprise from 39 to 100 nucleotides, preferably from 39 to 75 nucleotides.

[0052] In particular embodiments, the aptamer according to the invention may comprise a chemical modification selected from the group consisting of a chemical substitution at a sugar, a chemical substitution at a phosphate, and a chemical substitution at a nucleotide. This may be a modification at the 3' and / or 5' end intended to protect said end from degradation by nucleases. It may also be a modification intended to conjugate the aptamer with a compound enabling its detection, for example, conjugation with a fluorescent marker or biotin.

[0053] In particular embodiments, the aptamer according to the invention is conjugated with biotin, for example at its 3' end or its 5' end. Biotin is particularly suitable for detecting the presence of a non-covalent complex formed between the aptamer and CspZ (or the presence of a non-covalent complex formed between the aptamer and the bacterium of the genus Borrelia) with a labeled antibody or labeled streptavidin.

[0054] In particular embodiments, the aptamer according to the invention is conjugated with a fluorescent marker, for example, a FAM (fluorescein phosphoramidite) type marker or a FITC (fluorescein isothiocyanate) type marker. Conjugation with a fluorescent marker can occur at the 3' end or the 5' end. Fluorescent markers are particularly well-suited for detecting the presence of a non-covalent complex formed between the aptamer and Borrelia bacteria by flow cytometry.

[0055] Solid support according to the invention

[0056] According to a third aspect, the invention relates to a support on which an aptamer according to the invention is immobilized.

[0057] The solid support according to the invention can be of different kinds insofar as the aptamer can be immobilized on its surface and in particular used for the implementation of a diagnostic process from a biological sample.

[0058] The solid support can take a variety of shapes, from the simplest to the most complex. The solid support can be in the form of a strip, a plate, a disc, a rod, a particle, including a ball, a tube, a well, etc. In general, the solid support is relatively flat, such as a blade, but it can be spherical, such as a ball, or cylindrical (for example a column). Among the solid supports that can be used are microtiter wells, microscope slides, membranes, beads (e.g. paramagnetic beads, microbeads), filled paper, Langmuir-Blodgett films, silicon wafers and chips (e.g. microfluidic chips).

[0059] The solid support may include any material capable of providing physical support to the aptamers attached to its surface. The material is generally capable of withstanding the conditions related to the attachment of the aptamers to its surface and any subsequent treatment, handling, or transformation encountered during its use, for example, during its use in a diagnostic process.

[0060] The solid support can be natural or synthetic. Suitable solid supports may include silicon, graphite, reflective surfaces, laminates, ceramics, plastics (including polymers such as, for example, poly(vinyl chloride), polyethylene, polypropylene, and polypropylene), cyclo-olefin copolymers, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon, poly(vinyl butyrate)), germanium, gallium arsenide, silver, etc. Other solid supports may be considered, such as glass, which contains silica. It can also be a functionalized solid support that has one or more functional groups on its surface, such as an amine, carboxyl, thiol or hydroxyl functional group.Functional groups can, for example, be used to covalently immobilize aptamers on the solid support.

[0061] The nature of the solid support is not limiting insofar as the aptamer can be immobilized therein and retains its ability to bind its target when implementing a diagnostic process from a biological sample.

[0062] Several techniques for immobilizing an aptamer on a solid support are described in the literature. These may involve covalent or non-covalent immobilization.

[0063] The main technique for immobilizing aptamers on a solid support described in the prior art is based on the use of the biotin-streptavidin or biotin-avidin pair.

[0064] The prior art also describes several techniques for the covalent grafting of nucleic acids onto solid supports for the implementation of analytical methods. For example, the grafting of nucleic acid aptamers possessing a reactive amino group onto a sepharose support activated by cyanogen bromide has been described (Madru et al., 2009, Anal. Chem., Vol. 81: 7081-7086). The grafting of periodate-oxidized RNA aptamers onto an agarose support activated by adipic acid dihydrazide groups has also been described (Caputi et al., 1999, The EMBO). Journal, Vol. 18(14): 4060-4067). Techniques for grafting nucleic aptamers via bifunctional coupling agents such as SIAB are also known (Rehder et al., 2001, Electrophoresis, Vol. 22(17): 3759).

[0065] The prior art also discloses techniques for covalent coupling of nucleic acids, including aptamers, on solid supports such as silica or agarose comprising carboxylic acid groups pre-activated by N-hydroxysuccimmide (NHS) (Goss et al., 1990, J Chromatogr, Vol. 508: 279-287; Larson et al., 1992, Nucleic Acids research, Vol. 20(13): 3525, Allerson et al., 2003, RNA, Vol. 9: 364-374; EP2658864A1).

[0066] The chosen immobilization technique is not limiting insofar as the solid support on which the aptamers are immobilized can be used for the implementation of a diagnostic process from a biological sample.

[0067] The aptamer can be immobilized on the solid support by means of a spacer, for example, a nucleotide spacer or a non-nucleotide spacer. The spacer's function is to physically distance the aptamer from the surface of the solid support on which it is immobilized, thus allowing relative mobility of the aptamer with respect to the surface of said solid support. The spacer limits or prevents steric hindrance, due to excessive proximity of the solid support to the nucleic portion of the aptamer, from interfering with the binding events between said aptamer and its target. The spacer can be added to the 5' end or the 3' end of the aptamer. The choice of spacer is not limiting as long as the aptamer retains its ability to bind to its target.

[0068] Use of aptamers according to the invention

[0069] According to a fourth aspect, the invention relates to the use of an aptamer according to the invention or a solid support according to the invention to detect the CspZ protein in a biological sample.

[0070] According to a fifth aspect, the invention relates to the use of an aptamer according to the invention or a solid support according to the invention to detect a bacterium of the genus Borrelia in a biological sample.

[0071] According to a sixth aspect, the invention relates to an in vitro method for detecting the CspZ protein in a biological sample, said method comprises the following steps: a) to bring the biological sample into contact with an aptamer according to the invention or a solid support according to the invention in order to form a non-covalent complex between the CspZ protein and said aptamer; b) analyze the biological sample to detect the presence of the non-covalent complex formed in step a).

[0072] According to a seventh aspect, the invention relates to an in vitro method for detecting a bacterium of the genus Borrelia in a biological sample, said method comprises the following steps: a) to bring the biological sample into contact with an aptamer according to the invention or a solid support according to the invention in order to form a non-covalent complex between the bacterium of the genus Borrelia and said aptamer; b) analyze the biological sample to detect the presence of the non-covalent complex formed in step a).

[0073] According to an eighth aspect, the invention relates to an in vitro method for diagnosing infection by a bacterium of the genus Borrelia in a subject, said method comprises (i) the detection of the CspZ protein in a biological sample of said subject by implementing the method according to the sixth aspect of the invention, or (ii) the detection of a bacterium of the genus Borrelia in a biological sample of said subject by implementing the method according to the seventh aspect of the invention.

[0074] When the aptamer comes into contact with a biological sample, it binds specifically to the CspZ protein present in the sample, for example, to the CspZ protein naturally expressed on the surface of Borrelia bacteria. The aptamer may include a label that allows the presence of the non-covalent complex formed between the CspZ protein and the aptamer (or the presence of the non-covalent complex formed between the Borrelia bacterium and the aptamer) to be detected by determining the presence or absence of a signal provided by said label. For example, the aptamer may be labeled with a fluorescent compound, such as a FAM (fluorescein phosphoramidite) type label or a FITC (fluorescein isothiocyanate) type label. A fluorescent label may allow visualization of the aptamer bound to its target by fluorescence or laser scanning microscopy or by flow cytometry.Furthermore, particularly for detection purposes, the aptamer can be immobilized on conventional solid supports such as beads, enabling the detection of the non-covalent complex formed between the CspZ protein and the aptamer (or the presence of the non-covalent complex formed between Borrelia bacteria and the aptamer) and thus the diagnosis of infection with Borrelia bacteria. In addition, the aptamer can be biotinylated or coupled to streptavidin, avidin, or neutravidin for use in the specific detection of the presence of the non-covalent complex formed between the CspZ protein and the aptamer (or the presence of the non-covalent complex formed between Borrelia bacteria and the aptamer).

[0075] Methods that allow the presence of the non-covalent complex formed between the CspZ protein and the aptamer (or the presence of the non-covalent complex formed between the Borrelia bacterium and the aptamer) to be revealed in a sample The biological methods used in implementing the processes according to the invention can be of various kinds. Examples include immunogenic methods such as the ELISA (Enzyme-Linked Immunosorbent Assay), fluorescence detection methods such as flow cytometry, the Luminex system, or any other suitable method described in the literature, such as PCR, etc.

[0076] In particular embodiments, the biological sample is chosen from whole blood, blood serum, blood plasma, saliva, urine, cerebrospinal fluid, synovial fluid, genital secretions.

[0077] In particular embodiments, the bacterium of the genus Borrelia is chosen from a species that expresses the CspZ protein on its surface. Preferably, the bacterium of the genus Borrelia belongs to the genus Borrelia burgdorferi sensu lato, including, but not limited to, Borrelia burgdorferi sensu stricto, Borrelia garinii and / or Borrelia afzelii. In a particularly preferred embodiment, the bacterium of the genus Borrelia is Borrelia burgdorferi sensu stricto.

[0078] Diagnostic kit according to the invention

[0079] According to an eighth aspect, the invention relates to a diagnostic kit for infection by a bacterium of the genus Borrelia comprising an aptamer according to the invention or a solid support according to the invention.

[0080] The kit may also include reagents for revealing the presence of a non-covalent complex formed between (i) the Borrelia genus bacterium and the aptamer and / or (ii) the CspZ protein and the aptamer.

[0081] Processing method

[0082] The invention also relates to a treatment method comprising the following steps: a) bringing a biological sample into contact with an aptamer according to the invention or a solid support according to the invention in order to form a non-covalent complex between the bacterium of the genus Borrelia and said aptamer; b) analyze the biological sample to detect the presence of the non-covalent complex formed in step a); and c) treat the subject with appropriate treatment when the presence of the non-covalent complex between the bacterium of the genus Borrelia and said aptamer is detected in step b).

[0083] Appropriate treatment may be based on the administration of one or more antibiotic(s), such as one or more antibiotic(s) chosen from tetracyclines (doxycycline), fl-lactams (amoxicillin, ceftriaxone), second and third generation cephalosporins, macrolides (azithromycin).

[0084] For example, doxycycline (200 mg / day in one or two doses) or amoxicillin (1 to 2 g three times a day) can be administered as first-line treatment for 14 days. The therapeutic efficacy of these treatments is generally Excellent. Amoxicillin stands out from other antibiotics due to its relative safety, allowing its use in children under 8 years of age and pregnant women. Azithromycin can be considered as a second-line treatment alternative.

[0085] The same first-line treatment is recommended for erythema migrans with multiple lesions, without associated extracutaneous involvement, or in the treatment of Lyme lymphocytoma, but for a duration of 21 days. In cases of Lyme neuroborreliosis in adults, oral ceftriaxone or doxycycline antibiotic therapy for 21 days is recommended. Finally, for joint involvement, doxycycline antibiotic therapy for 28 days is recommended.

[0086] More targeted and specific treatments for Borrelia bacteria may also be considered, such as hygromycin A. Brief description of the figures

[0087] [Fig. 1] Dot blot analysis of the interaction of each candidate aptamer with different proteins: A - CspZ, B - FhbA, or C - BSA. D - After protein immobilization, biotinylated aptamers are revealed by HRP-conjugated polyclonal anti-biotin antibodies. The amount of each partner is constant in the different blots. Positive controls (+) are revealed using specific HRP-conjugated antibodies. E - Total intensity of each dot, measured with ImageJ, for CspZ, FhbA, and BSA proteins

[0088] [Fig.2] Analysis of the interactions between aptamers and CspZ by ELONA. A - Experimental setup. B - Screening of the 13 aptamer candidates at 2.5 pg / mL incubated with 10 pM CspZ.

[0089] [Fig. 3] SPR sensorgrams. Immobilized ligands: Aptamers Ctrl, 3, 4, 6, 7, 9, 10, and 11. A - Analytes: increasing concentrations of 6His-CspZ (from 0.25 pM to 20 pM depending on the aptamer). B - Analytes: increasing concentrations of 6His-FhbA (from 0.7 pM to 20 pM depending on the aptamer).

[0090] [Fig. 4] Analysis of Bbss labeling with several FAM-conjugated fluorescent aptamers by flow cytometry. For each experiment, 500 nM of aptamers were incubated with the bacteria for 8 days of growth. A - Aptamers considered not to interact with Bbss, reflecting AptaCtrl. B - Aptamers considered to interact with Bbss by comparison with AptaCtrl.

[0091] [Fig. 5] Recognition of Bbss by selected aptamers (mean fluorescence intensity assessed by flow cytometry). Bbss autofluorescence was assessed (incubation without aptamers, shown blank). The bacterial culture was then incubated with 500 nM of FAM-conjugated AptaCtrl, Apta7, Apta9, or AptalO. Error bars represent the standard deviation associated with the mean fluorescence intensity of 3 independent experiments. The statistical analysis used in this experiment is a two-sample t-test (* = p < 0.05 and *** = p < 0.001).

[0092] [Fig. 6] Flow cytometry analysis. Fluorescence intensity of B. garinii (A, B) and B. afzelii (C, D) induced by AptaCtrl, 7, 9 and 10 (A, C) conjugated with FAM and by two controls (B, D). The positive control (polyclonal antibody directed against CspZ revealed by a FITC-conjugated secondary antibody) is in light gray, the negative control (secondary antibody alone) in black.

[0093] [Fig. 7] Epifluorescence microscopy analysis of Bbss (40x magnification). Images were acquired without aptamers (Borrelia only) and using the FAM-conjugated aptamers AptaCtrl, Apta7, Apta9, and AptalO. The red circle highlights the fused image (phase contrast and fluorescence) in the center. The red arrows indicate some Borrelia exhibiting a fluorescence signal. B - Epifluorescence microscopy observation of B. garinii and B. afzelii bacteria (40x magnification). Detection was performed using primary antibodies directed against CspZ and FITC-conjugated secondary antibodies, or with FAM-conjugated aptamer probes: AptaCtrl (negative control), Apta9, and AptalO.

[0094] [Fig.8] A - Experiment format, B - Analysis of the interaction of complete or truncated aptamers by ELONA. Results obtained for aptamers 9 and 10, truncated (-) and complete (+). Examples

[0095] Example 1: Development of aptamers

[0096] Aptamers were able to be selected thanks to the development of a complex SELEX (Systematic Evolution of Ligands by EXponential enrichment) process implementing numerous steps of different kinds, including 12 successive selection cycles.

[0097] In short, after a negative selection cycle against bare beads to eliminate aptamers interacting with the solid support, three successive positive selection cycles were performed against histidine-tagged CspZ protein immobilized on Ni-NTA beads. Two additional cycles against GST-fused CspZ protein immobilized on GSH beads were applied to eliminate aptamers capable of recognizing the histidine tag. Four more rounds were performed against histidine-tagged CspZ protein, during which selection pressures were added (increased number of washes and reduced incubation time) to ensure enrichment in oligonucleotides affinity for the target.A spin on Bbss bacteria (strain B31) was then inserted with the aim of selecting aptamers that recognize CspZ as it is oriented on the bacterial surface (specific targeting of the effectively accessible epitopes of the protein). Finally, two... additional rounds against the histidine-tagged CspZ protein immobilized on Ni-NTA beads were performed.

[0098] Example 2: Characterization of aptamers by Dot Blot

[0099] 500 nM solutions of biotinylated aptamers (addition of a TEG-biot spacer at the 5' end) were prepared. The biotinylated aptamers that were tested are: - Aptamer with sequence SEQ ID NO: 5 (Apta9). - Aptamer of sequence SEQ ID NO: 6 (AptalO). - Aptamers identified by the SELEX process of example 1 (Aptal-8 and 11-13). - Biotinylated aptamer negative control (AptaCtrl).

[0100] The membranes were prepared by depositing 4.5 pg of CspZ (i.e., 164 pmol). The controls (FhbA and BSA) were deposited by also applying 164 pmol of each protein per dot onto the membrane. The aptamers were heated for 7 min at 95°C, then immediately cooled on ice for 7 min. Finally, each aptamer was left at room temperature for 7 min before use. After saturating the sensitized membrane (10% BSA in PBS), an incubation with the aptamers prepared as described was carried out for 1 h at room temperature without shaking. Anti-biotin antibodies diluted 1 / 5000 in B / W buffer (A150-111P, Bethyl Laboratories) were added for 1 h at room temperature. After washing, a Clarity™ Western Enhanced chemiluminescence (ECL) Substrate solution was added and a chemiluminescence reading was performed with the Chemidoc.Each exposure time was adjusted to allow for comparison across all experiments.

[0101] The results are presented in [Fig. 1].

[0102] Fig. 1 revealed interactions of varying strength for all aptamers with CspZ. As expected, the negative control AptaCtrl exhibited minimal signal intensity. Signals similar to AptaCtrl were observed for aptamers 1 and 2, although these were overrepresented in the SELEX selection process. In contrast, aptamers 3 to 13 were able to recognize CspZ. In particular, aptamers 3, 4, 6, 7, and 9 to 12 exhibited the strongest signals.

[0103] Regarding the functional mime FhbA (Figure IB and 1E), only aptamers 3 and 11 showed strong signals. The other aptamers (1, 2, 4 to 10, and 12 to 13) showed only weak (or even moderate) signals, suggesting weaker interactions. Finally, with the negative protein control BSA, no significant interactions were observed for any oligonucleotide, except for oligonucleotides 3 and 4, for which a weak signal was detectable (Figure IC and 1E).

[0104] Example 3: Characterization of aptamers by ELONA

[0105] Streptavidin (10 pg / mL in PBS) was immobilized overnight at 4°C on a 96-well plate. After three 5-minute washes with PBS containing 0.1% Tween20, saturation with BSA (10% for 1 hour at room temperature) was performed. Aptamers (see Example 2), at an optimized concentration of 2.5 pg / mL in B / W buffer, were incubated for 30 minutes at room temperature. From this point onward, washes and antibody / protein resuspensions were performed in B / W buffer. CspZ was then added at 10 pM and incubated for 1 hour at room temperature. After washing, an anti-His HRP-conjugated secondary antibody (reference sc-8036, 1 / 500 dilution) was incubated for 1 hour at room temperature. Finally, the ABTS substrate was added and the absorbance at 405 nm was measured after 15 minutes using the VANTAstar.

[0106] The results are presented in [Fig.2].

[0107] The results confirmed the data obtained by Dot Blot, highlighting a significant interaction between 7 aptamers (Apta3, 4, 6, 7, 9, 10, 11) and CspZ.

[0108] Example 4: Characterization of aptamers by SPR

[0109] The SPR experiments were carried out using a Biacore T100 instrument (Cytiva) at 25°C.

[0110] Streptavidin chips (SA, reference BR 100531, Cytiva) were used in all protein-aptamer interaction measurements. Before immobilization, the tracks were conditioned with 1 M NaCl in 50 mM NaOH following the Biacore assistant. Each aptamer was then resuspended in B / W buffer at a concentration of 1 pg / mL and heated for 7 min at 95 °C, then immediately cooled on ice for 7 min. Finally, each aptamer was left at room temperature for 7 min before use. The aptamers (aptamers Ctrl, 3, 4, 6, 7, 9, 10, and 11) were used as ligands by coupling via the streptavidin / biotin procedure until the target levels of 115 RU were reached. A control cell (blank) was prepared in the same way, but without injection of aptamers.

[0111] The proteins CspZ and FhbA were used as analytes. Kinetic measurements were performed by injecting various quantities of protein, ranging from 0.5 pM to 20 pM, at a flow rate of 20 pL / min at 25 °C. All injections were performed using Single Cycle Kinetics (SCK). The association time for each concentration was 1000 seconds, followed by a dissociation time of 30 minutes before regeneration. The surfaces were regenerated by two injections of 60 pL of 100 mM NaOH. The blank was subtracted from each sensorgram before data processing using the BIAcore T100 evaluation software, applying a 1:1 Langmuir model.

[0112] The sensorgrams are shown in [Fig.3] (Figures 3A and 3B respectively), and the kinetic parameters are listed in Table 1. The latter confirm a strong and stable binding of CspZ to the immobilized aptamers.

[0113] [Tables 1] Séquence Analyt e Kd (nM) ko„ (M 4 .s -1) k<(sa) x2 U-val ue AptaCtrl CspZ Pas d’interaction à 20 pM CspZ 3 CspZ 314.0 + 0.8 131.2 + 0.2 (312.0 + 0.9) x 107 1.9 2 FhbA 2777.4 + 12.3 18.3 + 0.1 (507.7 + 1.3) x 10 7 0.3 1 4 CspZ 45.7 + 0.4 146.3 + 0.1 (66.8 + 0.6) x 107 30.3 5 FhbA 1723.9 + 31.8 26.7 + 0.5 (460.1 + 3.0) x 107 17.5 2 6 CspZ 567.8 + 2.7 38.0 + 0.1 (215.6 + 0.9) x 107 25.5 3 FhbA 1604.2 + 9.2 20.0 + 0.1 (321.0 + 1.4) x 107 13.1 3 7 CspZ 328.5 + 1.2 399.4+ 1.2 (1312.0 + 2.7) x 10 7 3.0 1 FhbA 35131.8 + 1524. 8 2.5 + 0.1 (892.7 + 2.8) x 107 14.8 2 9 CspZ 446.9+ 1.3 92.1 + 0.1 (411.5 + 1.1) x 10 7 9.9 2 FhbA 764.1 + 13.2 510.8 + 6.9 (3903.0 + 42.0) x 107 9.7 1 10 CspZ 454.2+ 1.4 44.3 + 0.1 (201.4 + 0.5) x 107 24.6 2 FhbA 2830.1 + 16.8 42.2 + 0.2 (1194.0 +2.8) x 10 7 13.3 1 11 CspZ 208.2 + 0.6 344.8 + 0.6 (717.9 + 1.6) x 107 5.3 2 FhbA 543.5 + 2.8 80.9 + 0.2 (439.6 + 2.1) x 107 35.4 4

[0114] Table 1: Quantitative analysis of interactions between immobilized aptamers and CspZ or FhbA proteins by SPR.

[0115] As expected, AptaCtrl showed no detectable binding, regardless of the protein marker used, namely CspZ, FhbA, or BSA. The aptamer AptalO (chosen arbitrarily) was also tested against 20 pM BSA, and no interaction was observed. The interactions of all the selected aptamers with CspZ were confirmed.

[0116] Example 5: Recognition of CspZ on Borrelia bacteria

[0117] In order to evaluate the ability of the selected aptamers (Aptal-13) to bind to epitopes available on the surface of bacteria, flow cytometry experiments The analyses were performed using a CytoFlex flow cytometer (Beckman Coulter Life Sciences, Indianapolis, USA) and CytExpert software. For the analysis of one sample, after 8 days of growth in BSK-H medium at 33°C, 50 pL of Borrelia culture supplemented with 450 pL of B / W buffer were centrifuged at 8,000 g for 5 minutes. The cell pellet was resuspended in 1 mL of B / W buffer. After a second centrifugation (8,000 g, 5 min), the cells were resuspended in 25 pL of B / W buffer. Anti-CspZ antibodies were used as a positive control: a 25 pL volume of bacteria prepared as directed was incubated with 100 pL of anti-CspZ polyclonal antibodies (rabbit antibody, Rockland, #200-401-C19, 1 / 200 dilution), and then detected with a mouse secondary antibody against rabbit constant regions conjugated to FAM (Invitrogen / Thermofisher, #31584, 1 / 750 dilution). The secondary antibody alone was used as a negative control.

[0118] The binding of FAM aptamers to Borrelia was tested by mixing 25 pL of bacteria prepared as described above with 100 pL of FAM-conjugated oligonucleotides, for a final aptamer concentration of 500 nM. After incubation for one hour at room temperature (RT) under static conditions, the bacteria were washed once as described previously in B / W buffer.

[0119] The fluorescence of the samples was then measured by flow cytometry: more than 150,000 events corresponding to Borrelia cells were recorded for each treatment at a flow rate of 10 pL per minute. The fluorescence emitted following excitation of FAM at 488 nm was collected using a 525 / 40 nm bandpass filter. A threshold was applied for an SSC value greater than 1000 (height), and the gains were set as follows: FSC = 165, SSC = 400, and FITC = 240. The data were analyzed using Cytexpert 2.0 software. The measurements were performed in triplicate.

[0120] Results obtained with Borrelia bursdorferi ([Fig.4] and [Fig.5]J

[0121] As illustrated in Figure 4B, six aptamers, namely aptamers 3, 6, 9, and 10, induced a shift in fluorescence intensity, as did aptamers 4 and 12 to a lesser extent.

[0122] Aptamers 9 and 10 showed the largest shift, comparable to the shift caused by recognition with the commercial anti-CspZ antibody.

[0123] In contrast, Aptal, 2, 5, 8, and 13, just like Apta7 and 11 previously studied, completely overlap with AptaCtrl (Figure 4A), suggesting that the aptatope they target on the bacterial surface is not accessible.

[0124] It should be emphasized that the signal generated by commercial antibodies results from amplification, since polyclonal primary antibodies are revealed by fluorescent secondary antibodies. In contrast, the signal generated by the Aptamers 9 and 10 is direct and does not benefit from such amplification, suggesting that aptamers allow for better detection of the bacterium.

[0125] To further validate the screening experiment and confirm the specific binding of Apta9 and AptalO to CspZ, a quantitative analysis of the fluorescence signal derived from flow cytometry experiments was then performed. Bacteria and AptaCtrl served as negative controls. Apta7, which binds to recombinant CspZ but not to whole bacteria, was included as an additional control. The mean fluorescence intensity of FAM-Apta-labeled Borrelia from three independent experiments is shown in [Fig. 5].

[0126] The results suggest that Apta9 and AptalO exhibit significantly higher mean fluorescence (4112 ± 348 AU and 3641 ± 889 AU, respectively) compared to AptaCtrl (1171 ± 257 AU). In contrast, Apta7 showed no significant difference (1736 ± 699 AU). These results corroborate the initial screening analysis, where Apta9 and AptalO showed binding to Borrelia, unlike Apta7. This also strengthens the hypothesis that the epitope targeted by Apta7 is inaccessible on the bacterial surface. Collectively, these results provide a strong proof of concept for the development of novel diagnostic probes targeting pathogen surface proteins.

[0127] Results obtained with Borrelia garinii and Borrelia afzelii ([Fig.6] )

[0128] In order to evaluate whether the CspZ aptamers also recognize European Borrelia species, the aptamers inducing the most pronounced shift (Apta 9 and 10) were incubated with B. afzelii BO23 and B. garinii CIP 103362. These Borrelia species, mostly found in the French territory, possess CspZ proteins with an amino acid sequence identity of approximately 80% to CspZ of Borrelia burgdorferi sensu stricto (strain B31).

[0129] Figure 6 shows the fluorescence intensity shift as a function of the aptamers tested. A fluorescence shift was observed for Apta9 and 10 for each strain. In contrast, and as observed for Bb B31, AptaCtrl and Apta7 are identical and showed no fluorescence change.

[0130] Thus, Apta9 and AptalO confirmed their ability to recognize CspZ on the bacterial surface in two European species of Borrelia.

[0131] Example 7: Recognition of Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii by epifluorescence microscopy

[0132] To validate the flow cytometry data, epifluorescence microscopy experiments were carried out on the 3 strains: Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii.

[0133] Epifluorescence microscopy experiments were performed using the same preparations as those used for flow cytometry experiments.

[0134] The results are presented in [Fig.7].

[0135] Figure 7A shows images taken on B. burgdorferi ss without aptamer and in the presence of the aptamers AptaCtrl, Apta7, 9 and 10.

[0136] Figure 7B shows images taken on B. garinii and B. afzelii in the presence of a positive control (Primary and Secondary Antibody) and AptaCtrl, Apta9 and AptalO.

[0137] For all strains, microscopy data confirmed the ability of Apta9 and AptalO to interact with CspZ on the bacterial surface. Furthermore, the signal obtained with the antibody was amplified, as induced by the use of secondary antibodies, which was not the case for the signals obtained with Apta9 and 10. Moreover, the observed fluorescence distribution was similar to the recognition profile obtained with the anti-CspZ antibody, confirming that the aptamers are capable of recognizing CspZ on the bacterial surface.

[0138] Finally, no signal was detected with AptaCtrl and Apta7 for B. burgdorferi. Apta7 was not tested on the two strains B. garinii and B. afzelii.

[0139] Example 8: Role of flanking regions

[0140] To study the role of flanking and random regions, aptamers 9 and 10 containing only random regions, i.e. A9- and A10- of sequence SEQ ID NO: 2 and SEQ ID NO: 3 respectively, were synthesized and ELONA measurements were carried out (see protocol of Example 3).

[0141] The results are presented in [Fig. 8]. The results show that the truncated aptamers A9- and A10-, lacking their flanking regions, retain affinity for their target, although decreased compared to the complete aptamers A9+ and A10+ of sequence SEQ ID NO: 7 and SEQ ID NO: 8 respectively (40% with 0.159 AU versus 0.259 AU; and 30% with 0.295 AU versus 0.410 AU, respectively) (Figure 8B). This signal decrease could be attributed to reduced stability of the aptamer-target complex or to intrinsic destabilization of the truncated aptamer. Sequence listing

[0142] SEQ ID NO: Denomination Séquence 1 Formule I TGGCAATGGN1GGGGTGGTN2GGAGGG GGN3N4N5N6N7N8N9GGGTTNi0Ni! In the following Ni, Nio and Nu have their own choice with A, T, G, C or a délétion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently chosen from among A, T, G or C. 2 Apt9 core TGGCAATGGAGGGGTGGTTGGAGGGGGGAATGT GGGGTT 3 AptlO core TGGCAATGGGGGGTGGTCGGAGGGGGTTTACGT GGGTTCG 4 Formula II ATACCAGCTTATTCAATTTGGCAATGGNiGGG GTGGTN2GGAGGGGGN3N4N5N6N7N8N9GGG TTNioNnAGATAGTAAGTGCAATCT in which Ni, Nio and Nu are independently chosen from among A, T, G, C or a délétion ; N2, N3, N4, N5, N6, N7, N8 and N9 are independent of each other: A, T, G or C.5 Apt9 ATACCAGCTTATTCAATTTGGCAATGGAGGGGT GGTTGGAGGGGGGAATGTGGGGTTAGATAGTAA GTGCAATCT 6 AptlO ATACCAGCTTATTCAATTTGGCAATGGGGGGTG GTCGGAGGGGGTTTACGTGGGTTCGAGATAGTA AGTGCAATCT 7 CspZ MKKSFLSIYMLISISLLSCDVSRLNQRNINELKIFV EKAKYYSIKLDAIYNECTGAYNDIMTYSEGTFSDQS KVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSK LIDDFAIELDQAVDNDVSNARHVADSYKKLRKS VVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFV . EENDLIALECIVKTIGDMVNDREINSRSRYNNFYKK EADFLGAAVELEGAYKAIKQTLL 8 Apt3 core GAACCGGGATGGGAGGGAGGGGGTGGAGGA GGCAGTTCAA 9 Apt4 core CACTTGGTGGTGGTGGCGGGATGGGATGGGTTG GGTTTGT 10 Apt6 core TGGGGCAAGGGAGGGCGGGGGCAGCGGCGGTAC GAATTGA 11 Apt3 ATACCAGCTTATTCAATTGAACCGGGATGGGAG GGAGGGGGTGGAGGAGGCAGTTCAAAGATAGTA AGTGCAATCT 12 Apt4 ATACCAGCTTATTCAATTCACTTGGTGGTGGTG GCGGGATGGGATGGGTTGGGTTTGTAGATAGTA AGTGCAATCT 13 Apt6 ATACCAGCTTATTCAATTTGGGGCAAGGGAGGG CGGGGGCAGCGGCGGTACGAATTGAAGATAGTA AGTGCAATCT

Claims

Demands

1. Aptamer that binds to CspZ, said aptamer comprises a nucleotide sequence SEQ ID NO: 1 of formula (I) or a nucleotide sequence having at least 90% identity with the nucleotide sequence of formula (I): 5'-TGGCAATGGNjGGGGTGGTNzGGAG GGGGN3N4N5N6N7N8N9GGGTTNi0Ni r3' (I) in which Ni, Nio and Nu are independently selected from A, T, G, C or a deletion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently selected from A, T, G or C.

2. Aptamer according to claim 1, said nucleotide sequence of formula (I) is selected from: (i) the sequence 5'-TGGCAATGGAGGGGTGGTTGGAG GGGGGAATGTGGGGTT-3' (SEQ ID NO: 2) or a sequence having at least 90% identity with SEQ ID NO: 2; or (ii) the sequence 5'-TGGCAATGGGGGGTGGTCGGAG GGGGTTTACGTGGGTTCG-3' (SEQ ID NO: 3) or a sequence having at least 90% identity with SEQ ID NO:

3.

3. Aptamer according to any one of the preceding claims, said aptamer comprises a nucleotide sequence SEQ ID NO: 4 of formula (II) or a nucleotide sequence having at least 90% identity with the nucleotide sequence of formula (II): 5'-ATACCAGCTT ATTCAATTTGGCAATGGNi GGGGTGGTN2GGAGGGGGN3N4N5N6N7N8N9 GGGTTNioNnAGATAGTAAGTGCAATCT-3' (II) in which Ni, Nio and Nu are independently selected from A, T, G, C or a deletion; N2, N3, N4, N5, N6, N7, N8 and N9 are independently selected from A, T, G or C.

4. Aptamer according to claim 3, said nucleotide sequence of formula (II) is selected from: (i) the sequence 5' - AT ACCAGCTT ATTCAATTTGGCAATGGAG GGGTGGTTGGAGGGGGGAATGTGGGGTTA (ii) the sequence 5' - AT ACCAGCTT ATTCAATTTGGCAATGGGGG GTGGTCGGAGGGGGTTTACGTGGGTTCGAGA TAGTAAGTGCAATCT-3' (SEQ ID NO: 6) or a sequence having at least 90% identity with SEQ ID NO:

6.

5. Aptamer according to any one of the preceding claims, said aptamer binds to CspZ with a KD less than 1 pM measured by SPR (Surface Plasmon Resonance), preferably a KD less than 500 nM measured by SPR.

6. Aptamer that competes for binding to CspZ with an aptamer of sequence 5'- AT ACCAGCTT ATTCAATTTGGCAATGGAG GGGTGGTTGGAGGGGGGAATGTGGGGTTA GATAGTAAGTGCAATCT-3' (SEQ ID NO: 5) or with an aptamer of sequence 5'- ATACCAGCTTATTCAATTTGGCAATGGGGGGTGGTCGGAG GGGGTTTACGTGGGTTCGAGATAGTAAGTGCAATCT-3' (SEQ ID NO: 6).

7. Solid support on which an aptamer is immobilized according to any one of claims 1 to 6.

8. Use of an aptamer according to any one of claims 1 to 6 or of a solid support according to claim 7 to detect the CspZ protein in a biological sample.

9. Use of an aptamer according to any one of claims 1 to 6 or of a solid support according to claim 7 to detect a bacterium of the genus Borrelia in a biological sample.

10. An in vitro method for detecting the CspZ protein in a biological sample, said method comprising the following steps: a) contacting the biological sample with an aptamer according to any one of claims 1 to 6 or a solid support according to claim 7 in order to form a non-covalent complex between the CspZ protein and said aptamer; b) analyzing the biological sample to detect the presence of the non-covalent complex formed in step a).

11. An in vitro method for detecting a bacterium of the genus Borrelia in a biological sample, said method comprising the following steps: a) bringing the biological sample into contact with an aptamer according to any one of claims 1 to 6 or a solid support according to claim 7 in order to form a non-covalent complex between the bacterium of the genus Borrelia and said aptamer; b) analyzing the biological sample to detect the presence of the non-covalent complex formed in step a).

12. An in vitro method for diagnosing infection with a bacterium of the genus Borrelia in a subject, said method comprising (i) detecting the CspZ protein in a biological sample of said subject by implementing the method according to claim 10, or (ii) detecting a bacterium of the genus Borrelia in a biological sample of said subject by implementing the method according to claim 11.

13. A method according to any one of claims 10 to 12 or a use according to any one of claims 8 or 9, wherein the biological sample is selected from whole blood, blood serum, blood plasma, saliva, urine, cerebrospinal fluid, synovial fluid, genital secretions.

14. A method according to any one of claims 11 to 13 or use according to claim 9, wherein the bacterium of the genus Borrelia belongs to the genus Borrelia burgdorferi sensu lato, such as Borrelia burgdorferi sensu stricto, Borrelia garinii and / or Borrelia afzelii, preferably Borrelia burgdorferi sensu stricto.

15. Diagnostic kit for infection by a bacterium of the genus Borrelia comprising an aptamer according to any one of claims 1 to 6 or a solid support according to claim 7.

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