Glycosylated YGHJ polypeptide from uropathogenic E. coli

Abstract

The present invention relates to YGHJ polypeptides having novel glycosylation patterns. The glycosylated polypeptides are expressed in avirulent recombinant strains of naturally occurring pathogenic bacteria, which produce glycosylated YGHJ polypeptides that resemble the wild-type polypeptide to a higher degree than polypeptides expressed in E. coli production strains. The present invention also relates to uses of such polypeptides and production strains.

Description

[Technical Field]

[0001] The present invention relates to immunogenic glycosylated YGHJ polypeptides from or derived from uropathogenic Escherichia coli (UPEC). In particular, the present invention relates to compositions or vaccines comprising the glycosylated polypeptides and their applications in immunization, vaccination, and therapy. The present invention also relates to engineered production strains for such polypeptides. [Background technology]

[0002] Complicated urinary tract infections (UTIs) are serious conditions associated with a significant burden of morbidity and mortality in risk groups, including those with diabetes, kidney stones, and focal spinal cord dysfunction, as well as surgical patients. The risk of developing complicated UTIs increases dramatically in individuals over the age of 60 (Zhao et al., 2020). Complicated UTIs are often healthcare-associated UTIs (HAUTIs), which are the result of the intervention of another disease or condition. The risk of developing a hospital-acquired UTI upon hospitalization has been reported to range from 1.7 to 4.8% (Mitchell et al., 2016).

[0003] This group of UTIs is associated with a significantly greater risk of developing into more serious and costly infections with potentially fatal outcomes. The Centers for Disease Control and Prevention (CDC) reported in 2002 that urinary tract infections (UTIs) accounted for the highest number of hospital-acquired infections (HAIs) (>560,000) compared to other HAIs, and with a mortality rate of 2.3%, were responsible for 13,000 deaths.

[0004] Additionally, HAUTIs are associated with an average increase in hospital length of stay of 4 days (Mitchell et al., 2016) and an average direct cost of 5,700 euros per case (Cassini et al., 2016; Vallejo-Torres et al., 2018). Escherichia coli (E. coli) is the most prevalent bacterium causing HAUTIs, with UPEC responsible for over one-third (26%-47%) of HAUTIs (Cek et al., 2014; The European Centre for Disease Prevention and Control, 2017; Medina and Castillo-Pino, 2019; Zhao et al., 2020).

[0005] A conservative assessment of the current UPEC UTI burden is >25,000 deaths and a staggering €9.5 billion per year in direct costs in European and North American hospitals alone. Despite the enormous unmet need, there is no vaccine against UPEC UTI that successfully reaches patients.

[0006] WO 2006 / 089264(A2) discloses various open reading frames from strains of Escherichia coli that cause neonatal meningitis (MNEC), and a subset of these that are of particular interest for preparing compositions for immunization against MNEC infection.

[0007] WO 2011 / 007257(A1) discloses a detoxified Escherichia coli immunogen.

[0008] Nesta et al. (PLOS Pathogens; May 2014 | Volume 10 | Issue 5 | e1004124) disclose antibodies that confer in vitro mucin-degrading enzyme activity and in vivo colonization by both intestinal and extraintestinal E. coli strains.

[0009] WO 2017 / 059864(A1) discloses glycosylated YGHJ polypeptides from enterotoxigenic E. coli (ETEC).

[0010] Thorsing et al. (Frontiers in Cellular and Infection Microbiology. August 2021|Volume 11|Article 705468) disclose a link between O-linked glycosylation of bacterial proteins and relative immunogenicity, further highlighting the importance of this observation in considering ETEC proteins for inclusion in future broad-spectrum subunit vaccine candidates.

[0011] Tapader et al. (Microbial Pathogenesis, vol. 105, 16 February 2017, pp. 96-99) disclose the pathogenic potential of YghJ in sepsis pathophysiology, but also demonstrate the enterotoxic potential of YghJ.

[0012] Therefore, improved treatments for uropathogenic E. coli (UPEC) would be advantageous, particularly more effective and / or reliable vaccines against uropathogenic E. coli (UPEC). Summary of the Invention

[0013] The present invention is based on the identification that the YGHJ polypeptides according to the present invention are more potent as vaccines when expressed in their native host compared to when expressed in different expression hosts. Furthermore, the present team identified a unique glycosylation pattern in full-length YGHJ from UPEC (see, e.g., Example 2), which is believed to be responsible for its ability to enhance immune responses and produce very strong protection when used as a vaccine against infection in the bladder, as documented in a pig study exemplified in Example 4.

[0014] From a manufacturing standpoint, expressing immunogens in naturally pathogenic hosts is generally considered undesirable due to the risk of infection to personnel handling purification processes, waste management, and post-manufacturing device handling. Therefore, polypeptide vaccines against bacterial pathogens are conventionally expressed in non-pathogenic production strains that do not require high biosafety level manufacturing facilities.

[0015] To overcome this problem, the present team also developed a detoxified and non-pathogenic production strain, which is capable of producing polypeptides with glycosylation patterns associated with the naturally occurring pathogenic bacteria (see Example 3).

[0016] The present team also identified an optimized tag for purification purposes (see Example 5).

[0017] Furthermore, the present team confirmed that the polypeptides and compositions according to the present invention can produce antibodies with higher avidity compared to the YGHJ version previously isolated from an E. coli expression strain (Example 6).

[0018] Furthermore, the present team confirmed that expression from the production strains of the present invention resembles expression from the wild-type pathogen chromosome to a greater extent than expression from standard E. coli production strains, including in terms of glycan:protein ratio (Example 7).

[0019] Accordingly, improved vaccines and production strains for such vaccines are disclosed herein.

[0020] In WO 2006 / 089264, proteins are cloned and expressed in bacteria (in non-pathogenic laboratory E. coli hosts or in Bacillus such as B. subtilis or B. megaterium). Thus, WO 2006 / 089264 is silent about peptides being expressed in these pathogenic natural hosts.

[0021] WO 2011 / 007257(A1) mentions that expression of the peptide can occur in E. coli strains, however, preferably from a heterologous host for expression. The heterologous host can be a prokaryotic (e.g., bacterium) or eukaryotic organism. Suitable hosts include, but are not limited to, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, and mycobacteria (e.g., M. tuberculosis) (see WO 2011 / 007257(A1) , p. 13). Therefore, WO 2011 / 007257(A1) is silent regarding the peptides expressed in these pathogenic natural hosts. From the data presented in WO 2011 / 007257(A1), it appears that all peptides are expressed in E. coli BL21(DE3).

[0022] Nesta et al. (data overlapping with WO 2011 / 007257(A1)) also used E. coli BL21(DE3) (Invitrogen) for expression of His-tagged fusion proteins. Therefore, Nesta et al. did not describe peptides expressed in these recombinant non-pathogenic native hosts.

[0023] WO 2017 / 059864(A1) does not describe peptides expressed in pathogenic natural UPEC hosts.

[0024] It is therefore an object of the present invention to provide an improved vaccine against UPEC.

[0025] It is therefore an object of the present invention to provide a bacterial production strain that overcomes the above-mentioned problems of the prior art regarding maintaining the correct glycosylation pattern.

[0026] Thus, one aspect of the present invention is a method for producing a medicament comprising: a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; and / or c) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or d) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; Including, The polypeptide has a glycosylation pattern defined by being glycosylated at at least 15 positions in SEQ ID NO: 1 selected from the group set forth in Table 2, such as at least 20 positions or at least 40 positions. Concerning polypeptides.

[0027] Another aspect of the present invention relates to a composition comprising a polypeptide according to the present invention and / or a plurality of polypeptides having a glycosylation pattern according to the present invention.

[0028] A further aspect of the present invention is to provide a polypeptide according to the present invention or a composition according to the present invention for use as a medicament, such as a vaccine.

[0029] A further aspect relates to a polypeptide according to the invention or a composition according to the invention for use in the treatment, prevention and / or alleviation of an E. coli infection, preferably a urinary tract infection (UTI), such as a bladder infection.

[0030] However, the aspect is FimH; PapG; and WaaL The present invention relates to a genetically modified E. coli (E. coli) that does not express the

[0031] Yet another aspect of the present invention is a method for producing a glycosylated polypeptide of interest, said method comprising: a) expressing said polypeptide of interest in a recombinant E. coli according to the invention; and b) purifying the glycosylated polypeptide of interest from said bacterium; The method includes:

[0032] Aspects also relate to glycosylated polypeptides obtained or obtainable by the methods according to the invention.

[0033] Furthermore, the present invention relates to antibodies specific for the polypeptides according to the invention, such as monoclonal or polyclonal antibodies. [Brief explanation of the drawings]

[0034] [Figure 1]Number of glycosylated peptides identified as a function of input sample amount (μg). The total number of glycopeptides derived from hyperglycosylated full-length YGHJ (GPV02) isolated from a production strain (open circles) or full-length YGHJ isolated from an E. coli overexpression strain (open squares) is plotted as a function of sample input. The filled circles represent the total number of identified glycopeptides in 12 BEMAP analyses using GPV02 (full-length YGHJ isolated from a production strain as defined in the present invention). The filled squares represent the number of glycopeptides identified in 1 BEMAP analysis of full-length YGHJ isolated from a conventional E. coli overexpression strain. [Figure 2] Figure 1 shows the relative abundance of glycopeptides derived from GPV02 isolated from either the production strain defined in the present invention or full-length YGHJ isolated from a conventional E. coli overexpression strain. Triangles indicate the relative abundance of 4BEMAP analysis from GPV02 isolated from the production strain. Filled squares indicate the relative abundance of full-length YGHJ peptide isolated from the E. coli overexpression strain. Peptides not identified in any of the five tests are shown at an abundance of 0.0001%. [Figure 3] Schematic diagram of lipopolysaccharide (LPS) biosynthesis in E. coli showing the gene products involved in synthesis, which were targeted for deletion to obtain a strain producing O-antigen-free LPS. O-antigen subunits are synthesized on the inner side of the cytoplasmic membrane on an undecaprenyl diphosphate carrier and flipped to the outer side by Wzx. Here, the subunits are polymerized into long chains by Wzy and Wzz and ligated to lipid A-core molecules by WaaL to generate the complete LPS molecule. The undecaprenyl carrier is recycled to the inner side of the cytoplasmic membrane and reused in another step. The entire LPS molecule is transported across the periplasmic space and outer membrane and incorporated into the outer leaflet. Figure adapted from (Wang and Quinn, 2010). [Figure 4]Western blot analysis of O-antigen levels in culture supernatants from UTI89 wild-type and its Δc4349-c4351 mutant. Strains were grown to the same optimal density, and bacteria-free supernatant samples were collected. Samples were separated by SDS-PAGE and transferred to PVDF membranes. O-antigen was detected using a primary porcine antibody against the O18 serotype and a secondary HRP-conjugated antibody against porcine IgG. Antibody complexes were visualized by chemiluminescence after wetting with Immobilon Forte Western HRP substrate. [Figure 5] Western blot of O-antigen levels in culture supernatants from UTI89 wild type and its ΔWaaL mutant prepared as described in Figure 4. To aid in estimating relative signal intensities between wild type and mutant, wild-type samples were serially diluted 2-fold before SDS PAGE. [Figure 6] Western blot analysis of full-length YGHJ levels in culture supernatants (upper panel) and intracellular levels of σE (lower panel) in UTI89 wild-type and ΔwaaL yghJ::3xFLAG-10xHHis. Sample collection and analysis were as described in Figure 4; bacterial pellets were harvested for intracellular levels. YGHJ was detected using a primary rabbit antibody against full-length YGHJ and a secondary HRP-conjugated anti-rabbit antibody, and σE was detected using a primary mouse anti-σE antibody and a secondary HRP-conjugated anti-mouse antibody. [Figure 7] Adhesion assay showing the relative ability of UPEC wild-type, a production strain as defined in this invention, and a non-pathogenic E. coli strain to adhere to human bladder cells in vitro. Adhesion ability is relative to wild-type (100%). Bars represent the mean and SD of four biological replicates, each containing eight technical replicates. p-values ​​were determined using a two-tailed unpaired Student's t-test assuming a Gaussian distribution. [Figure 8]Figure 1 shows the number of colony-forming units (CFU) associated with pig bladder tissue on the final day of the challenge experiment. 19 CFU counts for vaccinated pigs are plotted as filled circles. 17 CFU counts for control pigs are plotted as filled triangles. Geometric means are shown. Statistical analysis: two-tailed unpaired Student's t-test (Mann-Whitney test) not assuming Gaussian distribution. Exact P values ​​are shown. [Figure 9] Figure 1 shows the number of colony-forming units (CFU) in pig urine on day 1 post-infection. 19 CFU counts from vaccinated pigs are plotted as filled circles. 17 CFU counts from control pigs are plotted as filled triangles. Geometric means are shown. Statistical analysis: two-tailed unpaired Student's t-test (Mann-Whitney test) not assuming Gaussian distribution. Bars indicate geometric means. Exact P values ​​are indicated. [Figure 10] Serum IgG avidity against 4 pigs vaccinated with either glycosylated or non-glycosylated GPV02 is shown. Statistical analysis: two-tailed paired Student's t-test assuming Gaussian distribution. Means and standard deviations are plotted. Exact P values ​​are indicated. [Figure 11] The glycan to protein ratio is shown for GPV02 isolated from either the wild-type UTI89 strain, the production strain defined in the present invention, or a conventional E. coli production strain. Glycan content was determined using FTIR (Fourier transform infrared spectroscopy).

[0035] The invention is described in more detail below. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition Before discussing the present invention in more detail, the following terms and conventions will first be defined:

[0037] Uropathogenic E. coli (UPEC) Uropathogenic E. coli (UPEC) is a major cause of urinary tract infections (UTIs). In ascending infections, the bacteria colonize the urethra and spread down the urinary tract to the bladder and kidneys (causing pyelonephritis) or to the prostate in men. Because women have shorter urethras than men, they are more likely to suffer from ascending UTIs.

[0038] Uropathogenic E. coli (UPEC) is part of the extraintestinal pathogenic E. coli (ExPEC) pathotype.

[0039] GPV02 In this context, the term "GPV02" refers to the hyperglycosylated full-length YGHJ polypeptide according to the present invention.

[0040] Thus, in an embodiment, GPV02 is obtained by purification from a purified strain as defined in the present invention. In the Examples section, GPV02 is obtained from a production strain according to Example 3.

[0041] Glycosylation The term "glycosylation" refers to O-linked glycosylation, which is the attachment of a sugar molecule to the hydroxyl oxygen of either a serine or threonine side chain (hence, an O-linkage) in a protein.

[0042] Sequence identity The term "sequence identity" refers to a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length. The two sequences being compared should accommodate the insertion of gaps and, optimally, truncations at the ends of protein sequences. Sequence identity can be measured by:

[0043]

number

[0044] can be calculated as, dif is the total number of non-identical residues in the two sequences when aligned, and N refis the number of residues in one of the sequences. Thus, the DNA sequence AGTCAGTC has 75% sequence identity with the sequence AATCAATC (N dif = 2 and N ref A gap is counted as a non-identity of a particular residue, i.e., the DNA sequence AGTGTC has 75% sequence identity with the DNA sequence AGTCAGTC (N dif = 2 and N ref =8). Alternatively, sequence identity can be calculated using a BLAST program, for example the BLASTP program (WR Pearson and DJ Lipman (1988)). In one embodiment of the invention, alignments are performed using the sequence alignment method ClustalW using default parameters as described by JD Thompson et al (1994), available at http: / / www2.ebi.ac.uk / clustalw / .

[0045] When comparing a polypeptide fragment with a longer amino acid sequence, in order to calculate sequence identity, the polypeptide fragment is aligned with the segment of the longer amino acid sequence.The polypeptide fragment and the segment of the longer amino acid sequence can be substantially the same length.Therefore, the polypeptide fragment and the segment of the longer amino acid sequence can be the same length.After aligning the polypeptide fragment with the segment of the longer amino acid sequence, the sequence identity is calculated by computer as described above.

[0046] A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

[0047] Accordingly, one embodiment of the present invention relates to a polypeptide as described herein, wherein the polypeptide or polypeptide fragment has at least 80% sequence identity to the full-length sequence of SEQ ID NO:1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

[0048] An embodiment of the invention is a polypeptide as described herein, wherein said polypeptide or polypeptide fragment has at least 90% sequence identity to SEQ ID NO:1.

[0049] Immunogenic Polypeptides An immunogenic polypeptide is defined as a polypeptide that elicits an immune response. The immune response may be monitored by one of the following methods:

[0050] In vitro cellular responses are determined by the release of relevant cytokines, such as IFN-γ, from lymphocytes taken from animals or humans currently or previously infected with UPEC, or by detecting the proliferation of these T cells. 1 x 10 per well 5 cells ~3×10 5Induction is performed by adding the polypeptide or immunogenic portion to a cell-containing suspension. Cells are isolated from either blood, spleen, liver, or lung, and the polypeptide or immunogenic portion is added to achieve a concentration of 20 μg or less per ml of suspension. Stimulation is performed for 2 to 5 days. To monitor cell proliferation, cells are pulsed with radiolabeled thymidine, and proliferation is detected by liquid scintillation counting after 16 to 22 hours of incubation. A positive response is a response greater than background plus two standard deviations. IFN-γ release can be determined by ELISA, a method well known to those skilled in the art. A positive response is a response greater than background plus two standard deviations. When monitoring immunogenic responses to polypeptides such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, and TGF-β, cytokines other than IFN-γ may be relevant.

[0051] An alternative and more sensitive method for determining the presence of cytokines (e.g., IFN-γ) is the ELISPOT assay, in which 1–4 × 10 cells isolated from either blood, spleen, liver, or lung are used. 6 The cell suspension is then diluted to a desired concentration of 1-2 x 10 cells / ml and incubated for 18-22 hours in the presence of the polypeptide or an immunogenic portion of the polypeptide to obtain a concentration of 20 µg / ml or less. 6 The cells are diluted to 1 / ml and transferred to anti-IFN-γ coated Maxisorp plates, preferably incubated for 4-16 hours. IFN-γ-producing cells are determined by using a labeled secondary anti-IFN antibody and the relevant substrate, which produces spots that can be enumerated using a dissecting microscope. PCR techniques can also be used to determine the presence of mRNA encoding the relevant cytokine. Typically, one or more cytokines are measured, for example, using PCR, ELISPOT, or ELISA. Those skilled in the art will recognize that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used to assess the immunoreactivity of the polypeptide.

[0052] In vitro cellular responses may be determined using T cell lines derived from immune individuals or UPEC-infected individuals, which were driven with either live UPEC, extracts from bacterial cells, or filtrates of cultures supplemented with IL-2 for 10-20 days. Up to 20 μg of polypeptide per ml of suspension was administered at 1 × 10 per well. 5 cells ~3×10 5 Induction is performed by adding the antibody to a T cell line containing the cells, followed by incubation for 2-6 days. Induction of IFN-γ or release of other relevant cytokines is detected by ELISA. T cell stimulation can also be monitored by detecting cell proliferation using radiolabeled thymidine as described above. For both assays, a positive response is a response + 2 standard deviations above background.

[0053] In vitro humoral responses are determined by specific antibody responses in immunized or infected individuals. The presence of antibodies may be determined by ELISA techniques or Western blots, in which the polypeptide or immunogenic portion is absorbed onto either a nitrocellulose membrane or a polystyrene surface. Serum, preferably diluted 1:10 to 1:100 in PBS, is added to the absorbed polypeptide and incubated for 1 to 12 hours. By using labeled secondary antibodies, the presence of specific antibodies can be determined by measuring the presence or absence of specific label, for example, by ELISA; a positive response is a response greater than background plus two standard deviations or a visible response in a Western blot.

[0054] Another relevant parameter is the measurement of protection induced in animal models after vaccination with polypeptides in adjuvants or DNA vaccination.Suitable animal models include primates, guinea pigs, or mice, which are challenged with UPEC infection.The readout for induced protection can be a reduction or absence of bacterial load in target organs compared with non-vaccinated animals, a prolonged survival time and reduced weight loss compared with non-vaccinated animals, or morbidity compared with non-vaccinated animals.

[0055] Thus, a glycosylated polypeptide as described herein is immunogenic if one of the above tests is positive.

[0056] Polypeptides As outlined above and in the Examples section, the present team has engineered an E. coli expression strain capable of producing GPV02, a non-pathogenic yet highly glycosylated YGHJ immunogenic protein that has been found to be highly effective in pig immunization studies (see Example 4). Thus, aspects of the present invention include: a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; and / or c) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or d) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; Including, The polypeptide has a glycosylation pattern defined by being glycosylated at at least 15 positions in SEQ ID NO: 1 selected from the group set forth in Tables 2A-B, such as at least 20 positions, or at least 40 positions. Concerning polypeptides.

[0057] In embodiments, the polypeptide is glycosylated at at least 15 positions, such as at least 20 positions, such as all of the positions shown in Table 5B.

[0058] In embodiments, the polypeptide is glycosylated at at least 40 positions, such as at least 50 positions, such as all of the positions set forth in Tables 5A-B. As shown in Example 2, Table 2 provides a complete list of all identified glycosylation sites.

[0059] In another embodiment, the polypeptide is glycosylated at at least 15 positions selected from the group set forth in Tables 3A-B, such as at least 20 positions, such as at least 30 positions, or such as all of the positions set forth in Tables 3A-B.

[0060] In yet another embodiment, the polypeptide is glycosylated at at least 15 positions selected from the group set forth in Table 3B, such as at least 20 positions, such as at least 30 positions, or all of the positions set forth in Table 3B. Table 3 lists 41 sites (all of which are part of Table 2) that are considered to be the most abundantly identified sites (see also Example 2).

[0061] In yet another embodiment, the polypeptide is glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or all of the positions set forth in Table 4. Table 4 lists previously unidentified newly identified sites (all of Tables 2 and 3) (see also Example 2).

[0062] The most abundantly identified glycosylation sites are S 152, S 154 and S 164. Thus, in a preferred embodiment, the polypeptide is glycosylated at least at positions S152, and / or S154 and / or S164, such as S152 and S154, such as S154 and S164, such as S152 and S164, or such as S152, S154 and S164.

[0063] Other highly abundantly identified glycosylation sites are T592 and S594 and S597. Thus, in another preferred embodiment, the polypeptide is glycosylated at least at positions T592, and / or S594 and / or S597, such as T592 and S594, such as S594 and S597, such as T592 and S597, or such as T592, S594 and S597.

[0064] In a further embodiment, the amino acid sequence according to b) has at least 85% sequence identity with SEQ ID NO:1, such as at least 90% sequence identity with SEQ ID NO:1, such as at least 95%, such as at least 99% sequence identity.

[0065] In a further embodiment, the amino acid sequence according to c) or d) is a fragment of at least 1100 contiguous amino acids from SEQ ID NO: 1, such as at least 1200 contiguous amino acids, such as at least 1300 contiguous amino acids, such as at least 1400 contiguous amino acids or at least 1500 contiguous amino acids from SEQ ID NO: 1. SEQ ID NO: 1 has a length of 1520 AA, therefore it is believed that larger glycosylated fragments may also be able to generate an immunogenic response.

[0066] In an embodiment, the polypeptide is from an ExPEC, such as a UPEC strain. In an embodiment, the polypeptide is from a production strain according to the invention.

[0067] In another embodiment, the polypeptide is immunogenic, such as a vaccine.

[0068] In a preferred embodiment, the polypeptide is SEQ ID NO:1.

[0069] The tag may improve purification of the polypeptide. Thus, in an embodiment, the polypeptide preferably comprises a Flag tag and / or a His tag at the C-terminus.

[0070] In a preferred embodiment, the polypeptide comprises a 3xFLAG-6xHis tag or a 3xFLAG-10xHis tag, preferably at the C-terminus. As shown in Example 5, improved tags have been designed for the polypeptides according to the invention.

[0071] In a preferred embodiment, the present invention provides: a) the amino acid sequence set forth in SEQ ID NO: 1 Includes; the polypeptide has a glycosylation pattern defined by being glycosylated at at least 15 positions in SEQ ID NO:1 selected from the group set forth in Table 2B; and glycosylated at at least two of positions S152, S154 and S164; and glycosylated at at least two of positions T592, S594 and S597; The present invention relates to a polypeptide comprising:

[0072] In another preferred embodiment, the present invention provides: a) the amino acid sequence set forth in SEQ ID NO: 1 Includes; The polypeptide has a glycosylation pattern defined by being glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or such as all of the positions set forth in Table 4. Concerning polypeptides.

[0073] The polypeptide has a glycan to protein ratio of at least 0.020 (preferably measured by Fourier transform infrared spectroscopy, such as described in Example 7), such as at least 0.025, such as at least 0.030, such as at least 0.034, such as in the range of 0.025-0.034, such as in the range of 0.020-0.030, such as in the range of 0.020-0.025, by weight.

[0074] In a further preferred embodiment, the polypeptide has a glycan to protein ratio of at least 0.025 by weight, preferably at least 0.030, such as at least 0.020 by weight, or in the range of 0.020-0.050 by weight, preferably in the range of 0.030-0.050, more preferably in the range of 0.030-0.04. As shown in Example 7, a high glycan to protein ratio is important for similarity to YghJ produced in the wt strain.

[0075] In embodiments, the glycan to protein ratio is determined by Fourier transform infrared spectroscopy (FTIR). In Example 7, FTIR was used.

[0076] In a further preferred embodiment, the polypeptide is an ExPEC-derived polypeptide, such as a UPEC-derived polypeptide.

[0077] In another preferred embodiment, the polypeptide has a glycosylation pattern defined by being glycosylated at least at 40 positions in SEQ ID NO:1 selected from the group shown in Tables 2A-B.

[0078] composition The present invention also relates to a composition comprising a polypeptide according to the invention. Accordingly, another aspect of the present invention relates to a composition comprising a polypeptide according to the invention and / or a plurality of polypeptides having a glycosylation pattern according to the invention.

[0079] It should be understood that in this embodiment of the invention, a single polypeptide in a composition may not contain all of the glycosylation specified for a polypeptide according to the invention, but the glycosylation may be distributed among different polypeptides, because of the way bacteria attack glycosylation, and although the pattern described in claim 1 is indeed expected, each unique polypeptide may not have the same glycosylation pattern.

[0080] Thus, in an embodiment, a plurality of polypeptides have an overall glycosylation pattern according to the present invention. The overall glycosylation pattern may be determined using the BEMAP method described in Example 2.

[0081] In embodiments, the composition is a pharmaceutical composition.

[0082] In another embodiment, the composition further comprises a pharmaceutically acceptable carrier, diluent, and / or adjuvant.

[0083] In a preferred embodiment, the adjuvant is selected from the group consisting of dmLT, Litevax CMS, and combinations thereof. These adjuvants were used in a pig study (Example 4 - Pig Challenge Study).

[0084] "Litevax CMS" is an adjuvant containing a "carbohydrate monosulfate / squalane / polysorbate 80 emulsion in PBS at 40 mg / mL CMS." Thus, "Litevax CMS" is a synthetic carbohydrate fatty acid monosulfate derivative ("CMS") immobilized on nanodroplets of a squalane-in-water emulsion. The concentration is expressed in mg of CMS, and the weight / weight ratio of CMS / polysorbate 80 / squalane is 1:1:2. (See also WO 2016 / 013938 - Adjuvants and (Hilgers et al., 2017)).

[0085] The adjuvant "LT(R192G / L211A)" or "dmLT" is a detoxified version of the heat-labile enterotoxin of Escherichia coli that contains two mutations in its A-subunit that remove the enterotoxin but preserve the adjuvanticity of the molecule. DmLt is also described in U.S. Patent No. 6,033,673.

[0086] In another embodiment, the adjuvant is selected from the group consisting of dimethyloctadecylammonium bromide (DDA), dimethyloctadecenylammonium bromide (DODAC), Kir A, Poly I:C, aluminum hydroxide, Freund's incomplete adjuvant, IFN-γ, IL-2, IL-12, monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), trehalose dibehenate, and muramyl dipeptide (MDP).

[0087] Pharmaceutical compositions comprising the polypeptides described herein may be administered in a physiologically acceptable medium (e.g., demineralized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, protein solution, mannitol, aqueous glucose, vegetable oil, or the like).

[0088] Accordingly, an embodiment of the present invention relates to a composition comprising a polypeptide as described herein, comprising the polypeptide comprising the polypeptide as described herein, comprising a pharmaceutical composition.

[0089] In particular, the medium is buffered at a pH generally in the range of about 5-10, the buffer generally in the range of about 50-250 mM salt concentration, and may also include buffers with salt concentrations generally in the range of about 5-500 mM, physiologically acceptable stabilizers, and the like.

[0090] The compounds may be lyophilized for convenient storage and transport.

[0091] Therefore, in a further embodiment of the invention, the composition comprises one or more excipients, diluents and / or carriers.

[0092] Aqueous suspensions may contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.

[0093] Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents may be natural phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain fatty alcohols, such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as polyethylene sorbitan monooleate.

[0094] Vaccines are administered in a manner compatible with the dosage form and in an amount that is prophylactically or therapeutically effective and immunogenic. The amount administered depends on the subject being treated, including, for example, the capacity of the individual's immune system to mount an immune response and the degree of protection desired. Suitable dosage ranges are in the range of several hundred microorganisms of a polypeptide of the invention per vaccination, with a preferred range of about 0.1 μg to 1000 μg, such as in the range of about 1 μg to 300 μg, particularly in the range of about 10 μg to 100 μg. Suitable regimens for initial administration and booster vaccinations are also variable, but are typically based on an initial administration, followed by subsequent vaccinations or other administrations.

[0095] The application method can vary widely. Any of the conventional methods of vaccine administration can be applied. These include oral, nasal, or mucosal administration, either parenterally, by subcutaneous, intradermal, or intramuscular injection, or by transdermal application, for example, in the form of a solid (e.g., pill, suppository, or capsule) or physiologically acceptable dispersion, such as a spray, powder, or liquid, containing the active ingredient. The vaccine dosage will depend on the route of administration and will vary depending on the age and, to a lesser extent, the size of the person being vaccinated. Currently, most vaccines are administered intramuscularly by needle, and this is likely to continue as the standard route. However, vaccine formulations that induce mucosal immunity have been developed, typically via oral or nasal delivery. One of the most commonly tested delivery systems for eliciting mucosal immunity contains cholera toxin (CT) or its B subunit. This protein enhances mucosal immune responses and induces IgA production when administered in a vaccine formulation. An advantage is the ease of oral or nasal vaccine delivery. Modified heat-labile toxins from Gram-negative bacteria or modified toxins from other microbial species that have reduced toxicity but retain immunostimulatory capacity, such as staphylococcal enterotoxins, may also be used to produce similar effects. These molecules are particularly suitable for mucosal administration.

[0096] Vaccines are conventionally administered parenterally, for example, by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include suppositories, and in some cases, oral formulations. For suppositories, traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides; such suppositories can be made from mixtures containing 0.5% to 10%, preferably 1 to 2%, of the active ingredient. Oral formulations include commonly employed excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, and advantageously contain 10 to 95%, preferably 25 to 70%, of the active ingredient.

[0097] In embodiments, the composition is a pharmaceutical composition, such as an immunogenic composition, such as a vaccine.

[0098] In a further embodiment, the composition is formulated for intradermal, transdermal, subcutaneous, intramuscular or mucosal application, preferably subcutaneous application.

[0099] Genetically modified E. coli As outlined in Example 3 - Non-pathogenic Production Strain, the present team has engineered a non-pathogenic UPEC strain for the production of YGHJ that retains its ability to efficiently glycosylate YGHJ. Thus, aspects of the present invention include: FimH; and / or PapG; and / or WaaL The present invention relates to a genetically modified E. coli (E. coli) that does not express the

[0100] In a preferred embodiment, the genetically modified E. coli does not express FimH and WaaL. As shown in Example 3, such constructs also have reduced adhesion to bladder cells.

[0101] In an even more preferred embodiment, the genetically modified E. coli comprises: FimH; PapG; and WaaL does not express.

[0102] In embodiments, the gene is knocked out by a method selected from the group consisting of in-frame deletion, introduction of a stop site and total gene removal.In the example section, expression is stopped (knocked out) by CRISPR / CAS and Datsenko & Wanner (Datsenko and Wanner, 2000).Of course, those skilled in the art can use other methods to knock out the same gene.

[0103] The recombinant E. coli can express an (endogenous) protein of interest or can be adapted to be transfected with a gene of interest. Thus, in an embodiment, the recombinant E. coli expresses a glycosylated polypeptide of interest, such as an immunogenic vaccine; or It is adapted to express a glycosylated polypeptide of interest, such as an immunogen / vaccine.

[0104] In this context, the term "adapted to express" should be understood as a strain that is adapted to be subsequently modified to express a gene construct of interest that is not present in the strain at this stage; therefore, the strain is "adapted to express".

[0105] In embodiments, the genetically modified E. coli is an ExPEC, such as a UPEC, such as UTI 89. In a preferred embodiment, the genetically modified E. coli is a UPEC E. coli, such as UTI 89.

[0106] In embodiments, the genetically modified E. coli encodes a polypeptide that can be expressed in the E. coli and has a glycosylation pattern similar to a non-genetically modified version of the E. coli (e.g., UTI89).

[0107] In another embodiment, the genetically modified E. coli encodes a polypeptide according to the present invention.

[0108] In a related embodiment, the recombinant E. coli encodes a polypeptide comprising a glycosylation pattern as defined for the present invention.

[0109] In embodiments, the polypeptide is under the control of an endogenous promoter. This may be the case if it is a wild-type protein that is expressed (e.g., with a tag). Thus, the protein of interest may not be located on a plasmid.

[0110] In embodiments, the polypeptide is expressed from the genome, e.g., is an endogenous polypeptide, and is not expressed from an exogenous vector, such as a plasmid. In the Examples section, this is wild-type YGHJ expressed from the bacterial genome.

[0111] However, the polypeptide of interest may also be expressed from an exogenous vector, such as a plasmid. Thus, in an embodiment, the polypeptide is expressed from an exogenous vector, such as a plasmid.

[0112] As outlined in Example 3 (see also Figure 7), the production strain is considered non-pathogenic, e.g., by having very low adherence to bladder cells. Thus, in embodiments, the genetically modified E. coli is avirulent and / or non-pathogenic and / or not capable of causing disease.

[0113] In a related embodiment, the genetically modified E. coli has lower adherence to human bladder cells, such as human bladder cell line 5637, than the corresponding wild-type strain.

[0114] In another related embodiment, the genetically modified E. coli has lower adherence to human bladder cells, such as human bladder cell line 5637, than E. coli K-12 MG1655.

[0115] In embodiments, the production strain ensures the same or similar glycan to protein ratio as the wild-type UPEC strain, as shown in Example 7 and Figure 11.

[0116] In a preferred embodiment, the genetically modified E. coli comprises: a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and / or c) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or d) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; Including, The polypeptide has a glycosylation pattern defined by glycosylation at at least 15 positions in SEQ ID NO: 1 selected from the group set forth in Tables 2A-B, such as at least 20 positions, or at least 40 positions. Expressing an ExPEC-derived polypeptide, such as a UPEC-derived polypeptide.

[0117] Method for producing polypeptides In an aspect, the present invention provides a method for producing a glycosylated polypeptide of interest and / or a plurality of glycosylated polypeptides of interest, said method comprising: a) expressing said polypeptide of interest in a recombinant E. coli according to the invention; and b) purifying said glycosylated polypeptide of interest and / or said plurality of glycosylated polypeptides of interest from said bacteria, such as from a lysate and / or culture medium and / or supernatant, preferably from said culture medium and / or supernatant; The present invention relates to a method, comprising:

[0118] As outlined above, although the amino acid sequence of purified polypeptides may be the same, the exact glycosylation pattern may vary between individual polypeptides due to the nature of the glycosylation process in bacteria. Thus, in embodiments, the polypeptide of interest and / or a plurality of glycosylated polypeptides of interest is a polypeptide or pool of polypeptides according to the invention.

[0119] In an embodiment, in step b), the glycosylated polypeptide is purified from the culture medium and / or supernatant, preferably without a lysis step. As shown in Example 5, it is possible to purify the protein from the supernatant / culture medium.

[0120] Product of the method A further aspect of the present invention relates to a glycosylated polypeptide and / or a plurality of glycosylated polypeptides obtained or obtainable by the method according to the invention. As also outlined in the Examples section, it can be difficult to accurately establish the glycosylation pattern of an individual polypeptide.

[0121] In an embodiment, the glycosylated polypeptide relates to a composition comprising a polypeptide according to the invention or a plurality of polypeptides having a glycosylation pattern according to the invention.

[0122] Medical Use As outlined throughout the present application, polypeptides or compositions according to the present invention are effective vaccines (see Example 4 - Pig Challenge Study). Accordingly, aspects of the present invention relate to polypeptides and / or compositions according to the present invention for use as pharmaceuticals, such as vaccines.

[0123] In a similar aspect, the invention relates to a polypeptide and / or composition according to the invention for generating an immune response in a patient, such as being a vaccine.

[0124] In a further aspect, the present invention relates to a polypeptide and / or composition according to the present invention for use in the treatment, prevention and / or alleviation of E. coli infection.

[0125] In embodiments, the E. coli infection is an extraintestinal pathogenic E. coli (ExPEC) infection.

[0126] In embodiments, the polypeptides or compositions are for use in the treatment, prevention, and / or alleviation of extraintestinal pathogenic E. coli (ExPEC) infections, such as UPEC infections, such as urinary tract infections (UTIs), including bladder and / or kidney infections. Again, as shown in Example 4—Pig Challenge Studies, the polypeptides are highly effective against bladder infections. Without being bound by theory, it is believed that the glycosylation pattern of the polypeptides according to the invention resembles that of more highly pathogenic strains by being produced in a non-pathogenic version of the WT strain. Thus, the polypeptides are not produced in standard E. coli production strains.

[0127] In embodiments, the polypeptide or composition is for use in preventing extraintestinal pathogenic E. coli (ExPEC) infections, such as UPEC infections, from entering the bloodstream. In related embodiments, the polypeptide and / or composition according to the invention is for use in the treatment, prevention and / or alleviation of sepsis.

[0128] In a preferred embodiment, the polypeptide or composition is for use in the treatment, prevention and / or alleviation of UPEC infection.

[0129] antibody As outlined in Example 6 - Antibody Data and shown in Figure 10, immunization with hyperglycosylated polypeptides and / or compositions according to the present invention induces the production of antibodies with higher avidity compared to non-glycosylated versions isolated from conventional E. coli expression strains, e.g., BL21(DE3) or MG1655.

[0130] Thus, in an aspect, the present invention relates to an antibody specific for a polypeptide according to the present invention.

[0131] In embodiments, antibodies include polyclonal antibodies, monoclonal antibodies, antibodies in which the heavy and light chains are linked by a flexible linker, Fv molecules, antigen-binding fragments, Fab fragments, Fab' fragments, F(ab')2 molecules, single domain antibodies (sdAB) such as nanobodies, fully human antibodies, humanized antibodies, and chimeric antibodies.

[0132] The antibodies may be suitable for generating chimeric and / or human versions that may be suitable for human in vivo use.

[0133] Thus, the present invention also relates to the polypeptides described herein for use in animals to produce antisera, such as for diagnostic and therapeutic purposes.

[0134] Antibodies obtained from animals exposed to the polypeptides described herein may be used to treat or diagnose bacterial infections, such as UPEC infections.

[0135] Therefore, in a further aspect, the present invention relates to in vitro uses of the polypeptides, compositions and antibodies according to the invention.

[0136] In a related aspect, the invention relates to the use of a glycosylated polypeptide according to the invention for raising antibodies against the glycosylated polypeptide.

[0137] Other Aspects An aspect of the present invention relates to a method for immunizing a subject, said method comprising administering to said subject a polypeptide according to the present invention or a composition according to the present invention.

[0138] Another aspect relates to a method for treating a subject infected with UPEC, said method comprising administering to said subject a polypeptide according to the invention and / or a composition according to the invention and / or an antibody according to the invention.

[0139] In embodiments, the subject is a mammal, such as a lifestock, pet or racing animal, preferably a human.

[0140] It should be noted that embodiments and features described in the context of one aspect of the invention also apply to the other aspects of the invention.

[0141] Throughout this specification, unless the context requires otherwise, the terms "comprise" or "comprises" or variations thereof such as "comprising" will be understood to mean the inclusion of a stated integer or group of integers but not the exclusion of other integers or groups of integers.

[0142] Item of the present invention 1. Polypeptides are: a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; and / or c) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or d) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; Including, The polypeptide has a glycosylation pattern defined by glycosylation at at least 15 positions in SEQ ID NO:1 selected from the group set out in Tables 2A-B, such as at least 20 positions or at least 40 positions.

[0143] 2. The polypeptide of item 1, wherein the polypeptide is glycosylated at at least 15 positions, such as at least 20 positions, such as all of the positions shown in Table 2B.

[0144] 3. The polypeptide is glycosylated at at least 15 positions selected from the group set forth in Tables 3A-B, such as at least 20 positions, such as at least 30 positions, or such as all of the positions set forth in Tables 3A-B; and / or The polypeptide is glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or such as all of the positions set forth in Table 4; 3. The polypeptide according to item 1 or 2.

[0145] 4. The polypeptide is glycosylated at least at positions S152, and / or S154 and / or S164, such as positions S152 and S154, such as positions S154 and S164, such as positions S152 and S164 or positions S152, S154 and S164; and glycosylated at least at positions T592, and / or S594 and / or S597, such as at positions T592 and S594, such as at positions S594 and S597, such as at positions T592 and S597, or such as at positions T592, S594 and S597, 4. The polypeptide according to any one of items 1 to 3.

[0146] 5. A polypeptide, the polypeptide comprising: a) the amino acid sequence set forth in SEQ ID NO: 1 Includes; The polypeptide has a glycosylation pattern defined by being glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or such as all of the positions set forth in Table 4. Polypeptide.

[0147] 6. A composition comprising a polypeptide according to any one of items 1 to 5 and / or a composition comprising a plurality of polypeptides having a glycosylation pattern according to any one of items 1 to 5.

[0148] 7. A genetically modified E. coli, comprising: -FimH; - PapG; and -WaaL A recombinant E. coli that does not express

[0149] 8. The recombinant E. coli according to item 7, which is an ExPEC, such as a UPEC, such as UTI89.

[0150] 9. The genetically modified E. coli according to item 7 or 8, which is avirulent and / or non-pathogenic and / or incapable of causing disease.

[0151] 10. A method for producing a glycosylated polypeptide of interest and / or a plurality of glycosylated polypeptides of interest, said method comprising: a) expressing the polypeptide of interest in the recombinant E. coli according to any one of items 1 to 9; and b) purifying said glycosylated polypeptide of interest and / or said plurality of glycosylated polypeptides of interest from said bacteria, such as from a lysate and / or culture medium and / or supernatant, preferably from said culture medium and / or supernatant; A method comprising:

[0152] 11. A glycosylated polypeptide and / or a plurality of glycosylated polypeptides obtained or obtainable by the method according to item 10.

[0153] 12. A polypeptide according to any one of items 1 to 5 and / or a composition according to item 6 and / or a glycosylated polypeptide and / or a plurality of glycosylated polypeptides according to item 11 for use as a pharmaceutical, such as a vaccine.

[0154] 13. The polypeptide according to any one of items 1 to 5 or the composition according to item 6 and / or the glycosylated polypeptide and / or glycosylated polypeptides according to item 11 for use in the treatment, prevention and / or alleviation of an E. coli infection, such as an extraintestinal pathogenic E. coli (ExPEC) infection, such as a UPEC infection.

[0155] 14. A polypeptide according to any one of items 1 to 5 or a composition according to item 6 and / or a multiply glycosylated polypeptide according to item 11 for use in the treatment, prevention and / or alleviation of UPEC bladder infections.

[0156] 15. An antibody specific to the polypeptide according to any one of items 1 to 5.

[0157] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0158] The present invention is further illustrated in the following non-limiting examples. [Example]

[0159] Example 1 - Materials and Methods Bacterial strains and growth conditions Escherichia coli UTI89 (Infect Immun. 2001, 69:4572) was used as the wild-type UPEC strain and served as the basis for the production strain. For cloning purposes, strains were routinely grown with aeration at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg / ml ampicillin (Amp), 40 μg / ml kanamycin (Kan), or 30 μg / ml chloramphenicol (Cml) as needed.

[0160] For production of full-length YGHJ, all strains were grown in LB+M9 medium (Clark and Maaloe, J Mol Biol. 1967, 23:99) supplemented with 0.2% glucose and 0.4% casamino acids (1:1 vol / vol).

[0161] For adhesion assays, we used UTI89 yghJ::3xFLAG (Kan R ) was used and compared with UTI89 ΔwaaL ΔfimH ΔpapG yghJ::3xFLAG-10xHis::FRT (the production strain defined in this invention) and E. coli K-12 MG1655 (a non-pathogenic conventional production strain) harboring pNDM220 (Mol Microbiol. 1998, 29:1065) derivatives or pKD46, respectively. Strains were grown statically overnight at 37°C in LB medium before the adhesion assay.

[0162] For pig challenge studies, wild-type UTI89 was grown statically overnight in LB medium at 37°C. The culture was diluted 4000-fold into fresh LB medium and incubated statically overnight at 37°C. Serial passage and static growth ensure that the majority of the population expresses type 1 pili, which are necessary for causing infection (Hung et al. Nat Protoc. 2009, 4:1230).

[0163] DNA manipulation His tag The UTI89 yghJ gene was tagged at its 3' end by the method described by Uzzau et al. (Proc Natl Acad Sci USA 2001, 98:15264). Briefly, a linear dsDNA molecule was prepared by PCR containing an epitope followed by a kanamycin resistance cassette (kan) flanked by FRT sites (FLP recombinase recognition sites; this allows for removal of drug resistance once tagged clones are obtained). The tag-FRT-kan-FRT product was flanked by sequences (60-400 bp) that are homologous to the final portion of yghJ and the region immediately downstream from the gene. The linear PCR product was transformed into the target strain carrying an easily curable helper plasmid (pKD46) expressing the phage lambda Red recombinase, which promotes recombination between the homologous regions. Clones expressing the C-terminally tagged YGHJ protein can be identified by standard immunodetection methods (e.g., Western blotting), and antibiotic resistance can be removed using a curable helper plasmid, pCP20, which expresses the FLP recombinase (Proc Natl Acad Sci USA 2000, 97:6640). The primer sequences used to generate the yghJ-6xHis, -3xFLAG-6xHis, and -3xFLAG-10xHis clones are shown in Table 1.

[0164] deletion The Δc4349-4351 mutant was generated by the Datsenko and Wanner method (Datsenko and Wanner, 2000), which utilizes a linear PCR product containing phage lambda Red recombinase and an antibiotic resistance gene (cml) flanked by sequences homologous to chromosomal regions upstream and downstream of the region to be deleted, similar to the epitope tagging described above. Thus, a recombination event replaces the deleted region on the chromosome with the cml cassette. Additionally, we used this technique in attempts to delete the O-antigen cluster either completely (c2303-c2312) or partially (c2308-c2312 or c2303-c2307), but these efforts were unsuccessful. Primer sequences are listed in Table 1.

[0165] The ΔwaaL, ΔfimH, and ΔpapG mutations were generated using a CRISPR / Cas9 procedure adapted from Zhao et al. (Microb Cell Fact. 2016, 15:205). Briefly, this method relies on a single plasmid encoding the Cas9 endonuclease and sgRNA required to generate a double-stranded break in the target sequence; this plasmid also contains donor DNA (homologous sequences flanking the target region), phage lambda Red recombinase, and the recA gene required for DNA repair by homologous recombination. Due to the molecular plasmid design, it is only necessary to prepare custom guide sequences (N20, generated by annealing two DNA oligos) and donor DNA (approximately 300 bp homology arms, generated by PCR) for each target. The invariant components of the plasmid are amplified by PCR (part 1 and part 2), and the four parts are assembled by Golden Gate assembly using BsaI restriction enzyme and T4 DNA ligase. The primers and oligos used to construct the deletion mutants are shown in Table 1.

[0166] Cell line culture conditions The adhesion ability of the production strains was tested using the human bladder cell line ATCC 5637. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere and grown in Gibco RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and penicillin-streptomycin (Pen-Strep; 100 units / ml; 100 μg / ml) (Gibco). When 90% confluence was reached, 5637 cells were trypsinized for 5 min, diluted 1:4, and seeded into 12-well plates (Nunc).

[0167] Adhesion assay One hour before adding bacteria, 100% confluent 5637 cells (approximately 5 × 10 5 The cells (100 cells / well) were washed three times with 1x phosphate-buffered saline (PBS) and incubated with growth medium without Pen-Strep. A multiplicity of infection (MOI) of 0.5 was used. Bacteria and cells were incubated for 1 hour. Nonadherent bacteria were removed by washing the cells three times with PBS, with vigorous shaking of the plate on a plate shaker at each wash step. Relative adherence was determined by serial dilution and plating onto selective LA plates. CFU was determined the next day, and the adherence of each strain was normalized to that of the wild-type strain UTI89. Assays were performed in four independent biological replicates, with four wells per strain. Each well was serially diluted, and the dilutions were spotted in duplicate onto selective plates.

[0168] Western blot Denaturing SDS PAGE and Western blots were performed as described by Thorsing et al. (Thorsing et al., 2021). Antibodies used are specified in the figure legends.

[0169] Protein purification Glycosylated YGHJ was purified by His purification essentially as described by Riaz et al. (Riaz et al., 2021) with minor modifications. The filtered culture supernatant was adjusted to 200 mM NaCl and 0.05% Triton X-100 and incubated with TALON® resin to capture tagged YGHJ. After incubation, the resin was washed twice with Wash Buffer I (1x PBS, pH 7.2, containing 600 mM NaCl and 0.05% Triton X-100) and once with Wash Buffer II (0.5x PBS, containing 0.05% Triton X-100, pH 7.6). Glycosylated YGHJ was eluted by incubating the resin in Elution Buffer (0.5x PBS, containing 150 mM imidazole and 0.05% Triton X-100, pH 9.4). The eluate was concentrated by spin filtration and further purified by ion exchange on a Toyopearl NH2-750F 1 ml column (Tosoh Bioscience) using a ▲A▼KTA start (Cytiva) chromatography system. The concentrated eluate was loop-loaded onto the column and washed with 10 column volumes (CV) of buffer A (20 mM K sodium phosphate buffer, pH 6.0, 75 mM NaCl, 0.01% Triton X-100), followed by 10 CV of buffer A containing an additional 260 mM NaCl, and eluted with 14 CV of buffer A containing an additional 1.425 M NaCl. The purified protein was quantified against a bovine serum albumin standard using a BCA Micro assay (Thermo Fischer Scientific).

[0170] [Table 1A]

[0171] [Table 1B]

[0172] Example 2 - Production-dependent glycosylation patterns Test objectives To demonstrate that purification of full-length YGHJ from a detoxified, avirulent production strain results in efficient protein glycosylation (GPV02, as defined in the present invention). In contrast, when full-length YGHJ is isolated from conventional E. coli production strains, such as BL21(DE3) or MG1655, it results in a protein with significantly less glycosylation and a different glycosylation pattern compared to the production strains according to the present invention.

[0173] material and method Full-length glycosylated YGHJ (GPV02) was isolated from a production strain according to the present invention and compared with full-length YGHJ isolated from a conventional E. coli overexpression strain (conventional YGHJ antigen). Using the BEMAP protocol, O-linked glycosylated tryptic peptides within the two YGHJ protein variants were identified (Boysen et al., 2016). For BEMAP analysis of GPV02, 3 μg to 138 μg of purified protein was used as sample input. For BEMAP analysis of the conventional YGHJ antigen, 300 μg of purified protein was used as sample input. The relative abundance of each identified glycopeptide within the BEMAP analysis was also determined using the Minora Feature detector in the Proteome Discoverer software program. Standard settings for the Minora tool were used. Briefly, the Minora tool measures the area under each identified glycopeptide peak. By summing all areas, the relative abundance of each peptide can be calculated as a percentage (area of ​​peptide / total area of ​​all peaks x 100).

[0174] result A total of 12 BEMAP analyses using different sample amounts of GPV02 were performed to identify glycopeptides. For GPV02, we observed that the number of identified glycopeptides correlated with increasing amounts of sample used in the BEMAP analysis. For example, when 9.6 μg, 31.8 μg, 82 μg, and 138 μg of GPV02 sample were used as input, 16, 39, 54, and 66 glycopeptides were identified, respectively. For a subset of glycopeptides, the same glycosylation pattern was observed regardless of the amount of sample used for the BEMAP analysis. For another subset of glycopeptides, these were identified only when the highest sample amount was used for analysis. The combination of all BEMAP analyses of GPV02 identified a total of 102 modified residues, see Table 2AB, and established additional glycosylation patterns, see Tables 3AB and 4.

[0175] [Table 2]

[0176] [Table 3]

[0177] [Table 4]

[0178] To compare the glycosylation pattern of GPV02 with conventional YGHJ antigens (such as those produced and tested in Nesta et al. (Nesta et al., 2014)), a BEMAP analysis of one of the conventional YGHJ antigens was performed.

[0179] To maximize the number of identified glycopeptides from the conventional YGHJ antigen, 300 μg of protein was used as input to the BEMAP protocol. However, despite this large amount of protein, the BEMAP analysis identified only 11 glycopeptides. The correlation between the number of identified glycopeptides (for both GPV02 and the conventional YGHJ antigen) and sample amount is plotted and shown in Figure 1.

[0180] Using the Minora Feature Detector software tool, we determined the relative abundance of all identified glycopeptides from GPV02. As noted above, when analyzing GPV02, a small subset of peptides was almost always identified. These sequences were also those with the highest abundance. For example, as shown in Figure 2, four BEMAP analyses identified 39, 31, 54, and 66 glycopeptides when 31.8 μg, 54 μg, 82 μg, and 138 μg GPV02 samples were used as input, respectively. In these four analyses shown in Figure 2, the relative abundance of two peptides averaged 66% and 13%, respectively. The analysis also determined that five peptides had abundances greater than 1%, while the remaining peptides were less than 1%. Some peptides were found to have relative abundances approaching 0.001%. We emphasize that the dynamic range of the analysis spans five orders of magnitude.

[0181] For comparison, we also determined the relative abundance of 11 identified glycopeptides derived from conventional full-length YGHJ (see Figure 2). The relative abundance of these peptides differed from that determined when analyzing GPV02. For example, a peptide with an average abundance of 66% in GPV02 was not detected in any of the conventional full-length YGHJ-derived samples. Conversely, one peptide derived from conventional full-length YGHJ with an abundance of 61.4% was determined to be approximately 400-fold less abundant in GPV02. At a global level, the analysis shows that the abundance of 8 peptides from 11 sequences is not similar when comparing the two antigens, and only 3 peptides are similar.

[0182] conclusion GPV02 is a 1520 amino acid polypeptide containing 237 Ser / Thr residues. Using 138 μg of GPV02, a total of 102 modified residues were identified (Table 2). This number indicates that GPV02 is hyperglycosylated. It is unlikely that all 237 residues are modified, as many Ser / Thr sites are buried within the tertiary structure and therefore inaccessible to glycosyltransferases.

[0183] This is supported by the BEMAP analysis shown in Figure 2, which shows what appears to be an upper limit on the number of sites that can be modified. The results of glycopeptide analysis of the conventional full-length YGHJ antigen, as produced in Nesta et al. (Nesta et al., 2014), were quite surprising. Despite using 300 μg of protein sample as input, only 11 peptides were identified. One would have expected to identify at least a similar number of glycopeptides compared to GPV02 in an analysis using a larger sample volume. However, this was not the case, indicating that the choice of strain used for protein antigen production is important when specifically looking for protein glycosylation. Relative glycopeptide abundances were determined for the two antigens. Surprisingly, the relative abundances and patterns were not very similar, indicating that the protein glycosylation machinery must be a mechanistically distinct function in the two strains.

[0184] We conclude that GPV02 protein glycosylation is highly dependent on the choice of host strain. This has serious implications for production. Expressing the immunogen in a naturally pathogenic host is generally considered undesirable due to the risk of infection for individuals handling the purification process, waste management, and post-production equipment handling. Therefore, to obtain a glycosylated GPV02 vaccine, a production strain must be engineered that is capable of producing antigens with the glycosylation pattern of the naturally pathogenic bacterium, as defined in this invention, while at the same time being nonpathogenic and capable of being grown in a low-level biosafety laboratory. We have shown that this can be achieved by deleting genes involved in lipopolysaccharide production and human cell colonization (Example 3).

[0185] Example 3 - Non-pathogenic production strain Test objectives To obtain a non-pathogenic UPEC strain for production of a full-length YGHJ antigen (tagged to allow purification) that retains its ability to efficiently glycosylate YGHJ. Genes for editing were selected based on their importance to pathogenicity as well as undesirable immunogenicity. We focused on the removal of adhesins to hinder the pathogen's ability to adhere to host cells, as well as the reduction of the pathogen's pyrogenic O-antigen.

[0186] material and method His tagged: The UTI89 yghJ gene was tagged at its 3' end by the method described by Uzzau et al. (2001). Briefly, a linear dsDNA molecule was prepared by PCR containing an epitope followed by a kanamycin resistance cassette (kan) flanked by FRT sites (FLP recombinase recognition sites; this allows for removal of drug resistance once tagged clones are obtained). The tag-FRT-kan-FRT product was flanked by sequences (60-400 bp) that are homologous to the final portion of yghJ and the region immediately downstream from the gene. The linear PCR product was transformed into the target strain carrying an easily curable helper plasmid (pKD46) expressing the phage lambda Red recombinase, which promotes recombination between the homologous regions. Clones expressing C-terminally tagged YGHJ proteins are identified by standard immunodetection methods (e.g., Western blotting), and antibiotic resistance can be cured using a curable helper plasmid, pCP20, which expresses the FLP recombinase (Datsenko and Wanner, 2000). The primer sequences used to generate the yghJ-6xHis, -3xFLAG-6xHis, and -3xFLAG-10xHis clones are shown in Table 1.

[0187] deletion The Δc4349-4351 mutant was generated by the Datsenko and Wanner method (Datsenko and Wanner, 2000), which utilizes a linear PCR product containing phage lambda Red recombinase and an antibiotic resistance gene (cat) flanked by sequences homologous to the chromosomal regions upstream and downstream of the region to be deleted, similar to the epitope tagging described above. Thus, the recombination event replaces the deleted region on the chromosome with the cat cassette. Additionally, we used this technique in attempts to delete the O-antigen cluster either completely (c2303-c2312) or partially (c2308-c2312 or c2303-c2307), but these efforts were unsuccessful. Primer sequences are listed in Table 1.

[0188] The ΔwaaL, ΔfimH, and ΔpapG mutations were generated using a CRISPR / Cas9 procedure adapted from Zhao et al. (Zhao et al., 2016). Briefly, this method relies on a single plasmid encoding the Cas9 endonuclease and sgRNA required to generate a double-stranded break in the target sequence; this plasmid also contains donor DNA (homologous sequences flanking the target region), phage lambda Red recombinase, and the recA gene required for DNA repair by homologous recombination. Due to the molecular plasmid design, it is only necessary to prepare custom guide sequences (N20, generated by annealing two DNA oligos) and donor DNA (approximately 300 bp homology arms, generated by PCR) for each target. The invariant components of the plasmid are amplified by PCR (part 1 and part 2), and the four parts are assembled by Golden Gate assembly using BsaI restriction enzyme and T4 DNA ligase. The primers and oligos used to construct the deletion mutants are shown in Table 1.

[0189] result For purification purposes, the yghJ gene of UTI89 was modified to encode YGHJ with a C-terminal 3xFLAG-10xHis tag (see Example 5 below for epitope tag selection). We achieved this using the Datsenko-Wanner method (Datsenko and Wanner, 2000), which leaves some "scar" sequence downstream of the edited region after FLP-mediated excision of the antibiotic selection marker, although other methods (e.g., CRISPR / Cas) could also be used to introduce the tag.

[0190] O-antigen shedding Deletion of the wzx and wxy genes To shed O-antigens from the cell surface, we attempted to delete the wzx and wxy genes, which are involved in O-antigen transport and polymerization, respectively (Figure 3). In E. coli, genes involved in O-antigen biosynthesis were generally found in a cluster between the galF and gnd genes, which also support UTI89 (Samuel & Reeves, 2003). (In this rfbE / c2308 cluster, genes c2303-c2312 are putative O-antigen transporters, and c2307 encodes a putative O-antigen polymerase.) However, our efforts to delete all or part of this cluster (c2308-c2312 or c2303-c2307) were unsuccessful, and the most likely deletions were lethal. Therefore, we attempted a novel strategy: at distal loci on the chromosome, wzxE (c4349) and wzyE (c4351) are annotated as putative O-antigen flippase and O-antigen polymerase, respectively. We were able to delete both of these genes along with c4350, which encodes WecF, involved in enterobacterial common antigen (ECA) biosynthesis. However, as shown in Figure 3, the Δc4349-4351 mutant exhibited approximately half the O-antigen in the culture supernatant compared to the wild type, which was insufficient for further use of the mutant as a production strain.

[0191] WaaL deletion: In an alternative approach, we used CRISPR / Cas9 to perform a deletion in the WaaL gene (c4167), annotated as an O-antigen ligase (Figure 3), resulting in an in-frame deletion of amino acids 35–385 of WaaL. This mutant had approximately 500-fold less O-antigen in the culture supernatant than the wild-type strain (Figure 5), and the shift in migration pattern on SDS gels indicated that the remaining O-antigen polymers in the ΔwaaL mutant were generally shorter. Western blot analysis of the culture supernatant using an antibody against glycosylated YGHJ showed that the amount of exported YGHJ was comparable between the wild-type and the ΔwaaL mutant, but significantly, exported YGHJ from the deletion mutant appeared as two bands rather than one (Figure 6, upper panel). Considering that knockout of WaaL leads to outer membrane instability either directly due to the absence of O-antigen or indirectly by interfering with other biosynthetic pathways (e.g., peptidoglycan, capsule, or ECA synthesis) that depend on recycling of the general undecaprenyl phosphate carrier (Fig. 3), we investigated σ as an indicator of envelope stress. E We analyzed the intracellular levels of and found comparable levels in the wild type and the ΔwaaL mutant (Fig. 6 , lower panel).

[0192] Adhesin shedding: Deletion of fimH(c5017) and papG(c4887): The UTI89 ΔwaaL yghJ::3xFLAG-10xHis::FRT strain was further edited to reduce its virulence by removing two adhesins encoded by fimH (c5017) and papG (c4887), both of which are involved in early infection pathogenesis. Using CRISPR / Cas9 (Zhao et al., 2016) methods, we sequentially introduced in-frame deletions to remove amino acids 19–290 of FimH and amino acids 20–238 of PapG. When testing the resulting production strain (UTI89 ΔwaaL ΔfimH ΔpapG yghJ::3xFLAG-10xHis::FRT) in an in vitro adhesion assay with the human bladder cell line 5637, we found that the adhesion of the mutant was significantly reduced compared to the wild type and even lower than that of the nonpathogenic laboratory strain, E. coli K-12 MG1655 (Figure 7).

[0193] conclusion A production strain for expression of YghJ-3xFLAG-10xHis was established based on the natural pathogenic host, E. coli UTI89. The production strain was rendered unable to colonize by in-frame deletions of the genes encoding the adhesins, FimH and PapG. The production strain was further engineered to reduce the O-antigen binding to lipopolysaccharide (LPS), the most prominent component of the outer leaflet of the outer membrane. This was achieved by deletion of the waaL gene, which encodes the O-antigen ligase and reduces O-antigen levels 500-fold compared to the wild-type strain (Figure 5).

[0194] The phenotypic significance of these deletions was confirmed by in vitro adhesion assays, which showed that the adherence of the production strain to human bladder cells was comparable to that of the non-pathogenic control and significantly lower than that of the wild-type strain (Figure 7). The phenotype of the production strain defined in this invention was characterized by reduced adherence not only compared to wild-type and conventional production strains, but also compared to the single KO mutants ΔwaaL and ΔfimH (data not shown).

[0195] Example 4 - Pig Challenge Study Test objectives To evaluate the degree of protection afforded by GPV02 produced in a production strain according to the invention (Example 3) against UPEC bladder infection in pigs.

[0196] material and method The experimental pig model (immunization and bladder infection) uses 9-week-old female LYD pigs that are challenged in the bladder with UPEC UTI89 (Nielsen et al., 2019). 1 × 10 in a total volume of 100 ml PBS buffer. 4 All pigs were challenged with a dose of 100 CFU of UPEC UTI89 and grown as described above. This dose was experimentally determined to be the minimum dose required for 100% of animals to develop bladder infection (Stārk et al., 2022). The experiment consisted of 40 pigs split into two separate trials. The first and second limbs of the trial were stopped 8 and 4 days post-infection, respectively.

[0197] Animals were immunized subcutaneously in the neck three times, spaced 2 weeks apart. When the vaccinated pigs achieved a predetermined antibody titer threshold on day 28, all animals were scheduled to be inoculated on day 42. In each of the two studies, 10 pigs were immunized with GPV02 antigen or mock-immunized with saline buffer. For immunization with GPV02, the dose consisted of 25 μg of GPV02 and 0.5 μg of dmLT and 8 mg of Litevax CMS adjuvant in a total volume of 1.5 ml. Urine samples were collected pre-inoculation, 1 day post-infection, and on the final day of the study. The number of CFUs in urine and the number of bacteria associated with bladder tissue were determined by serial dilution and plate counting. IgG and IgA antibody endpoint titers were determined in blood and vaginal swab samples isolated throughout the challenge study using an ELISA in which plates were coated with GPV02. A summary of the challenge study is shown in Table 5.

[0198] [Table 5]

[0199] result Immunization and bladder infection were performed as described above. On day 43 (1 day post-infection (DPI)), urine from 3 of 19 pigs in the vaccinated group contained no bacteria (P=0.168), indicating sterilizing immunity. In contrast, urine samples from all control group animals contained variable bacteria, demonstrating bladder infection. Bacterial counts associated with bladder tissue were tabulated for both groups at the end of the experiment. As can be seen in Figure 8, GPV02 vaccination significantly reduces the ability of UTI89 to colonize bladder tissue. Bacterial counts in urine were determined 1 day post-infection for both groups. As shown in Figure 9, bacterial counts in urine are reduced in vaccinated animals compared to the control group.

[0200] conclusion The data presented demonstrate the biological relevance of GPV02 as a vaccine candidate. GPV02 conferred sterilizing immunity against bladder infection in 15.8% of pigs, with significantly fewer CFU in bladder tissue at termination and fewer CFU counts in urine at day 1 post-infection from vaccinated animals compared to controls (P=0.0026 and P=0.139, respectively).

[0201] In a previous study, the full-length YGHJ / SSLE antigen failed to protect against bladder infection in a mouse model (Nesta et al., 2014). To our knowledge, our results are the first to demonstrate a YGHJ / SSL-derived antigen that confers protection in an animal model of urinary tract infection. The differentiating feature between GPV02 and the antigen used in Nesta et al., 2014, is hyperglycosylation.

[0202] As outlined above, the antigens used in Nesta et al. were produced in conventional production strains (and not in their original hosts), which likely resulted in suboptimal antigens due to different glycosylation patterns.

[0203] Example 5 - Tagging For the purpose of antigen production from culture supernatants, the yghJ gene was edited to encode an in-frame polyhistidine tag (His tag) at the end of the open reading frame. The His tag was placed at the C-terminus while YGHJ has an N-terminal signal peptide that targets the protein for export.

[0204] Initially, we used a 6xHis tag, but we were unable to detect binding to TALON® resin under the test conditions. Next, we added a triple FLAG tag between YGHJ and the 6xHis tag, which acted as a 22-amino acid spacer, providing some distance between YGHJ and the His tag to make interaction with the resin more accessible. The YghJ-3xFLAG-6xHis fusion bound to TALON® resin, albeit only weakly. Therefore, to enhance the strength of the interaction, the production strain was edited to further encode 10 histidine residues in the tag. This modification significantly improved the interaction between the YghJ-3xFLAG-10xHis fusion and TALON® resin (see Table 6). In a similar setup, we tested a construct using a 4x glycine-serine linker (GS linker) (4 glycines followed by 1 serine) before the His tag instead of the 3xFLAG tag and found it to be effective for purification purposes (Table 6). This setup was tested as it was thought to reduce linker immunogenicity.

[0205] [Table 6]

[0206] conclusion To improve the purification method, a YghJ-3xFLAG-10xHis fusion construct was prepared and used in the production strain, as defined in the present invention.

[0207] Example 6 - Antibody Data Test objectives Immunization with hyperglycosylated GPV02 elicits the production of antibodies with higher avidity compared to the non-glycosylated full-length YGHJ version isolated from conventional E. coli expression strains, such as BL21(DE3) or MG1655.

[0208] material and method Animal Immunization: Glycosylated GPV02 and non-glycosylated full-length YGHJ antigens were purified from each strain using the protocol described (Thorsing et al., 2021). Nine-week-old female LYD pigs were used for the experiment. The study consisted of eight pigs divided into two groups of four animals each. Animals were immunized subcutaneously in the neck twice, two weeks apart. Groups received either GPV02 or the non-glycosylated full-length YGHJ variant. The dose consisted of 25 μg of antigen and 0.5 μg of dmLT and 8 mg of Litevax CMS adjuvant in a total volume of 1.5 ml. Two weeks after the final dose, serum was isolated from the animals. Serum samples were used for IgG and IgA antibody endpoint titer determination and avidity assays.

[0209] Avidity assay The avidity assay was ELISA-based and performed essentially as described (Luo et al., 2016) with as few modifications as possible. Briefly, ELISA plates were coated overnight at 4°C with either 0.15 μg / ml GPV02 or 0.3 μg / ml nonglycosylated full-length YGHJ in PBS buffer. The starting dilution of pre-immune samples was 50x, while the starting serum sample dilution from immunized animals was 400x before addition to the plate. All serum samples were serially diluted 2-fold in the plate. Serum samples were tested on plates coated with the same antigens used for immunization, GPV02 and nonglycosylated full-length YGHJ, respectively. After 1 h of serum incubation at room temperature, the plate was washed with PBS buffer (PBS + 0.05% Tween 20). PBS buffer with or without 6 M urea was then added to the plate for 75 minutes at room temperature. After washing the plate with PBS buffer, a 16,000-fold diluted secondary porcine IgG-HRP-conjugated antibody was added to the wells for 1 hour at room temperature. After washing the plate with PBS buffer, the reaction was determined by kinetic ELISA. Avidity was calculated as the kinetic ELISA slope (Vmax) + UREA / - UREA.

[0210] result Avidity indices were calculated for both groups of animals and are plotted in Figure 10. The analysis shows that pigs immunized with GPV02 produced antibodies with significantly higher functional affinity to the antigen compared to animals vaccinated with the non-glycosylated full-length YGHJ antigen.

[0211] conclusion Memory B cells are a major component of long-term antibody-mediated protective immunity after infection or vaccination. High antibody avidity has previously been found to correlate with the presence of antigen-specific memory B cells in several human bacterial pathogens (Alam et al., 2013; Luo et al., 2016). Therefore, antibody avidity may be a marker for predicting whether a vaccine will provide protective immunity.

[0212] Our analysis shows that vaccination with a glycosylated antigen such as GPV02 results in significantly higher antibody avidity compared to a non-glycosylated but otherwise identical variant, suggesting that GPV02 isolated from a production strain may provide protective immunity to a greater extent than antibodies currently generated with conventional industrial production strains.

[0213] Example 7 - Glycan to Polypeptide Ratio Test objectives To further characterize YghJ isolated from a wild-type UPEC strain, a standard E. coli K12 production strain and a production strain according to the present invention, we used the services of biotech company Spectralys Biotech, which uses FTIR (Fourier Transform Infrared Spectroscopy) to analyze proteins for their glycan-to-protein ratio.

[0214] material and method The sample input to the Spectralys analysis is i) wild-type UPEC strains (yghJ-FLAG tag, depending on chromosomal expression level); ii) production strains dependent on chromosomal expression level (ΔwaaL, ΔfimH, ΔpapG, yghJ-GS linker-10xHis tag) and iii) Artificially induced protein expression from a plasmid in a standard E. coli K12 genetic background The first was YghJ isolated from

[0215] To determine the glycan / peptide ratio, Fourier transform infrared spectroscopy was performed on the sample input to obtain FTIR spectra. The spectra were then analyzed from 1182 to 1002 cm. -1The peak area ratio, which reflects the mass ratio between carbohydrates and proteins, was obtained by integrating the peaks at 1740–1478 m (absorption of glycans) and 1740–1478 m (absorption of proteins). For more detailed information, the method is described in Derenne et al. ((2021). Analysis of Glycoproteins by ATR-FTIR Spectroscopy: Comparative Assessment. In: Delobel, A. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol. 2271. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-1-0716-1241-5_25).

[0216] [Table 7]

[0217] result Results from the Spectralys analysis are shown in Figure 11. The analysis shows that YghJ isolated from either the wild-type UPEC strain or the production strain has an identical glycan-to-protein ratio of 0.034. In contrast, plasmid expression in a conventional E. coli production strain resulted in the lowest level of glycosylation, with a ratio of only 0.018.

[0218] conclusion The current industry standard for protein expression in conventional E. coli yields proteins that are dissimilar to those produced "in nature" when considering the glycan-to-protein ratio. This assertion is supported by Figure 1, which shows that YghJ protein isolated from a standard E. coli production strain yielded a much smaller number of unique glycopeptides. Further support for this assertion is shown in Figure 2, which shows that the relative glycopeptide abundance and patterns are less similar between the production strains according to the present invention and the standard E. coli expression strains. This is further supported by the data in Example 7 and Figure 11, which also show that the glycans relative to the protein vary between the standard production strains and wt strains and the production strains according to the present invention.

[0219] In summary, the data indicate that current industrial production standards result in a low-efficacy vaccine. On the other hand, YghJ isolated from a production strain with an intact protein glycosylation apparatus yields a protein whose total glycan-to-protein content is similar to that of wild-type UPEC strains.

[0220] array SEQ ID NO: 1: (1520 AA)

[0221] References Alam, M.M., Arifuzzaman, M., Ahmad, S.M., Hosen, M.I., Rahman, M.A., Rashu, R., et al. (2013). Study of avidity of antigen - specific antibody as a means of understanding development of long - term immunological memory after Vibrio cholerae O1 infection. Clin. Vaccine Immunol. 20, 17 - 23. doi:10.1128 / CVI.00521 - 12. Boysen, A., Palmisano, G., Krogh, T.J., Duggin, I.G., Larsen, M.R., M▲o▼ller - Jensen, J., et al. (2016). A novel mass spectrometric strategy “BEMAP” reveals Extensive O - linked protein glycosylation in Enterotoxigenic Escherichia coli. Sci. Rep. 6, 32016. doi:10.1038 / srep32016. Cassini, A., Plachouras, D., Eckmanns, T., Abu Sin, M., Blank, H.P., Ducomble, T., et al. (2016). Burden of Six Healthcare - Associated Infections on European Population Health: Estimating Incidence - Based Disability - Adjusted Life Years through a Population Prevalence - Based Modelling Study. PLoS Med. 13, 1 - 16. doi:10.1371 / journal.pmed.1002150. Cek,M.,Tando▲g▼du,Z.,Wagenlehner,F.,Tenke,P.,Naber,K.,and Bjerklund-Johansen,T.E.(2014)。Healthcare-associated urinary tract infections in hospitalized urological patients-a global perspective:results from the GPIU studies 2003-2010。World J.Urol.32,1587-1594.doi:10.1007 / s00345-013-1218-9。 Datsenko,K.A.,and Wanner,B.L.(2000)。One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products。Proc.Natl.Acad.Sci.U.S.A.97,6640-6645.doi:10.1073 / pnas.120163297。 Hilgers,L.A.T.,Platenburg,P.P.L.I.,Bajramovic,J.,Veth,J.,Sauerwein,R.,Roeffen,W.,et al.(2017)。Carbohydrate fatty acid monosulphate esters are safe and effective adjuvants for humoral responses。Vaccine 35,3249-3255.doi:10.1016 / j.vaccine.2017.04.055。 Luo,Q.,Vickers,T.J.,and Fleckenstein,J.M.(2016)。Immunogenicity and protective efficacy against enterotoxigenic Escherichia coli colonization following intradermal,sublingual,or oral vaccination with EtpA adhesin。Clin.Vaccine Immunol.23,628-637.doi:10.1128 / CVI.00248-16。 Medina,M.,and Castillo-Pino,E.(2019)。An introduction to the epidemiology and burden of urinary tract infections。Ther.Adv.Urol.11,3-7。 Mitchell,B.G.,Ferguson,J.K.,Anderson,M.,Sear,J.,and Barnett,A.(2016)。Length of stay and mortality associated with healthcare-associated urinary tract infections:A multi-state model。J.Hosp.Infect.93,92-99.doi:10.1016 / j.jhin.2016.01.012。 Nesta,B.,Valeri,M.,Spagnuolo,A.,Rosini,R.,Mora,M.,Donato,P.,et al.(2014)。SslE Elicits Functional Antibodies That Impair In Vitro Mucinase Activity and In Vivo Colonization by Both Intestinal and Extraintestinal Escherichia coli Strains。PLoS Pathog.10.doi:10.1371 / journal.ppat.1004124。 Nielsen,T.K.,Petersen,N.A.,St▲a▼rk,K.,Gr▲o▼nnemose,R.B.,Palarasah,Y.,Nielsen,L.F.,et al.(2019)。A Porcine Model for Urinary Tract Infection。Front.Microbiol.10,1-12. doi:10.3389 / fmicb.2019.02564。 Riaz,S.,Steinsland,H.,Thorsing,M.,Andersen,A.Z.,Boysen,A.,and Hanevik,K.(2021)。Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ(SslE)。Front.Immunol.12,1-11.doi:10.3389 / fimmu.2021.760135。 St▲a▼rk,K.,Andersen,M.▲O▼.,and Andersen,T.E.(2022)。Uropathogenic Escherichia coli can cause cystitis at extremely low inocula in a pig model。J.Med.Microbiol.71,1-5.doi:10.1099 / jmm.0.001537。 The European Centre for Disease Prevention and Control(2017)。ECDC:SURVEILLANCE REPORT。Surveillance of antimicrobial resistance in Europe 2016.doi:10.2900 / 296939。 Thorsing,M.,Krogh,T.J.,Vitved,L.,Nawrocki,A.,Jakobsen,R.,Larsen,M.R.,et al.(2021)。Linking inherent O-Linked Protein Glycosylation of YghJ to Increased Antigen Potential。Front.Cell.Infect.Microbiol.11,1-10.doi:10.3389 / fcimb.2021.705468。 Vallejo-Torres,L.,Pujol,M.,Shaw,E.,Wiegand,I.,Vigo,J.M.,Stoddart,M.,et al.(2018)。Cost of hospitalised patients due to complicated urinary tract infections:A retrospective observational study in countries with high prevalence of multidrug-resistant Gram-negative bacteria:The COMBACTE-MAGNET,RESCUING study。BMJ Open 8,1-9.doi:10.1136 / bmjopen-2017-020251。 Wang,X.,and Quinn,P.J.(2010)。Lipopolysaccharide:Biosynthetic pathway and structure modification。Prog.Lipid Res.49,97-107.doi:10.1016 / j.plipres.2009.06.002。 Zhao,D.,Yuan,S.,Xiong,B.,Sun,H.,Ye,L.,Li,J.,et al.(2016)。Development of a fast and easy method for Escherichia coli genome editing with CRISPR / Cas9。Microb.Cell Fact.15,1-9.doi:10.1186 / s12934-016-0605-5。 Zhao,X.,Wang,L.,Wei,N.,Zhang,J.,Ma,W.,Zhao,H.,et al.(2020)。Epidemiological and clinical characteristics of healthcare-associated infection in elderly patients in a large Chinese tertiary hospital:A 3-year surveillance study。BMC Infect.Dis.20,1-7.doi:10.1186 / s12879-020-4840-3。

Claims

1. A genetically modified E. coli comprising: does not express FimH; and - does not express PapG and / or WaaL, Genetically modified E. coli.

2. The recombinant E. coli is does not express FimH; and - does not express WaaL, 2. The recombinant E. coli of claim 1.

3. The recombinant E. coli is - does not express FimH; - does not express PapG; and - does not express WaaL, The recombinant E. coli according to claim 1 or 2.

4. 4. The recombinant E. coli of claim 1, wherein the recombinant E. coli is an ExPEC, such as a UPEC, such as UTI89.

5. The genetically modified E. coli according to any one of claims 1 to 4, wherein the genetically modified E. coli is avirulent and / or non-pathogenic and / or incapable of causing disease.

6. 6. The genetically modified E. coli of any one of claims 1 to 5, wherein the gene is knocked out by a method selected from the group consisting of in-frame deletion, introduction of a stop site, and total gene removal.

7. The recombinant E. coli is - expressing a glycosylated polypeptide of interest, such as an immunogenic vaccine; or - adapted to express a glycosylated polypeptide of interest, such as an immunogen / vaccine, The recombinant E. coli according to any one of claims 1 to 6.

8. 8. The genetically modified E. coli of any one of claims 1 to 7, wherein the genetically modified E. coli encodes a polypeptide that can be expressed in the E. coli and has a glycosylation pattern similar to that of a non-genetically modified version of the E. coli, such as UTI89.

9. 9. The genetically modified E. coli of claim 8, wherein the polypeptide is under the control of its endogenous promoter.

10. 10. The genetically modified E. coli of claim 8 or 9, wherein the polypeptide is expressed from the genome, i.e., is an endogenous polypeptide, and is not expressed from an exogenous vector, such as a plasmid.

11. 10. The genetically modified E. coli of claim 8 or 9, wherein the polypeptide is expressed from the genome and not from an exogenous vector such as a plasmid.

12. 10. The recombinant E. coli of claim 8 or 9, wherein the polypeptide is expressed from an exogenous vector, such as a plasmid.

13. 12. The recombinant E. coli of any one of claims 8 to 9 and 11, wherein the polypeptide is expressed from a plasmid.

14. 14. The genetically modified E. coli of any one of claims 1 to 13, wherein the genetically modified E. coli is avirulent and / or non-pathogenic and / or incapable of causing disease.

15. 15. The recombinant E. coli of any one of claims 1 to 14, wherein the recombinant E. coli has lower adherence to human bladder cells, such as human bladder cell line 5637, than the corresponding wild-type strain.

16. 16. The recombinant E. coli of any one of claims 1 to 15, wherein the recombinant E. coli has lower adherence to human bladder cells, such as human bladder cell line 5637, than E. coli K-12 MG1655.

17. The recombinant E. coli is a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and / or c) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or d) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; expressing an ExPEC-derived polypeptide, such as a UPEC-derived polypeptide, comprising The polypeptide has a glycosylation pattern defined by being glycosylated at at least 15 positions in SEQ ID NO: 1 selected from the group set forth in Tables 2A-B, such as at least 20 positions, or at least 40 positions. The recombinant E. coli according to any one of claims 1 to 16.

18. 1. An ExPEC-derived polypeptide, such as a UPEC-derived polypeptide, said polypeptide comprising: a) the amino acid sequence set forth in SEQ ID NO: 1; and / or b) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and / or a) an amino acid sequence that is a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; and / or b) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 and comprising a fragment of at least 1000 consecutive amino acids from SEQ ID NO: 1; Including, The polypeptide has a glycosylation pattern defined by being glycosylated at at least 15 positions in SEQ ID NO: 1 selected from the group shown in Tables 2A-B, such as at least 20 positions or at least 40 positions. Polypeptide.

19. 19. The polypeptide of claim 18, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

1.

20. 19. The polypeptide of claim 18, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1, and has a glycosylation pattern defined by being glycosylated at at least 40 positions in SEQ ID NO:1 selected from the group set forth in Tables 2A-B.

21. The polypeptide according to any one of claims 18 to 20, wherein the polypeptide is derived from a UPEC strain.

22. 22. The polypeptide of any one of claims 18 to 21, wherein the polypeptide is glycosylated at at least 15 positions, such as at least 20 positions, such as all of the positions shown in Table 2B.

23. 23. The polypeptide of any one of claims 18-22, wherein the polypeptide is glycosylated at at least 15 positions selected from the group set forth in Tables 3A-B, such as at least 20 positions, such as at least 30 positions, or such as all of the positions set forth in Tables 3A-B.

24. 24. The polypeptide of any one of claims 18 to 23, wherein the polypeptide is glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or such as all of the positions set forth in Table 4.

25. the polypeptide is glycosylated at least at position S152, and / or S154 and / or S164, such as at positions S152 and S154, such as at positions S154 and S164, such as at positions S152 and S164, or at positions S152, S154 and S164; and glycosylated at least at positions T592, and / or S594 and / or S597, such as at positions T592 and S594, such as at positions S594 and S597, such as at positions T592 and S597, or such as at positions T592, S594 and S597, A polypeptide according to any one of claims 18 to 24.

26. A polypeptide according to any one of claims 18 to 25, or a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; The polypeptide has a glycosylation pattern defined by being glycosylated at at least 10 positions selected from the group set forth in Table 4, such as at least 15 positions, such as at least 20 positions, or such as all of the positions set forth in Table 4. Polypeptide.

27. The polypeptide according to any one of claims 18 to 26, wherein the polypeptide has a glycan to protein ratio of at least 0.020, such as at least 0.025 by weight, preferably at least 0.030, or in the range of 0.020 to 0.050 by weight, preferably in the range of 0.030 to 0.050, more preferably in the range of 0.030 to 0.

04.

28. 28. The polypeptide of claim 27, wherein the glycan to protein ratio is determined by Fourier transform infrared spectroscopy (FTIR).

29. A composition comprising a polypeptide according to any one of claims 18 to 28.

30. 1. A method for producing a glycosylated polypeptide of interest and / or a plurality of glycosylated polypeptides of interest, said method comprising: a) expressing the polypeptide of interest in the recombinant E. coli according to any one of claims 1 to 17; and b) purifying said glycosylated polypeptide of interest and / or said plurality of glycosylated polypeptides of interest from said bacteria, such as from the lysate and / or culture medium and / or supernatant, preferably from said culture medium and / or supernatant; Including, method.

31. 31. The method according to claim 30, wherein in step b) the glycosylated polypeptide is purified from the culture medium and / or supernatant, preferably in the absence of a lysis step.

32. 32. A glycosylated polypeptide and / or a plurality of glycosylated polypeptides obtained or obtainable by the method of claim 30 or 31.

33. A polypeptide according to any one of claims 18 to 28 and / or a composition according to claim 29 and / or a glycosylated polypeptide and / or a plurality of glycosylated polypeptides according to claim 32 for use as a pharmaceutical, such as a vaccine.

34. 32. The polypeptide of any one of claims 18 to 28 or the composition of claim 29 and / or the glycosylated polypeptide and / or glycosylated polypeptides of claim 32 for use in the treatment, prevention and / or alleviation of E. coli infections, such as extraintestinal pathogenic E. coli (ExPEC) infections, such as UPEC infections.

35. A polypeptide according to any one of claims 18 to 28 or a composition according to claim 29 and / or a glycosylated polypeptide and / or a plurality of glycosylated polypeptides according to claim 32 for use in the treatment, prevention and / or alleviation of UPEC bladder infections.

36. An antibody specific for a polypeptide according to any one of claims 18 to 28 or 32.

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