Histagged FIMH protein for use as a vaccine against e. coli

Abstract

The invention pertains to His tagged FimHLD R60P protein for use as a medicament, in particular as an antigen in a vaccine to protect against an infection with E. coli.

Description

[0001] HISTAGGED FIMH PROTEIN FOR USE AS A VACCINE AGAINST E. COLI

[0002] TECHNICAL FIELD

[0003] The invention pertains to the field of vaccines, in particular vaccines to protect an animal (including humans) against an infection with Escherichia coli E. coli), further in particular a vaccine using the FimH adhesin as protective antigen.

[0004] BACKGROUND

[0005] Urinary-tract infections are among the more wide-spread bacterial diseases that occur in animals, including human. The main causative agent in these infections is (uropathogenic) E. coli. Studies demonstrated that recurrent infections may originate not only from fecal and vaginal flora but also from intracellular bacterial communities within epithelial cells of the bladder. Biofilm formation by such bacterial communities allows the bacteria to persist a host immune response and even an antibiotic treatment. Vaccine available for recurrent UTIs are formulated mainly with whole inactivated bacterial cells and administered orally. This type of vaccine has a limited success.

[0006] It is known since more than 20 years that the FimH adhesin of E. coli can protect against an infection with E. coli (Langerman et al: Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coif, in J Infect Dis. 2000 Feb; 181 (2)774-8). FimH is a major determinant of the adhesive subunit of Type 1 fimbriae, which has high tropism for urinary tract receptors. It binds to monomannose. Additional studies after the publication of Langerman have confirmed that it is a good candidate for a vaccine because of its critical role in cystitis pathogenesis (Loubet et al: Sec. Antimicrobials, Resistance and Chemotherapy, in Front. Microbiol., 03 July 2020, Volume 11 - 2020). For example, studies on mice with an intranasal and intramuscular administration also showed a protection against cystitis, but the intranasal method produced a stronger immune response. In a more recent study, a recombinant protein MrpH.FimH (consisting of a combination between two adhesins, FimH from UPEC and MprH from P. mirabilis) was injected with a transurethral instillation and the authors demonstrated a high immune response and a protection against E. coli and P. mirabilis (Habibi et al., 2016: Transurethral instillation with fusion protein MrpH.FimH induces protective innate immune responses against uropathogenic Escherichia coli and Proteus mirabilis-, APMIS 124, 444-452).

[0007] For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. For FimH, Rabbani has demonstrated in 2018 (J. Biol. Chem, 23(5) 1835 - 1849) that adherence to the urothelial surface is mediated by the adhesin FimH located at the tip of bacterial type 1 pili. Full-length FimH (FimHFL) is composed of two domains: the N-terminal lectin domain (FimHLD) which is connected to the C-terminal pilin domain (FimHpo) by a short linker. FimHLD contains the carbohydrate recognition domain, which is responsible for binding to the highly mannosylated uroplakin 1a (UP1a) on the urothelial cell surface. Interestingly, the FimHLD R60P mutant mimicked the conformational and kinetic behaviors of the full-length FimH protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. The low-affinity conformation of mutant R60P was shown to result from a slightly altered clamp loop. A bacterial adhesion assay to show the correct (low affinity) formation of a FimH protein is known from Thomas et al (Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shea; in J. Mol. Biol. 2022 September 15; 434(17)). All in all, this shows that FimH LD R60P represents a minimalistic allosteric system of FimH, useful as a preferred antigen in a vaccine to protect against an infection with E. coli.

[0008] SUMMARY OF THE INVENTION

[0009] It is an object of the invention to improve the FimHLD R60P protein, such that an even better conformation is obtained, which shows as an improved mannose binding function, and thus even better mimicking the conformational and kinetic behaviours of the natural full-length FimH protein. To this end, it was found that a when the FimHLD R60P protein is provided with a his tag at its C terminal end, the mannose binding function is even further improved. This came as a surprise since such a tag is typically regarded an inert feature, not interfering with the biologic function of a protein. However, his tagged FimHLD R60P protein was found to have a significantly improved mannose binding function and hence, a conformation that even better mimicks the conformational and kinetic behaviours of natural the full-length FimH protein. It is essential that the His tag is a permanent tag, and not a cleavable tag as is commonly known from the prior art (i.a. US 2019 / 275135). Only this way, the protein with the tag will be available in the subject that is vaccinated for raising an appropriate antibody response. A permanent tag will lead to an anti-His antibody response in animals vaccinated with the new antigen.

[0010] The invention also pertains to FimHLD R60P protein provided with a permanent His tag at its C terminal end for use as an antigen in a vaccine to protect an animal against an Escherichia coli infection, to the use of FimHLD R60P protein provided with a permanent His tag at its C terminal end for the manufacture of a vaccine to protect an animal against an Escherichia coli infection, and lastly to a method of protecting an animal against an infection with Escherichia coli, by administering a vaccine to the animal comprising FimHLD R60P protein provided with a permanent His tag at its C terminal end as an antigen.

[0011] DEFINITIONS

[0012] The FimHLD protein is the N-terminal lectin domain of the full-length FimH protein. FimHLD is a commonly known 160 amino acid protein that contains the carbohydrate recognition domain (Rabbani, 2018; supra).

[0013] The FimHLD R60P protein is FimHLD wherein at position 60, the amino acid Arginine is replaced by a Proline. This position is shown in Rabbani, figure 7A. The 60 position is between the 59 Gin and 61 Gly amino acid.

[0014] A His tag is an amino acid motif that typically consists of multiple histidine (His) residues, often at the N- or C-terminus of the protein. The total number of histidine residues may vary in the tag from as low as two, to as high as 10 or more His residues. The most used number is six histidine residues, also known as a hexa histidine-tag, 6xHis-tag, or His6 tag. When a protein is provided with a his tag, the protein is said to be his tagged.

[0015] A vaccine is a constitution suitable for application to an animal, having an acceptable safety, comprising one or more antigens in an immunologically effective amount, i.e. capable of stimulating the immune system of the target animal sufficiently to induce an immune response, such as antibodies, against the antigens and therewith against the corresponding naturally occurring antigens and therewith potentially the naturally occurring pathogen, typically combined with a pharmaceutically acceptable carrier (i.e. a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the subject animal, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine) such as a liquid containing water and / or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), optionally comprising immunostimulating agents (adjuvants), which upon administration to the animal induces an immune response that is able to protect the animal against a (post-vaccinating) infection.

[0016] To protect an animal against an infection with a pathogen means aiding in preventing, ameliorating or curing a pathogenic infection with the pathogen, or aiding in preventing, ameliorating or curing a disorder arising from that infection, for example to prevent or reduce one or more clinical signs resulting from the infection with the pathogen.

[0017] DESCRIPTION OF EMBODIMENTS

[0018] In a first further embodiment of the invention the the His tag comprises 2-12 histidine molecules, preferably 6-12 histidine molecules, and more preferably 6 histidine molecules. In the latter embodiment, the tag is a simple, commonly used his-his-his-his-his-his tag, for example present at the C terminal of the FimHi_D R60P protein.

[0019] The invention will now be further explained using the following specific example. EXAMPLE

[0020] The basis for the determination of FimH binding is the assay as known form Thomas 2022 (supra). The mechanics of this assay are as follows: FimH constructs are captured in an ELISA plate by pre-coating with the FimH. The naturally occurring glycosylations of horseradish peroxidase (HRP) can bind to the lectin domain of FimH and this interaction is leveraged in the assay to detect the presence of functional FimH by colorimetric reaction of HRP and TMB. The assay shows FimH binding to HRP and that the co-addition of mannose (2% w / v) during the HRP incubation step, reduces HRP binding. Absorbance is read at 650 nm.

[0021] In particular, incubations of the ELISA plates were static and sealed with adhesive plate sealers. After incubations plates were washed three times with 300pl / well of phosphate buffered saline with 0.05% (v / v) of Tween20 (PBS-T). Volume of reagents were 10Opl / well except for the blocking buffer which was used at 200pl. 1 :50 dilutions of periplasmic extracts were diluted in carbonate coating buffer (0.05M bicarbonate, pH 9.6), added to 96 well, flat bottom ELISA plates (Nunc, 439454) and incubated at 4°C overnight. Plates were washed and then blocked with casein blocking buffer (ThermoFisher) and incubated at 37°C for 1 hr. Plates were washed, and 100pg / mL horseradish peroxidase (HRP) diluted in casein blocking buffer was incubated for 1 hr at 37°C. Plates were washed aggressively to remove non-specific HRP binding. TMB (SeraCare) was added for an HRP induced colorimetric reaction to occur for 20 minutes at room temperature. The reaction is stopped with 1 % SDS (Invitrogen) and absorbance at 650 nm was determined using a plate reading spectrophotometer.

[0022] Here below, the absorbance of two forms of FimHi_D R60P protein is indicated at various dilutions. The two forms are His tagged FimHi_D R60P protein (His6 tag provided at the C terminal end of the FimHi_D R60P protein) and FimHi_D R60P protein without a His tag, as known form Rabbani 2018. Table 1 Free HRP binding to FimH (OD at 650 nm)

[0023] The results indicate that His tagged FimHLD R60P protein has a superior binding capacity and thus seem to better mimic the conformational and kinetic behaviours of natural the full- length FimH protein. This means that when this protein is used as antigen, antibodies are elicited that are better able to bind to the natural the full-length FimH protein. Since the effect is very substantial it is believed that any His tag having between 2 and 12 histidine molecules leads to an improvement.

Claims

CLAIMS1 . FimHLD R60P protein provided with a permanent His tag at its C terminal end for use as a medicament.

2. His tagged FimHLD R60P protein for use according to claim 1 , characterised in that the His tag comprises 2-12 histidine molecules.

3. His tagged FimHLD R60P protein for use according to claim 1 or 2, characterised in that the His tag comprises 6-12 histidine molecules.

4. His tagged FimHLD R60P protein for use according to any of the claims 1 to 3, characterised in that the His tag comprises 6 histidine molecules.

5. His tagged FimHLD R60P protein for use according to any of the claims 1 to 4, characterised in that the His tag is present at the C terminal of the FimHLD R60P protein.

6. FimHLD R60P protein provided with a permanent His tag at its C terminal end for use as an antigen in a vaccine to protect an animal against an Escherichia coli infection.

7. Use of a FimHLD R60P protein provided with a permanent His tag at its C terminal end for the manufacture of a vaccine to protect an animal against an Escherichia coli infection.

8. A method of protecting an animal against an infection with Escherichia coli, by administering a vaccine to the animal comprising a FimHLD R60P protein provided with a permanent His tag at its C terminal end as an antigen.

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