Modified trypanosoma cruzi TC24 polypeptide

Abstract

The invention provides a modified Trypanosoma cruzi Tc24 polypeptide comprising an amino acid substitution at one or more of amino acid positions: 23, 98, 141, or any combination thereof, relative to the parent Trypanosoma cruzi Tc24 polypeptide. The invention further provides a diagnostic test comprising the modified Trypanosoma cruzi Tc24 polypeptide and a method of diagnosing an individual with Chagas disease using the diagnostic test of claim.

Description

[0001] Modified Trypanosoma cruzi Tc24 polypeptide

[0002] Technical Field of the Invention

[0003] The present invention relates to a modified Trypanosoma cruzi Tc24 polypeptide. In particular, the invention relates to a heat stable modified Trypanosoma cruzi Tc24 polypeptide for the diagnosis of Chagas disease.

[0004] Background to the Invention

[0005] Chagas disease is a chronic life-threatening disease, caused by the parasite Trypanosoma cruzi (T. cruzi), which is endemic in Latin American countries and represents a significant global health burden worldwide. Chagas disease has an annual incidence of about 12,000 deaths, with 7 million people estimated to be infected worldwide and a further 140 million individuals at risk of contracting the disease. As a result of human migration and climate change, Chagas disease continues to expand beyond endemic zones (including countries in North America, Europe and Australia), and it has been estimated that current levels of Chagas disease cause global economic hardship of $7.19 billion per year worldwide.

[0006] Most people with Chagas disease are asymptomatic and therefore unaware of the underlying high risk and remain untreated. Current treatment methods are effective for the acute infection but have toxic side-effects and are poorly effective against chronic infection. Crucially, no vaccine nor robust and affordable diagnostic test is presently available for Chagas disease. Consequently, the World Health Organization has confirmed Chagas disease as a priority in its 2021-2030 Neglected Tropical Diseases roadmap. Current diagnostic tests have significant limitations including that they often detect infection too late after signs of the disease have already occurred, when current therapies are no longer effective. Some tests are also limited by shelf-life and require cold storage, resulting in diminishing sensitivity. Additional limitations include cost, in the case of peptide-based tests, a lack of broad strain specificity, and a requirement for specialised personnel in health care settings (i.e. Elecsys Chagas test). The latter is often logistically difficult to implement in rural areas and is not affordable for some developing world economies.

[0007] Thus, a practical and affordable solution is needed to address the health time bomb that is Chagas disease. A new diagnostic test that can be easily employed in low-middle income countries, where cold chain storage of diagnostic reagents is not practical, would be of particular benefit.

[0008] Additionally, the development of rapid and affordable at-home or in-clinic diagnostic tests for Chagas disease would not only enhance privacy for receiving results, but it also addresses the stigma associated with the condition, enabling individuals to detect the disease discreetly and seek timely medical intervention accordingly. More broadly, the tests could be used by travellers, military personnel and peacekeepers in endemic zones, without the need of any resource.

[0009] The Trypanosoma cruzi (T. cruzi ) Tc24 protein has been identified as a potential candidate for the development of Chagas disease diagnosis and therapeutics. Tc24 is a calcium sensor in the flagellum of the parasite T. cruzi- The Tc24 polypeptide is conserved in all strains of T. cruzi which cause Chagas disease. Owing to such high conservation, the Tc24 polypetide is an ideal target for the development of a broad- spectrum diagnostic test, overcoming the current limitations of strain- specific tests. However, the wild-type (naturally occurring) Tc24 polypeptide has been previously reported to be prone to aggregation.

[0010] In WO2017160849A1, Tc24 protein expression attempts focussed on mutating 4 cysteines in the Tc24 polypeptide to alanine (3CS1_A, NCBI, https: / / www.ncbi.nlm.nih.gov / protein / 192988514). However, this resulted in very limited increase in stability.

[0011] The aim of the present invention is to address the above problem by developing new engineered variants of Tc24 for diagnostic applications. The new engineered variants aim to have increased thermal stability, improved solubility and improved expression profiles, without compromising immunological efficacy.

[0012] Further, the present invention aims to provide a universal, rapid, affordable, easy-to-use, and robust diagnostic test that is heat stable and has an extended shelf life compared to other tests currently available.

[0013] It is also an aim of embodiments to the invention to overcome or mitigate at least one problem of the prior art, whether disclosed herein or not.

[0014] Summary of the Invention

[0015] According to a first aspect of the invention, there is provided a modified Trypanosoma cruzi Tc24 polypeptide comprising an amino acid substitution at one or more of amino acid positions 23, 98, 141, or any combination thereof, relative to the parent Trypanosoma cruzi Tc24 polypeptide. The or each amino acid substitution may be in a position relative to the same amino acid position in the parent Trypanosoma cruzi Tc24 polypeptide. Sequence comparisons to determine the equivalent amino acid positions between the modified Trypanosoma cruzi Tc24 polypeptide and the parent Trypanosoma cruzi Tc24 polypeptide may be performed using EMBOSS Needle (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle) using Matrix: EBLOSUM62, Gap_penalty: 10.0, Extend_penalty: 0.5.

[0016] The parent Trypanosoma cruzi Tc24 polypeptide may comprise a wild-type Trypanosoma cruzi Tc24 polypeptide.

[0017] The modified Trypanosoma cruzi Tc24 polypeptide may comprise a parent polypeptide comprising SEQ ID NO: 1 or 2 comprising an amino acid substitution at one or more of amino acid positions: 23, 98, 141, or any combination thereof, relative to the same amino acid positions in SEQ ID NO: 1 or 2.

[0018] By amino acid substitution, it is meant that an amino acid at a specified position within the parent Trypanosoma cruzi Tc24 polypeptide is substituted by a naturally occurring or non-naturally occurring amino acid that is different to the amino acid that is present at the position in the parent Trypanosoma cruzi Tc24 polypeptide.

[0019] Throughout the description herein, the “modified Trypanosoma cruzi Tc24 polypeptide” is named “modified Tc24 polypeptide”, and the “wild-type Trypanosoma cruzi Tc24 polypeptide” is named “wild-type Tc24 polypeptide”.

[0020] The parent Tc24 polypeptide may be the full-length wild-type Tc24polypeptide. The modified Tc24 polypeptide may be derived from the full-length wild-type Tc24 polypeptide. The modified Tc24 polypeptide may be derived from a known strain of Tc24. The parent Tc24 polypeptide may be the known strain of NCBI PDB reference: 3CS1_A (https: / / www.ncbi.nlm.nih.gov / protein / 192988514), or NCBI reference sequence: XP_805575.1 (https: / / www.ncbi.nlm.nih.gov / protein / XP_805575.1). The parent Tc24 polypeptide may be SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitutions described herein relative to the same positions in SEQ ID NO: 1 or 2.

[0021] As mentioned above, the wild-type Tc24 polypeptide is found in a calcium sensor in the flagellum of Trypanosoma cruzi (T. cruzi). The wild-type Tc24 polypeptide is known to aggregate and form intermolecular disulfide bonds, making it difficult to develop for diagnostic and treatment purposes.

[0022] The modified Tc24 polypeptide of the present invention was found to have reduced aggregation, improved solubility, and improved thermal stability compared to the wild-type Tc24 polypeptide. The modified Tc24 polypeptide of the present invention also provides the benefit of a longer shelf life and is not affected by freeze thawing cycles. Thus, the modified Tc24 polypeptide of the present invention is much more well-suited for diagnostic and treatment purposes compared to the wild-type.

[0023] The modified Tc24 polypeptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid substitutions, relative to the parent Tc24 polypeptide. Preferably, the modified Tc24 polypeptide comprises at least 3 amino acid substitutions, relative to the parent Tc24 polypeptide.

[0024] The modified Tc24 polypeptide may comprise no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions, relative to the parent Tc24 polypeptide. Preferably, the modified Tc24 polypeptide comprises no more than 12 amino acid substitutions, relative to the parent Tc24 polypeptide.

[0025] The modified Tc24 polypeptide may comprise between 1-25 amino acid substitutions, preferably, between 2-15 amino acid substitutions, or more preferably between 3-13 amino acid substitutions, relative to the parent Tc24 polypeptide.

[0026] In some embodiments, the modified Tc24 polypeptide may comprise amino acid substitutions at amino acid positions 23, 98 and 141, relative to the parent Tc24 polypeptide.

[0027] In some embodiments, the modified Tc24 polypeptide comprises at least one of the following amino acid substitutions: K23S, S98A, A141T, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the following amino acid substitutions: K23S, S98A, and A141T, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. Alternative amino acid substitutions at these positions may be possible. In particular, the amino acid substitution may involve an alternative amino acid with similar properties, e.g. K23T instead of K23S.

[0028] The modified Tc24 polypeptide may comprise the amino acid substitution K23S and one or more amino acid substitution at position 98 or 141, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S98A and one or more amino acid substitution at position 23 or 141, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A141T and one or more amino acid substitution at position 23 or 98, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0029] The modified Tc24 polypeptide may comprise the amino acid sequence of SEQ ID NO: 4.

[0030] In some embodiments, the modified Tc24 polypeptide comprises an amino acid substitution at one or more of amino acid positions: 23, 98, 141, 44, 61, 72, 170, 194, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise amino acid substitutions at amino acid positions 23, 98, 141, 61, 72, 170 and 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0031] In some embodiments, the modified Tc24 polypeptide comprises at least one of the following amino acid substitutions: K23S, S98A, A141T, A44E, E61N, S72K, A170D, S194A, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the following amino acid substitutions: K23S, S98A, A141T, E61N, S72K, A170D, and S 194A, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0032] In some embodiments, the modified Tc24 polypeptide comprises an amino acid substitution at one or more of amino acid positions: 23, 98, 141, 44, 61, 72, 156, 170, 194, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise amino acid substitutions at amino acid positions 23, 98, 141, 61, 72, 156, 170 and 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0033] In some embodiments, the modified Tc24 polypeptide comprises at least one of the following amino acid substitutions: K23S, S98A, A141T, A44E, E61N, S72K, V156A, A170D, S194A, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the following amino acid substitutions: K23S, S98A, A141T, E61N, S72K, V156A, A170D, and S194A, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. Alternative amino acid substitutions at these positions may be possible. In particular, the amino acid substitution may involve an alternative amino acid with similar properties, e.g. K23T instead of K23S.

[0034] The modified Tc24 polypeptide may comprise the amino acid substitution K23S and one or more amino acid substitution at position 44, 61, 72, 98, 141, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A44E and one or more amino acid substitution at position 23, 61, 72, 98, 141, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution E61N and one or more amino acid substitution at position 23, 44, 72, 98, 141, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S72K and one or more amino acid substitution at position 23, 44, 61, 98, 141, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S98A and one or more amino acid substitution at position 23, 44, 61, 72, 141, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A141T and one or more amino acid substitution at position 23, 44, 61, 72, 98, 156, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution V156A and one or more amino acid substitution at position 23, 44, 61, 72, 98, 141, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A170D and one or more amino acid substitution at position 23, 44, 61, 72, 98, 141, 156, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S194A and one or more amino acid substitution at position 23, 44, 61, 72, 98, 141, 156, or 170, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0035] The modified Tc24 polypeptide may comprise the amino acid sequence of SEQ ID NO: 5.

[0036] In some embodiments, the modified Tc24 polypeptide comprises an amino acid substitution at one or more of amino acid positions: 23, 44, 61, 66, 72, 98, 141, 157, 161, 166, 170, 194, 196, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise amino acid substitutions at amino acid positions 23, 98, 141, 61, 72, 156, 170, 194, 44, 157, 161, 166 and 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise amino acid substitutions at amino acid positions 23, 98, 141, 61, 66, 72, 156, 170, 194, 44, 157, 161, 166 and 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0037] In some embodiments, the modified Tc24 polypeptide comprises at least one of the following amino acid substitutions: A44E, P157D, A161E, V166I, K23S, S98A, A141T, E61N, C66S, S72K, A170D, S194A, V196K, or any combination thereof, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the following amino acid substitutions: A44E, P157D, A161E, V166I, K23S, S98A, A141T, E61N, S72K, A170D, S194A and V196K, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the following amino acid substitutions: A44E, P157D, A161E, V166I, K23S, S98A, A141T, E61N, C66S, S72K, A170D, S194A and V196K, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. Alternative amino acid substitutions at these positions may be possible. In particular, the amino acid substitution may involve an alternative amino acid with similar properties, e.g. K23T instead of K23S.

[0038] The modified Tc24 polypeptide may comprise the amino acid substitution K23S and one or more amino acid substitution at position 44, 61, 66, 72, 98, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A44E and one or more amino acid substitution at position 23, 61, 66, 72, 98, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution E61N and one or more amino acid substitution at position 23, 44, 66, 72, 98, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution C66S and one or more amino acid substitution at position 23, 44, 61, 72, 98, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S72K and one or more amino acid substitution at position 23, 44, 61, 66, 98, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution S98A and one or more amino acid substitution at position 23, 44, 61, 66, 72, 141, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A141T and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 157, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution P157D and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 161, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A161E and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 157, 166, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution V166I and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 157, 161, 170, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution A170D and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 157, 161, 166, 194, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution SI 94 A and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 157, 161, 166, 170, or 196, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2. The modified Tc24 polypeptide may comprise the amino acid substitution V196K and one or more amino acid substitution at position 23, 44, 61, 66, 72, 98, 141, 157, 161, 166, 170, or 194, relative to the same amino acid positions in the parent Tc24 polypeptide of SEQ ID NO: 1 or 2.

[0039] The modified Tc24 polypeptide may comprise the amino acid sequence of SEQ ID NO: 6.

[0040] The modified Tc24 polypeptide may include one or more further substitutions, deletions, additions or other mutations, in addition to those substitutions disclosed and defined hereinabove. In some embodiments, the modified Tc24 polypeptide may comprise an N-terminal modification. The N-terminal modification may help with purification of the modified Tc24 polypeptide. The N-terminal modification may reduce aggregation. The N-terminal modification may improve the thermal stability of the modified Tc24 polypeptide.

[0041] The N-terminal modification may comprise a SpyTag peptide and / or a TEV protease cleavage site. The SpyTag peptide may have the amino acid sequence “AHIVMVDAYKPTK”. The TEV protease cleavage site may have the amino acid sequence “ENLYFQG”.

[0042] The N-terminal modification may comprise the amino acid sequence of SEQ ID NO: 3.

[0043] The modified Tc24 polypeptide may comprise the amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9.

[0044] The modified Tc24 polypeptide with N-terminal modification may comprise at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, or at least 230 amino acid residues. Preferably the modified Tc24 polypeptide with N-terminal modification comprises between 205-235 amino acid residues, or more preferably around 231 amino acid residues.

[0045] The modified Tc24 polypeptide (without any N-terminal modification) may comprise an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the wild-type Tc24 polypeptide, the parent Tc24 polypeptide, or SEQ ID NO: 1 or 2. Sequence identity may be determined using EMBOSS Needle (htps: / / www.ebi.ac.uk / jdispatcher / psa / emboss needle) using Matrix: EBLOSUM62, Gap_penalty: 10.0, Extend_penalty: 0.5.

[0046] Preferably, the modified Tc24 polypeptide (without any N-terminal modification) may comprise an amino acid sequence having at least 90% identity with the wild-type Tc24 polypeptide, the parent Tc24 polypeptide, or SEQ ID NO: 1 or 2.

[0047] The modified Tc24 polypeptide of the first aspect of the present invention is heat stable at a temperature of at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 56 °C, at least 57 °C, at least 58 °C, at least 59 °C, at least 60 °C, at least 61 °C, at least 62 °C, at least 63 °C, at least 64 °C, at least 65 °C, at least 66 °C, at least 67 °C, at least 68 °C, at least 69 °C, or at least 70 °C. Preferably, the modified Tc24 polypeptide is heat stable at a temperature of at least 60 °C, more preferably at least 65 °C, or even more preferably at least 67 °C. In most preferable embodiments, the modified Tc24 polypeptide is heat stable at a temperature of around 69 °C. The modified Tc24 polypeptide may be heat stable in solution at the temperatures identified above.

[0048] The modified Tc24 polypeptide is heat stable up to the melting temperature of the protein, at which point the secondary structure of the protein begin to unfold.

[0049] Embodiments of the modified Tc24 polypeptide comprising a greater number of the specified amino acid substitutions above may result in greater thermal stability.

[0050] The modified Tc24 polypeptide of the first aspect of the present invention is capable of detecting Tc24 specific antibodies (anti-Tc24 antibodies), preferably with high specificity and sensitivity. Thus, the immunological efficacy of the modified Tc24 polypeptides is not compromised. In some embodiments, the modified Tc24 polypeptide may be expressed by a vector. The vector may comprise a nucleic acid sequence that encodes the modified Tc24 polypeptide. The nucleic acid sequence that encodes the modified Tc24 polypeptides may be codon optimised. Methods of codon optimisation are known. For example, codon optimisation may be achieved using an online tool. The codon optimised nucleic acid sequence may comprise the nucleic acid sequence of SEQ ID NO: 12, 13 or 14.

[0051] According to a second aspect of the invention, there is provided a polypeptide comprising a parent Trypanosoma cruzi Tc24 polypeptide and an N-terminal modification comprising amino acid sequence SEQ ID NO: 3.

[0052] The parent Tc24 polypeptide may be as described in the first aspect of the invention.

[0053] The parent Tc24 polypeptide may have the amino acid sequence of SEQ ID NO: 1 or 2.

[0054] The polypeptide of the second aspect of the invention is easier to purify compared to the patent Tc24 polypeptide. The N-terminal modification reduces aggregation and has improved thermal stability.

[0055] The polypeptide of the second aspect of the invention may be heat stable at a temperature of at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 56 °C, at least 57 °C, at least 58 °C, at least 59 °C, at least 60 °C, or at least 62 °C. Preferably, the polypeptide may be heat stable at a temperature of at least 56, more preferably at least 58 °C, or most preferably around 60 °C. The polypeptide of the second aspect may be heat stable in solution at the temperatures identified above. In some embodiments, the polypeptide of the second aspect of the invention may be expressed by a vector. The vector may comprise a nucleic acid sequence that encodes the polypeptide of the second aspect of the invention. The nucleic acid sequence that encodes the polypeptide of the second aspect of the invention may be codon optimised.

[0056] According to a third aspect of the invention, there is provided a vector comprising a nucleic acid sequence that expresses the modified Tc24 polypeptide of the first aspect of the present invention, or the polypeptide of the second aspect of the invention.

[0057] The modified Tc24 polypeptide and its components may be as described for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0058] The polypeptide of the second aspect of the invention and its components may be as described for the second aspect and may include any optional feature or combination of optional features described for the second aspect.

[0059] The nucleic acid sequence that encodes the polypeptide of the second aspect, or the modified Tc24 polypeptides may be codon optimised. Methods of codon optimisation are known. For example, codon optimisation may be achieved using an online tool. The codon optimised nucleic acid sequence that encodes the modified Tc24 polypeptide of the first aspect of the invention may comprise the nucleic acid sequence of SEQ ID NO: 12, 13 or 14.

[0060] In some embodiments, the vector is a viral vector or a DNA plasmid vector. In specific embodiments, the vector may be a pET plasmid vector, more specifically a pET15b plasmid vector.

[0061] The pET plasmid vector that expresses the modified Tc24 polypeptide of the first aspect of the invention may comprise the nucleic acid sequence of SEQ ID NO: 16, 17, or 18.

[0062] The vector may be suitable for expression in a host cell. The host cell may be a bacterial host cell, for example, E. coli.

[0063] According to a fourth aspect of the invention, there is provided a diagnostic test comprising the modified Trypanosoma cruzi Tc24 polypeptide of the first aspect of the invention.

[0064] The modified Tc24 polypeptide and its components may be as described for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0065] The diagnostic test may be an ELISA, or similar.

[0066] The diagnostic test may be an antigen-based test.

[0067] The diagnostic test may be a lateral flow test, such as a rapid lateral flow test. In such embodiments, the modified Tc24 polypeptides may be deposited on a nitrocellulose membrane of the lateral flow test.

[0068] Thus, the diagnostic test may be simple and easy to use, produce rapid and easy-to-understand results, and can be used in an at-home test setting without the need for specialist equipment. In some embodiments, the diagnostic test may produce results in no more than 1 hour, no more than 50 minutes, no more than 40 minutes, no more than 30 minutes, no more than 25 minutes, no more than 20 minutes, no more than 15 minutes, no more than 10 minutes, no more than 9 minutes, no more than 8 minutes, no more than 7 minutes, no more than 6 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, no more than 1 minute, or no more than 30 seconds.

[0069] According to a fifth aspect of the invention, there is provided a method of diagnosing an individual with Chagas disease using with diagnostic test of the fourth aspect of the invention, and wherein the method comprises applying a sample taken from an individual with Chagas disease to the diagnostic test, and reading a positive signal produced by the diagnostic test.

[0070] The diagnostic test and its components may be as described for the fourth aspect of the invention and may include any optional feature or combination of optional features described for the fourth aspect.

[0071] The method of diagnosis may be used in the surveillance of Chagas disease. The individual may be a mammal. The individual may be a human, a cat, a camel, a racoon, a marsupial, a rabbit, or a dog.

[0072] In some embodiments, the sample taken from the individual is first diluted before applying to a sample well of the diagnostic test. The sample may be a sample of bodily fluid taken from the individual, for example, blood sample or serum. A blood sample may be taken, for example, from the finger of the individual. The positive signal may be a colour change, or the appearance or disappearance of an indicator. In some embodiments, diagnostic test may be connected to a visual display device and the positive signal may be shown on the device.

[0073] Detailed Description of the Invention

[0074] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0075] Figure 1 shows (A) the CD-melt temperatures of three different embodiments of the modified Tc24 polypeptides according to the first aspect of the invention (Tx74, Tx75, Tx76) and the control Tc24 polypeptide (Tx70); (B) the CD thermal melt curve of the control Tc24 polypeptide, Tx70; (C) the CD thermal melt curve of the modified Tc24 polypeptide, Tx74; (D) the CD thermal melt curve of the modified Tc24 polypeptide, Tx75; and (E) the CD thermal melt curve of the modified Tc24 polypeptide, Tx76;

[0076] Figure 2 shows the results of an TROSY-NMR experiment of wild- type Tc24 polypeptide (100 pM in PBS), performed at 20 °C at different time points. The experiment was recorded on a 600 MHz magnet;

[0077] Figure 3 shows the results of EEISA testing of the control Tc24 polypeptide (Tx70) and three different embodiments of the modified Tc24 polypeptides according to the first aspect of the invention (Tx74, Tx75 and Tx76), against human sera from Chagas patients from Northern and Southern hemispheres (0.7 mg / mL of polypeptide were used); Figure 4 illustrates the mapping of the mutations in each Tx24 variant (Tx74 (left), Tx75 (middle) and Tx76 (right)), during the protein engineering process, onto wild-type Tc243CS1 structure, using PyMOL;

[0078] Figure 5 are graphs showing hydrodynamic radius changes recorded after lyophilisation (Fig. 5A) and multiple freeze-thaw cycles of the modified Tc24 polypeptides (Tx75, Tx75 and Tx76) (Fig. 5B); and Figure 6 shows H-NMR analysis of stability in solution over time at 303K of Tc24 WT (A-B) and the Tc24 polypeptide variants (C-E);

[0079] Figure 7 shows a lateral flow diagnostic test strip which utilises the modified Tc24 polypeptides of the invention (A) and duplicate test strip results showing positive identification of Chagas disease (B).

[0080] The following examples demonstrate the development of three different embodiments of a modified Tc24 polypeptides according to the first aspect of the present invention. The three embodiments are named Tx74, Tx75 and Tx76 variants throughout the examples.

[0081] The examples also demonstrate the expression of the Tx74, Tx75 and Tx76 variants in E. coli, with the variants showing improved expression profiles.

[0082] Furthermore, the examples show the increased thermal stability of the Tx74, Tx75 and Tx76 variants compared to the control Tx70, and their ability to detect anti- Tc24 antibodies.

[0083] Example 1 - Design of modified Tc24 polypeptides: Two known parent Tc24 strains (3CS1_A and CL Brenner), their sequences shown in SEQ ID NO: 1 and 2 respectively, were used as parent Tc24 polypeptides for the development of modified Tc24 polypeptides with improved properties.

[0084] A computational design algorithm (PROSS2,

[0085]

[0086] was used to design modified Tc24 polypeptides with improved thermal stability, improved expression profiles compared to the control Tc24 polypeptide, but without compromising immunological efficacy.

[0087] The existing structure of Tc24 and the amino acid sequence of the 3CS1_A

[0088]

[0089] reference:

[0090] 3CS1_A, SEQ ID NO: 1) was used as template in PROSS2 to identify the set of mutations, predicted to stabilise the protein.

[0091] Three promising variants were identified, each with amino acid substitutions at key amino acid positions. The three identified variants can be described by their amino acid sequences as follows:

[0092] i. Tx74 variant - the 3CS1_A Tc24 polypeptide sequence as shown in SEQ ID NO: 1 with the following amino acid substitutions; K23S, S98A and A141T, relative to 3CS 1_A Tc24 polypeptide. Tx74 variant sequence shown in SEQ ID NO: 4.

[0093] ii. Tx75 variant - the 3CS1_A Tc24 polypeptide sequence as shown in SEQ ID NO: 1 with the following amino acid substitutions; K23S, E61N, S72K, S98A, A141T, V156A, A170D and S194A, relative to the 3CS1_A Tc24 polypeptide. Tx75 variant sequence shown in SEQ ID NO: 5. iii. Tx76 variant - the 3CS1_A Tc24 polypeptide sequence as shown in SEQ ID NO: 1 with the following amino acid substitutions; K23S, A44E, E61N, C66S, S72K, S98A, A141T, P157D, A161E, V166I, A170D, S194A and V196K, relative to the 3CS1_A Tc24 polypeptide. Tx76 variant sequence shown in SEQ ID NO: 6.

[0094] Sequence comparisons to determine the equivalent amino acid positions between the variants and the parent Tc24 polypeptide were performed using EMBOSS Needle (https: / / www.ebi. ac.uk / jdispatd er / psa / emboss needle) using Matrix: EBLOSUM62, Gap_penalty: 10.0, Extend_penalty: 0.5.

[0095] Figure 4 shows the mapping of the mutations of each variant onto the parent Tc24 structure.

[0096] Tx74, Tx75 and Tx76 were further modified with an N-terminal modification to produce Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) respectively.

[0097] For diagnostic tests on human sera, the Tc24 CL Brenner strain (NCBI reference sequence: XP 805575. L hitpsd / www.iicbiml. iih.goy / prot dXP 805575.1, SEQ ID NO: 2) comprising the N-terminal modification was used as a control. For biophysical tests, the Tc24 3CS1_A strain (NCBI PDB reference: 3CS1_A, https: / / www.ncbi.nlm.nih.gov / protein / 192988514) was used as a control (no N-terminal modification). The control Tc24 polypeptides are named “Tx70” herein.

[0098] The N-terminal modification consists of a SpyTag peptide sequence and a TEV protease cleavage site and has an amino acid sequence of SEQ ID NO: 3, comprising 20 amino acid residues. Consequently, the Tx74 (N-mod) variant has the amino acid sequence of SEQ ID NO: 7, the Tx75 (N-mod) variant has the amino acid sequence of SEQ ID NO: 8, and the Tx76 (N-mod) variant has the amino acid sequence of SEQ ID NO: 9. Tx74, Tx75 and Tx76, each variant comprises 231 amino acid residues.

[0099] Example 2 -Expression of modified Tc24 polypeptides in E. coli

[0100] The genes encoding Tx70, Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) were cloned into the plasmid, pET-15b (Addgene:

[0101]

[0102] databa e / 254 / ).

[0103] When cloning the genes into the pET-15b plasmid, the amino acid sequence of SEQ ID NO: 10 was added at the N-terminal of the amino acid sequences of Tx70, Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod).

[0104] The genes for cloning into the vector were codon optimised to increase protein expression. Codon optimisation was achieved using an online tool from TWIST Bioscience. The codon optimised nucleic acid sequence of the gene encoding the Tx70 has the sequence of SEQ ID NO: 11. The codon optimised nucleic acid sequence of gene encoding the Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) variants has the sequence of SEQ ID NO: 12, 13, and 14, respectively.

[0105] The resulting plasmids comprising the genes encoding the Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) have the nucleic acid sequence of SEQ ID NO: 16, 17, and 18, respectively.

[0106] The plasmids were transformed into BL21(DE3) pLysS E. coli competent cells. These cells were cultured overnight at 37 °C in 50 mL of culture media (2YT or Hyper Broth media with Nutrient Glucose Mix (500 mL) (AthenaES) supplied with 50 ug / mL ampicillin) and incubated in an INFORS 10 L fermenter (or ElectroLab SIP 10L Vessel with ElectroLab FerMac 360 Tower).

[0107] Once the culture reached an OD600 of 0.6, expression was induced using 0.5 mM IPTG for 21 h at 20 °C and under O2 (DO level of 30), pH 7.2 and stirring (250 rpm) conditions (stirring conditions automatically adjusted from a cascade setting to control DO level at 30). A bacterial pellet was then harvested from the 10 L culture by centrifugation at 4000xg for 15-20 min at room temperature and stored at -20 °C. The pellet was resuspended in 1 x PBS (50mM NaPi, 500mM NaCl, 25mM imidazole pH 7.4) with one protease inhibitor cocktail tablet per 50 mL (Roche).

[0108] The proteins were purified by affinity chromatography, washed with 50 mM NaPi, 500 mM NaCl, 25 mM imidazole (pH 7.4) and eluted in the same buffer supplemented with 0.25 M imidazole.

[0109] Protein expression was assessed by SDS-PAGE gel electrophoresis and Western blot, using anti-His6 (Invitrogen) or anti-Spytag (Biorad) antibodies.

[0110] The proteins were purified by size-exclusion chromatography with Superdex 200 Increase 10 / 300 GL, or similar.

[0111] Interestingly, the vectors expressing the Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) variants yielded greater than 10-fold increase in protein expression compared to the vector expressing the Tx70 (Tc24 CL Brenner strain with N-terminal modification).

[0112] Example 3 - Suitability of the modified Tc24 polypeptides as a diagnostic test with improved properties CD-melt experiments were conducted at beamline B24 at Diamond Light Source synchrotron. CD-melt experiments of the Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) variants show improved thermal stability as shown in Figures 1A-E.

[0113] Figures 1C-E show that Tx74 (N-mod) variant is stable up to around 60 °C, Tx75 (N-mod) is stable up to around 67 °C, and Tx76 (N-mod) is stable up to around 69 °C. Figure IB shows that Tx70 (Tc24 derived from CL Brenner strain with N-terminal modification) is stable up to around 60 °C.

[0114] For comparison, Figure 2 shows that the Tx70 (Tc24 derived from CL Brenner strain with N-terminal modification) is stable in solution for two weeks at 20 °C at 100 pM.

[0115] Fig. 5 shows FIDA analysis of the Tc24 variants after lyophilisation and multiple (5) freeze-thaw cycles. Figure 5A show hydrodynamic radius (nm) changes recorded across multiple flash-cooling thawing cycles with liquid N2 for Tc24 WT, Tc24 WT His-tagged and Tc24 variants Tx74, Tx75 and Tx76. A two-way RM ANOVA revealed no significant change of Rh by freeze-thaw cycle (F(2.441, 34.17) = 2.26, P = 0.11; Geisser-Greenhouse corrected). Figure 5B shows hydrodynamic radius (nm) changes recorded before (NL) and after lyophilisation (L) conditions. Data are presented as mean ± SEM. Statistical analysis was performed using Welsh’ sT-test (Tc24 WT His tagged t(3.856) = 5.906, P = 0.0046, two-tailed; Tx74 t(3.231) = 18.33, P = 0.0002; Tx75 t(2.437) = 6.946, P = 0.0116; Tx761(2.018) = 2.488, P = 0.0087). All experiments were performed in IX PBS pH 7.4. The results of Figure 5 show that stability is achieved by each of variants Tx74, Tx75 and Tx76 after lyophilisation and across 5 cycles of freeze-thawing, whereas the WT proteins were not stable or were less stable than the variants.

[0116] Figure 6 shows1H-NMR analysis of stability in solution over time of tagless Tc24 WT (TC243CSI TagieSS, Fig. 6A), Tc24 WT (Tc243CSlFig 6B) and Tc24 variants Tx74 (Fig 6C), Tx75 (Fig 6D) and Tx76 (Fig 6E). 'H-NMR experiments recorded on an 800 MHz spectrometer at 303 K at different time points in PBS buffer show stability up to 69 days. NMR experiments were performed at the Leicester Institute of Structural and Chemical Biology (LISCB, University of Leicester). Protein storage was at 278K for the entire length of the experiment. As can be seen from the graphs, the TC24-WT3CSI protein started unfolding after 12 days, whilst all of variants Tx74, Tx75 and Tx76 remained stable in solution for the same amount of time as indicated by the disappearance or broadening of the typical amide group peak at around 7 ppm over time.

[0117] Such improved thermal stability demonstrate that each variant is ideal for use in an antigen-based diagnostic test that does not require cold-storage and can be used in hot countries where Chagas Disease is endemic.

[0118] Furthermore, the Tx74 (N-mod), Tx75 (N-mod) and Tx76 (N-mod) variants were screened against Chagas patient sera versus healthy controls.

[0119] As shown in Figure 3, all variants gave a positive signal in ELISA across all T. cruzi genotypes and performed similarly to the control Tx70 (Tc24 derived from 3CS1_A strain with no N-terminal modification). This indicates the immunological efficacy of the variants, and that they are able to detect Tc24 specific antibodies (anti- Tc24 antibodies) in Chagas positive patients. Feasibility of diagnostic testing and retention of antibody detection from patients was assessed by immobilising Tc24 onto nitrocellulose strips in lateral flow test (LFT from Biorender)) cassettes and samples of patient sera were applied from the highest titre of Chagas disease from the southern region. In parallel the specificity to detect Chagas disease versus malaria and leishmaniasis was assessed. Figure 7A shows the LFT device, while Figure 7B shows positive identification of Chagas using the test strips.

[0120] These results validate each variant for use in broad-spectrum antigen-based diagnostic test.

[0121] The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

[0122]

[0123] KLEAWGAKVEDPAALFKELDKNGTGSVTFDEFAAWASAVKLDADGDPDNV PESA

[0124] SEQ ID NO: 2 ( CL Brenner strain Tc24) MGACGSKDSTSDKGLASDKDGKNAKDRKEAWERIRQAIPREKTAEAKQRRI ELFKKFDKNETGKLCYDEVHSGCLEVLKLDEFTPRVRDITKRAFDKARALGS KLENKGSEDFVEFLEFRLMLCYIYDFFELTVMFDEIDASGNMLVDEEEFKRAV PKLEAWGAKVEDPAALFKELDKNGTGSVTFDEFAAWASAVKLDADGDPDN VPESA

[0125] SEQ ID NO: 3 (N-terminal modification with the SpyTag peptide sequence underlined and the TEV protease cleavage site sequence in bold)

[0126]

[0127] Sl_A Tc24 including mutations K23S, S98A, A141T in bold) MGACGSKGSTSDKGLASDKDGKSAKDRKEAWERIRQAIPREKTAEAKQRRIE LFKKFDKNETGKI.. CYDEVYSGCLEVLKLDEFTSRVRDITKRAFDKARTT.. GSK LENKGSEDFVEFUiFRLMLCYIYDFFELTVMFDEIDTSGNMLVDEEEFKRAVP KLEAWGAKVEDPAALFKELDKNGTGSVTFDEFAAWASAVKLDADGDPDNV PESA

[0128] SEQ ID NO: 5 (Tx75; variant of3CSl_A Tc24 including mutations K23S, E61N, S72K, S98A, A141T, V156A, A170D, S194A in bold) MGACGSKGSTSDKGLASDKDGKSAKDRKEAWERIRQAIPREKTAEAKQRRIE LFKKFDKNNTGKLC YDE V YKGCLEVLKLDEFTS R VRDITKR AFD KARTLGS K LENKGSEDFVEFLEFRLMLCYIYDFFELTVMFDEIDTSGNMLVDEEEFKRAAP KLEAWGAKVEDPDALFKELDKNGTGSVTFDEFAAWAAAVKLDADGDPDNV PESA

[0129] SEQ ID NO: 6 (Tx76; variant of3CSl_A Tc24 including mutations K23S, A44E, E61N, C66S, S72K, S98A, A141T, P157D, A161E, V166I, A170D, S194A, V196Kin bold) MGACGSKGSTSDKGLASDKDGKSAKDRKEAWERIRQAIPREKTEEAKQRRIE LFKKFDKNNTGKLSYDEVYKGCLEVLKLDEFTSRVRD1TKRAFDKARTLGSK LENKGSEDFVEFLEFRLMLCYIYDFFELTVMFDEIDTSGNMLVDEEEFKRAVD KLEEWGAKIEDPDALFKELDKNGTGSVTFDEFAAWAAAKKLDADGDPDNV PSA SEQ ID NO: 7 (Tx74 variant with N -terminal modification (underlined)) AHIVMVDAYKPTKENLYFQGMGACGSKGSTSDKGLASDKDGKSAKDRKEA WERIRQAIPREKTAEAKQRRIELFKKFDKNETGKLCYDEVYSGCLEVLKLDEF TSRVRDITKRAFDKARTLGSKLENKGSEDFVEFLEFRLMLCYIYDFFELTVMF DEIDTSGNMLVDEEEFKRAVPKLEAWGAKVEDPAALFKELDKNGTGSVTFD EFAAWASAVKLDADGDPDNVPESA

[0130] SEQ ID NO: 8 (Tx75 variant with N-terminal modification (underlined)) AHIVMVDAYKPTKENLYFQGMGACGSKGSTSDKGLASDKDGKSAKDRKEA WERIRQAIPREKTAEAKQRRIELFKKFDKNNTGKLCYDEVYKGCLEVLKLDE FTSRVRDITKRAFDKARTLGSKLENKGSEDFVEFLEFRLMLCYIYDFFELTVM FDEIDTSGNMLVDEEEFKRAAPKLEAWGAKVEDPDALFKELDKNGTGSVTFD EFAAWAAAVKLDADGDPDNVPESA

[0131] SEQ ID NO: 9 (Tx76 variant with N-terminal modification (underlined)) AHIVMVDAYKPTKENLYFQGMGACGSKGSTSDKGLASDKDGKSAKDRKEA WERIRQAIPREKTEEAKQRRIELFKKFDKNNTGKLSYDEVYKGCLEVLKLDEF TSRVRDITKRAFDKARTLGSKLENKGSEDFVEFLEFRLMLCYIYDFFELTVMF DEIDTSGNMLVDEEEFKRAVDKLEEWGAKIEDPDALFKELDKNGTGSVTFDE FAAWAAAKKLDADGDPDNVP

[0132] SEQ ID NO: 10 HHHHHHSSGLVPRGSHM

[0133] SEQ ID NO: 11 (codon optimised Tx70 derived from 3CS1_A Tc24) GCCCATATTGTTATGGTGGATGCATATAAACCGACCAAAGAGAACCTGTA TTTTCAAGGTATGGGTGCATGTGGTAGCAAAGATAGCACCAGCGATAAAG GTCTGGCAAGTGATAAAGATGGCAAAAATGCCAAAGATCGTAAAGAAGC ATGGGAACGTATTCGTCAGGCAATTCCGCGTGAAAAAACCGCAGAAGCAA AACAGCGTCGTATTGAACTGTTCAAAAAGTTTGACAAAAACGAAACCGGC AAACTGTGCTATGATGAAGTTCATAGCGGTTGTCTGGAAGTTCTGAAACT GGATGAATTTACACCGCGTGTTCGTGATATTACCAAACGTGCATTTGATAA AGCACGTGCCCTGGGTAGCAAACTGGAAAATAAAGGTAGCGAAGATTTCG TCGAGTTTCTGGAATTTCGTCTGATGCTGTGCTATATCTATGATTTTTTTGA GCTGACCGTGATGTTCGATGAAATTGATGCAAGCGGTAATATGCTGGTTG ATGAAGAAGAATTTAAACGCGCAGTGCCGAAACTGGAAGCCTGGGGTGC AAAAGTTGAAGATCCGGCAGCACTGTTTAAAGAGCTGGATAAAAACGGC ACCGGTAGCGTTACCTTTGATGAATTTGCAGCATGGGCAAGCGCAGTTAA ACTGGACGCAGATGGTGATCCGGATAATGTTCCGGAAAGCGCA

[0134] SEQ ID NO: 12 (codon optimised Tx74 (N-mod)) GCTCACATCGTCATGGTGGACGCGTACAAGCCGACGAAAGAGAATTTATA TTTTCAGGGTATGGGCGCTTGCGGGAGTAAAGGCAGCACCAGCGATAAAG GCCTTGCTTCGGATAAGGATGGTAAGTCAGCCAAAGACCGCAAGGAGGCC TGGGAACGGATTCGGCAGGCGATCCCCCGGGAGAAAACGGCTGAGGCCA AACAACGGAGAATCGAGCTGTTTAAAAAATTTGACAAAAACGAAACCGG TAAACTTTGCTACGATGAAGTGTATTCCGGTTGTTTGGAGGTCTTGAAATT GGATGAGTTTACGTCTCGAGTTCGTGATATCACTAAGCGGGCTTTCGATAA AGCACGGACACTGGGGAGCAAGTTAGAAAACAAAGGCAGCGAGGACTTC GTGGAGTTCCTCGAATTTCGTCTCATGTTATGTTATATATACGATTTTTTTG AATTAACCGTTATGTTTGATGAAATAGACACCTCGGGCAATATGTTAGTTG ATGAAGAAGAATTCAAACGTGCTGTGCCTAAGTTGGAGGCCTGGGGGGCA AAAGTGGAAGACCCCGCTGCCCTGTTTAAGGAGTTAGACAAAAATGGGAC CGGTTCGGTAACCTTTGATGAATTTGCAGCCTGGGCGTCTGCAGTCAAGCT GGATGCCGACGGCGATCCCGATAATGTCCCGGAATCAGCGTAA

[0135] SEQ ID NO: 13 (codon optimised Tx75 (N-mod)) GCACATATTGTCATGGTGGATGCGTATAAACCTACAAAGGAAAATCTTTA TTTCCAAGGCATGGGGGCATGTGGCAGCAAGGGGTCGACTTCAGATAAAG GCCTCGCGTCCGACAAAGATGGTAAGAGCGCTAAAGATCGCAAAGAGGC GTGGGAGCGGATTCGTCAAGCCATACCTAGAGAGAAAACTGCTGAGGCA AAGCAACGCCGCATCGAGCTTTTCAAGAAATTCGATAAAAACAATACAGG CAAACTGTGCTATGATGAAGTATATAAAGGTTGTCTGGAAGTGCTCAAAC TTGATGAATTTACCAGTCGTGTGCGCGATATCACTAAGCGTGCATTTGATA AAGCACGTACGTTAGGCTCAAAACTCGAAAATAAAGGAAGTGAGGACTTC GTGGAATTTCTGGAATTCCGCCTGATGCTTTGCTACATCTACGATTTTTTTG AACTTACAGTAATGTTTGACGAGATCGATACATCAGGTAATATGCTTGTA GATGAAGAAGAATTTAAACGTGCCGCCCCGAAACTCGAGGCATGGGGTGC CAAAGTGGAAGATCCGGATGCACTTTTTAAAGAGCTGGATAAAAACGGAA CGGGCTCTGTCACCTTTGACGAATTTGCCGCTTGGGCTGCCGCCGTCAAAT TAGATGCCGATGGTGACCCGGACAATGTACCGGAATCAGCATAA

[0136] SEQ ID NO: 14 (codon optimised Tx76 (N-mod)) GCGCATATCGTAATGGTTGATGCGTATAAACCGACAAAAGAAAACTTATA TTTTCAGGGAATGGGGGCATGTGGAAGTAAAGGTAGCACCTCTGACAAGG GACTTGCGTCTGACAAGGATGGTAAATCTGCAAAGGACCGAAAAGAAGCT TGGGAAAGAATTCGCCAGGCTATACCGCGTGAAAAGACGGAGGAGGCCA AGCAGCGTCGGATTGAATTGTTTAAGAAATTCGATAAAAATAACACTGGA AAACTTTCATATGACGAAGTTTACAAAGGGTGCCTTGAGGTTCTTAAATTG GATGAATTCACCTCACGGGTACGCGATATCACGAAACGGGCATTCGATAA AGCTAGAACCTTAGGAAGTAAACTCGAAAACAAAGGATCTGAGGACTTTG TAGAATTCCTTGAGTTTCGGCTCATGCTGTGTTACATCTATGATTTTTTTGA ACTGACTGTTATGTTCGATGAAATTGACACATCCGGAAACATGTTAGTGG ATGAAGAAGAATTTAAAAGAGCGGTCGATAAACTTGAGGAGTGGGGAGC TAAGATTGAGGATCCGGATGCTCTCTTTAAGGAATTGGATAAAAATGGTA CAGGAAGTGTTACCTTCGATGAGTTTGCTGCGTGGGCGGCAGCGAAGAAA CTGGACGCTGACGGTGATCCTGATAATGTCCCCGAATCTGCCTAA

[0137] SEQ ID NO: 15 (pET Tx74) GCTCACATCGTCATGGTGGACGCGTACAAGCCGACGAAAGAGAATTTATA TTTTCAGGGTATGGGCGCTTGCGGGAGTAAAGGCAGCACCAGCGATAAAG GCCTTGCTTCGGATAAGGATGGTAAGTCAGCCAAAGACCGCAAGGAGGCC TGGGAACGGATTCGGCAGGCGATCCCCCGGGAGAAAACGGCTGAGGCCA AACAACGGAGAATCGAGCTGTTTAAAAAATTTGACAAAAACGAAACCGG TAAACTTTGCTACGATGAAGTGTATTCCGGTTGTTTGGAGGTCTTGAAATT GGATGAGTTTACGTCTCGAGTTCGTGATATCACTAAGCGGGCTTTCGATAA AGCACGGACACTGGGGAGCAAGTTAGAAAACAAAGGCAGCGAGGACTTC GTGGAGTTCCTCGAATTTCGTCTCATGTTATGTTATATATACGATTTTTTTG AATTAACCGTTATGTTTGATGAAATAGACACCTCGGGCAATATGTTAGTTG ATGAAGAAGAATTCAAACGTGCTGTGCCTAAGTTGGAGGCCTGGGGGGCA AAAGTGGAAGACCCCGCTGCCCTGTTTAAGGAGTTAGACAAAAATGGGAC CGGTTCGGTAACCTTTGATGAATTTGCAGCCTGGGCGTCTGCAGTCAAGCT GGATGCCGACGGCGATCCCGATAATGTCCCGGAATCAGCGTAAGGCTGCT AACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCT GAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCG GCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATG ACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCA ATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCA AACATGAGAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT TGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC CCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT GCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTT TAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT

[0138] GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGT AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGC TGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGT CAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCC CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA AGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACA CCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCC CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGC TCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCG TCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAG CGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTG TAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAG GATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAAC GTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAA AATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGC AGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTT CACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCC CGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCG TGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGA TGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACA GTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTA GCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCA CCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAA TCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTG ACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGA TCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGC CTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATG GGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCA GCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGT TTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCC GAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGT CCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGC ATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGC CCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAG ATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCAC TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCG GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTT TCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCC CTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAA AATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGG TATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGAC TCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAA ACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTG ATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGA CAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG ACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAAT ACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACAT TAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAG TTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGC TTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGC ACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTG CCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATC GCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTC ACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATC GTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGG GATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTA GTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCAC GCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCAT CGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTG ATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG AAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCC TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGC AGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGC

[0139] SEQ ID NO: 16 (pET Tx75) GCACATATTGTCATGGTGGATGCGTATAAACCTACAAAGGAAAATCTTTA TTTCCAAGGCATGGGGGCATGTGGCAGCAAGGGGTCGACTTCAGATAAAG GCCTCGCGTCCGACAAAGATGGTAAGAGCGCTAAAGATCGCAAAGAGGC GTGGGAGCGGATTCGTCAAGCCATACCTAGAGAGAAAACTGCTGAGGCA AAGCAACGCCGCATCGAGCTTTTCAAGAAATTCGATAAAAACAATACAGG CAAACTGTGCTATGATGAAGTATATAAAGGTTGTCTGGAAGTGCTCAAAC TTGATGAATTTACCAGTCGTGTGCGCGATATCACTAAGCGTGCATTTGATA AAGCACGTACGTTAGGCTCAAAACTCGAAAATAAAGGAAGTGAGGACTTC GTGGAATTTCTGGAATTCCGCCTGATGCTTTGCTACATCTACGATTTTTTTG AACTTACAGTAATGTTTGACGAGATCGATACATCAGGTAATATGCTTGTA GATGAAGAAGAATTTAAACGTGCCGCCCCGAAACTCGAGGCATGGGGTGC CAAAGTGGAAGATCCGGATGCACTTTTTAAAGAGCTGGATAAAAACGGAA CGGGCTCTGTCACCTTTGACGAATTTGCCGCTTGGGCTGCCGCCGTCAAAT TAGATGCCGATGGTGACCCGGACAATGTACCGGAATCAGCATAAGGCTGC TAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAAT AACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGC TGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCG GCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATG ACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCA ATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCA AACATGAGAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT TGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGA

[0140] AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC CCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGAT GGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT GCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTT TAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT

[0141] GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGT AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGC TGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGT CAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCC CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA AGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACA CCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCC CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGC TCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCG TCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAG CGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTG TAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAG GATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAAC GTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAA AATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGC AGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTT CACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCC CGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCG TGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGA TGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACA GTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTA GCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCA CCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAA TCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTG ACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGA TCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGC CTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATG GGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCA GCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGT TTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCC GAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGT CCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGC ATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGC CCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAG ATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCAC TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCG GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTT TCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCC CTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAA AATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGG TATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGAC TCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAA ACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTG ATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGA CAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG ACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAAT ACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACAT TAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAG TTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGC TTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGC ACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTG CCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATC GCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTC ACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATC GTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGG GATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTA GTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG GAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCAC GCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCAT CGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTG ATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG AAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCC TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGC AGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGC

[0142] SEQ ID NO: 17 (pET Tx76) GCGCATATCGTAATGGTTGATGCGTATAAACCGACAAAAGAAAACTTATA TTTTCAGGGAATGGGGGCATGTGGAAGTAAAGGTAGCACCTCTGACAAGG GACTTGCGTCTGACAAGGATGGTAAATCTGCAAAGGACCGAAAAGAAGCT TGGGAAAGAATTCGCCAGGCTATACCGCGTGAAAAGACGGAGGAGGCCA AGCAGCGTCGGATTGAATTGTTTAAGAAATTCGATAAAAATAACACTGGA AAACTTTCATATGACGAAGTTTACAAAGGGTGCCTTGAGGTTCTTAAATTG GATGAATTCACCTCACGGGTACGCGATATCACGAAACGGGCATTCGATAA AGCTAGAACCTTAGGAAGTAAACTCGAAAACAAAGGATCTGAGGACTTTG TAGAATTCCTTGAGTTTCGGCTCATGCTGTGTTACATCTATGATTTTTTTGA ACTGACTGTTATGTTCGATGAAATTGACACATCCGGAAACATGTTAGTGG ATGAAGAAGAATTTAAAAGAGCGGTCGATAAACTTGAGGAGTGGGGAGC TAAGATTGAGGATCCGGATGCTCTCTTTAAGGAATTGGATAAAAATGGTA CAGGAAGTGTTACCTTCGATGAGTTTGCTGCGTGGGCGGCAGCGAAGAAA CTGGACGCTGACGGTGATCCTGATAATGTCCCCGAATCTGCCTAAGGCTG CTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAA TAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG CTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACC GGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGAT GACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGC AATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTC AAACATGAGAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTT ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTT TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC AAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC CCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAA CGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTA TCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTC TCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGG ATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGAT AACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACG ATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT ACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATA AAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGT GCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT TTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA

[0143] GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTA ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCC GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG GGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTA CACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTAT CCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC GGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTC ACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG TTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGC CCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTC CCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTG TCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAA GCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCG CGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAA AGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCG TGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAG AGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGA ACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGA AAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGT TCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGG TGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCG AAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCG CTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAA CCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCAC CCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGG AGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTC ACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCG TTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCAT GCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTA CAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCC GTGACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTAAGAGCCGCGAG CGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCAT GGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATA ATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGC CCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAA CGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGAT TCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGC GGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGT TGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCG CGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTC GAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCT CACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGT TTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTG GCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGC GAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCT TCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCC GGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAA CCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTT GAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGA ATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGC CGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCA ATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAA ATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGG AACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCG GATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACC GCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACG CTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGA CGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGAC TGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCC GCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCC TGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGC GACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTC TTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGT GTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGC CCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCA TGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCAT ACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTT CCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCG CCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGAT CCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAA TTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGG GCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGC AGC

Claims

CLAIMS1. A modified Trypanosoma cruzi Tc24 polypeptide comprising an amino acid substitution at one or more of amino acid positions: 23, 98, 141, or any combination thereof, relative to the parent Trypanosoma cruzi Tc24 polypeptide.

2. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 1, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises at least one of the following amino acid substitutions: K23S, S98A, A141T, or any combination thereof, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

3. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 1 or claim 2, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions K23S, S98A, and A141T, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

4. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any preceding claim, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises an amino acid substitution at one or more of amino acid positions: 44, 61, 72, 170, 194, or any combinations thereof, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

5. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 4, wherein modified Trypanosoma cruzi Tc24 polypeptide comprises at least oneof the following amino acid substitutions: A44E, E61N, S72K, A170D, SI 94 A, or any combination thereof, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

6. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 5, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions: E61N, S72K, A170D, and S194A, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

7. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any one of claims 4 to 6, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises an amino acid substitution at one or more of amino acid positions: 44, 66, 156, 157, 161, 166, 196, or any combination thereof, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

8. The modified Trypanosoma cruzi Tc24 polypeptide of claim 7, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises at least one of the following amino acid substitutions: A44E, C66S, V156A, P157D, A161E, V166I, V196K, or any combination thereof, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

9. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 1, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions at positions: 23, 98 and 141, relative to the same amino acidpositions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

10. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 7, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions at positions: 23, 61, 72, 98, 141, 156, 170 and 194, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

11. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 7, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions at positions: 23, 44, 61, 66, 72, 98, 141, 157, 161, 166, 170, 194 and 196, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

12. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claims 3 and 6, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises the amino acid substitution V156A, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

13. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claims 3 and 6, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises amino acid substitutions: A44E, C66S, P157D, A161E, V166I and V196K, relative to the same amino acid positions in the parent Trypanosoma cruzi Tc24 polypeptide of SEQ ID NO: 1 or 2.

14. The modified Trypanosoma cruzi Tc24 polypeptide of any preceding claim wherein the parent Trypanosoma cruzi Tc24 polypeptide comprises SEQ ID NO: 1 or 2.

15. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 9, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

16. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 10, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

17. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 11, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

18. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any preceding claim, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises at least three and no more than 25 amino acid substitutions relative to the parent Trypanosoma cruzi Tc24 polypeptide.

19. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any preceding claim, wherein the modified Trypanosoma cruzi Tc24 polypeptide further comprises a N-terminal modification comprising the amino acid sequence of SEQ ID NO: 3.

20. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in claim 19, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises the amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9.

21. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any preceding claim, wherein the modified Trypanosoma cruzi Tc24 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 or 2.

22. The modified Trypanosoma cruzi Tc24 polypeptide as claimed in any preceding claim, wherein the modified Trypanosoma cruzi Tc24 polypeptide is capable of detecting Tc24 specific antibodies.

23. A polypeptide comprising a parent Trypanosoma cruzi Tc24 polypeptide and an N-terminal modification comprising amino acid sequence SEQ ID NO: 3.

24. A vector comprising a nucleic acid sequence that expresses the modified Trypanosoma cruzi Tc24 polypeptide as claimed in any one of claims 1 to 22.

25. The vector as claimed in claim 24, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 15, 16, or 17.

26. A diagnostic test comprising the modified Trypanosoma cruzi Tc24 polypeptide as claimed in any one of claims 1 to 22.

27. A method of diagnosing an individual with Chagas disease using the diagnostic test of claim 26, and wherein the method comprises applying a sample taken from an individual with Chagas disease to the diagnostic test, and reading a positive signal produced by the diagnostic test.

28. The method as described in claim 27, wherein the individual is a mammal.

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