Compositions and methods for producing immunity against bacterial infections

Abstract

The present disclosure relates, in part, to a polypeptide of formula (I) or a salt or solvate thereof, and a vaccine composition further comprising at least one pharmaceutically acceptable excipient. The disclosure also relates to isolated mRNA and / or isolated polynucleotides encoding the polypeptides of Formula (I), vectors and / or LNPs comprising the same, and pharmaceutical compositions thereof. The disclosure also relates to methods of treating, preventing and / or ameliorating bacterial infection, and / or generating immunity against infection by one or more pathogenic bacteria in a subject in need thereof, comprising administering to the subject at least one composition of the disclosure. In certain embodiments, the bacterial infection is urinary tract infection, sepsis (e.g., neonatal sepsis), or pneumonia.

Description

[0001] Cross-references to related applications Pursuant to 35 U.S.C., 119(e), this application claims priority to U.S. Provisional Patent Application No. 63 / 471,365, filed June 6, 2023, and U.S. Provisional Patent Application No. 63 / 535,969, filed August 31, 2023, the entire contents of which are incorporated herein by reference.

[0002] Reference to electronic sequence listing An XML file named "375836_7000WO1_SequenceListing.XML", created on June 5, 2024, contains 245 KB and is hereby incorporated in its entirety by reference. Background Technology

[0003] Bacterial infections (such as urinary tract infections (UTI), sepsis, and pneumonia) pose a significant threat to human health, resulting in high morbidity and mortality rates worldwide, including infant mortality (such as neonatal sepsis).

[0004] While infections such as UTIs, sepsis (e.g., neonatal sepsis), and pneumonia can be caused by a variety of bacteria, viruses, and / or fungi, these infections are most commonly caused by bacteria (e.g., Escherichia coli). Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ) and Proteus mirabilis ( Proteus mirabilis (This is generally considered the most common cause of UTIs). Therefore, antibiotics are often used as first-line treatments (such as ciprofloxacin). However, in addition to the risks associated with over-reliance on antibiotics (such as the development of antibiotic resistance), these antibiotics can also cause side effects, including but not limited to nausea, vomiting, stomach pain, heartburn, diarrhea, and fatigue.

[0005] Therefore, there is a need in the art for compositions and / or methods for preventing bacterial infections (e.g., UTIs, sepsis, and / or pneumonia) and / or generating immunity against one or more bacterial pathogens, non-limiting examples of which include *Escherichia coli*. This disclosure addresses this need. Summary of the Invention

[0006] In one aspect, the present invention provides a polypeptide comprising formula (I) or a salt or solvation thereof, wherein n and B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5 L 6 T1 T 2 and T 3 Each time it appears elsewhere in this article, it is defined as follows: (I).

[0007] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B11 B 12 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 and T 12 Defined elsewhere in this document.

[0008] In another aspect, the present invention provides a polypeptide comprising T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 L 1 L 2 L 3 L4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 and T 12 Defined elsewhere in this document.

[0009] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B9 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 and T 9 Defined elsewhere in this document.

[0010] In another aspect, the present invention provides a polypeptide comprising T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 and T 9 Defined elsewhere in this document.

[0011] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 1 -L 10 -B 6 -L 11 -T 2 -L 12 -B 7 -L 13 -T 3 -L 14 -B 8 -L 15 -T 4 -L 16 -B 9 -L 17 -T 1 -L 18 -B 10 -L 19 -T 2 -L 20 -B 11 -L 21 -T 3 -L 22 -B 4 -L 23 -T 4 B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 L 1 L 2 L 3 L 4 L 5 L6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 T 1 T 2 T 3 and T 4 Defined elsewhere in this document.

[0012] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 L 24 L 25 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Defined elsewhere in this document.

[0013] In another aspect, the present invention provides a polypeptide comprising T 1 -L 1 -T2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 -L 12 -T 13 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Defined elsewhere in this document.

[0014] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 B 1 、B 2 、B 3 、B 4 、B 5 、B 6 、B 7 、B 8 、B 9 、B 10 、B 11 、B 12 、B 13 、L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 、L 8 、L 9 、L 10 、L 11 、L 12 、L 13 、L 14 、L 15 、L 16 、L 17 、L 18 、L 19 、L 20L 21 L 22 L 23 L 24 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 and T 12 Defined elsewhere in this document.

[0015] In another aspect, the present invention provides a polypeptide comprising B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -T 1 -L 5 -T 2 -L 6 -T 3 -L 7 -T 4 -L 8 -B 5 -L 9 -B 6 -L 10 -B 7 -L 11 -B 8 -L 12 -T 5 -L 13 -T 6 -L 14 -T 7 -L 15 -T 8 -L 16 -B 9 -L 17 -B 10 -L 18 -B 11 -L 19 -B 12 -L 20 -T 9 -L 21 -T 10 -L 22 -T 11 -L 23 -T 12B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 and T 12 Defined elsewhere in this document.

[0016] In another aspect, the present invention provides a polypeptide comprising T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -B 1 -L 5 -B 2 -L 6 -B 3 -L 7 -B 4 -L 8-T 5 -L 9 -T 6 -L 10 -T 7 -L 11 -T 8 -L 12 -B 5 -L 13 -B 6 -L 14 -B 7 -L 15 -B 8 -L 16 -T 9 -L 17 -T 10 -L 18 -T 11 -L 19 -T 12 -L 20 -B 9 -L 21 -B 10 -L 22 -B 11 -L 23 -B 12 B 1 、B 2 、B 3 、B 4 、B 5 、B 6 、B 7 、B 8 、B 9 、B 10 、B 11 、B 12 、L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 、L 8 、L 9 、L 10 、L 11 、L 12 、L 13 、L 14 、L 15 、L 16 、L 17 、L 18 、L 19 、L 20 、L 21 、L 22 、L 23 、T1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 and T 12 Defined elsewhere in this document.

[0017] In another aspect, the present invention provides an isolated messenger ribonucleic acid (mRNA) encoding the polypeptide of the present invention.

[0018] In another aspect, the present invention provides an isolated deoxyribonucleic acid (DNA) encoding the polypeptide of the present invention.

[0019] In another aspect, the present invention provides an isolated polynucleotide encoding the mRNA of the present invention, wherein the polynucleotide comprises one or more promoters and / or polyadenylation signals operatively linked to a sequence encoding the mRNA.

[0020] In another aspect, the present invention provides a vector comprising isolated mRNA, isolated DNA, and / or isolated polynucleotides of the present invention.

[0021] In another aspect, the present invention provides a lipid nanoparticle (LNP) composition comprising isolated mRNA, isolated DNA, and / or isolated polynucleotides of the present invention.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising the LNP of the present invention and a pharmaceutically acceptable carrier.

[0023] In another aspect, the present invention provides a vaccine composition comprising the LNP of the present invention and / or the pharmaceutical composition of the present invention.

[0024] In another aspect, the present invention provides a vaccine composition comprising the polypeptide of the present invention and at least one pharmaceutically acceptable excipient.

[0025] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the polypeptide of the present invention to the subject.

[0026] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the isolated mRNA of the present invention to the subject.

[0027] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the isolated DNA of the present invention to the subject.

[0028] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the isolated polynucleotides of the present invention to the subject.

[0029] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the carrier of the present invention to the subject.

[0030] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the LNP of the present invention to the subject.

[0031] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the pharmaceutical composition of the present invention to the subject.

[0032] In another aspect, the present invention provides a method for treating, preventing, and / or improving bacterial infections in a subject in need, the method comprising administering the vaccine composition of the present invention to the subject.

[0033] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the polypeptide of the present invention to the subject.

[0034] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering to the subject the isolated mRNA of the present invention.

[0035] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the isolated DNA of the present invention to the subject.

[0036] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the isolated polynucleotide of the present invention to the subject.

[0037] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the carrier of the present invention to the subject.

[0038] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the LNP of the present invention to the subject.

[0039] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the pharmaceutical composition of the present invention to the subject.

[0040] In another aspect, the present invention provides a method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering the vaccine composition of the present invention to the subject. Attached Figure Description

[0041] The accompanying drawings illustrate various embodiments of this application by way of example rather than limitation.

[0042] Figure 1 A graph showing serum IL-6 concentrations in HLA-DR4 mice 24 hours after the second vaccination is provided, in which mice were vaccinated as follows: unvaccinated (i.e., naive; group 1); SEQ ID NO:127 (AlOH + dmLT adjuvant; group 2); SEQ ID NO:128 (AlOH + dmLT adjuvant; group 3); SEQ ID NO:127 (AlOH + CpG adjuvant; group 4); and SEQ ID NO:128 (AlOH + CpG adjuvant; group 5); according to the vaccination protocol described elsewhere in this document (Experiment 1).

[0043] Figures 2A-2D Provides serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination (i.e., day 35). Figure 2A ), IgG2b titer ( Figure 2B ) and serum IgA levels ( Figure 2C ) and urinary IgG(H) levels ( Figure 2D The figures are shown in Figure 1, where the second vaccination was performed using: unvaccinated (i.e., blank; group 1); SEQ ID NO:127 (AlOH + dmLT adjuvant; group 2); and SEQ ID NO:127 (AlOH + CpG adjuvant; group 4); as determined by standard ELISA, following the vaccination protocol described elsewhere in this document (Experiment 1).

[0044] Figures 3A-3B Provides serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination (i.e., day 35). Figure 3A ) and IgG2b titer ( Figure 3BThe figures are shown in Figure 1, where the second vaccination was performed using: unvaccinated (i.e., blank; group 1); SEQ ID NO: 128 (AlOH + dmLT adjuvant; group 3); and SEQ ID NO: 128 (AlOH + CpG adjuvant; group 5); as determined by standard ELISA, according to the vaccination protocol described elsewhere in this document (Experiment 1).

[0045] Figures 4A-4C Serum samples from HLA-DR4 mice were provided approximately 14 days after the second vaccination (i.e., day 35). Figure 4A ) and urine ( Figure 4B –4C)IgA( Figure 4A –4B) and IgG(H) Figure 4C The figures are plotted at the level of the second vaccination, where the second vaccination was performed as follows: unvaccinated (i.e., blank; group 1); SEQ ID NO:128 (AlOH + dmLT adjuvant; group 3); and SEQ ID NO:128 (AlOH + CpG adjuvant; group 5); and the results were determined by standard ELISA according to the vaccination protocol described elsewhere in this document (Experiment 1).

[0046] Figure 5 A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, 218-219, and 127-128) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using HLA-DR4 mouse serum approximately 14 days after the second vaccination, wherein the second vaccination was performed as follows: unvaccinated (Group 1); SEQ ID NO: 127 (AlOH + dmLT adjuvant; Group 2); and SEQ ID NO: 128 (AlOH + CpG adjuvant; Group 4); according to the vaccination protocol described elsewhere in this document (Experiment 1). For each type of coating shown on the x-axis (e.g., SEQ ID NO:17), three bars are given in the figure, where the left, middle, and right bars represent the data from groups 1, 2, and 4 of Experiment 1, respectively.

[0047] Figure 6A –6C provides a bar graph showing the production of Th1 / Th17 / pro-inflammatory cytokine IL-17A after restimulation of spleen cells from HLA-DR4 mice immunized with the following regimen using SEQ ID NO:127 and SEQ ID NO:128 approximately 22 days after the second vaccination (i.e., day 43). Figure 6A ), IL-2 Figure 6B ) and IL-6 ( Figure 6CThe immunization regimens were as follows: unvaccinated (i.e., blank; group 1); SEQ ID NO:127 (AlOH + dmLT adjuvant; group 2); SEQ ID NO:128 (AlOH + dmLT adjuvant; group 3); SEQ ID NO:127 (AlOH + CpG adjuvant; group 4); and SEQ ID NO:128 (AlOH + CpG adjuvant; group 5); cytokine levels were measured using a cell bead array (CBA) (BD Biosciences).

[0048] Figure 7A –7B provides a bar chart showing the production of Th1 / pro-inflammatory cytokines: TNF-α (…) after restimulation of spleen cells from HLA-DR4 mice immunized with the following regimen using SEQ ID NO:127 and SEQ ID NO:128 approximately 22 days after the second vaccination (i.e., day 43). Figure 7A ) and IFN-γ Figure 7B The immunization regimens were as follows: unvaccinated (i.e., blank; group 1); SEQ ID NO:127 (AlOH + dmLT adjuvant; group 2); SEQ ID NO:128 (AlOH + dmLT adjuvant; group 3); SEQ ID NO:127 (AlOH + CpG adjuvant; group 4); and SEQ ID NO:128 (AlOH + CpG adjuvant; group 5); assays were performed using a cell metering bead array (CBA) (BD Biosciences).

[0049] Figure 8A –8B provides a bar graph showing the production of the Th2 / anti-inflammatory cytokine IL-4 after restimulation of spleen cells from HLA-DR4 mice immunized with the following regimen using SEQ ID NO:127 and SEQ ID NO:128 approximately 22 days after the second vaccination (i.e., day 43). Figure 8A ) and IL-10 ( Figure 8B The immunization regimens were as follows: unvaccinated (i.e., blank; group 1); SEQ ID NO:127 (AlOH + dmLT adjuvant; group 2); SEQ ID NO:128 (AlOH + dmLT adjuvant; group 3); SEQ ID NO:127 (AlOH + CpG adjuvant; group 4); and SEQ ID NO:128 (AlOH + CpG adjuvant; group 5); assays were performed using a cell metering bead array (CBA) (BD Biosciences).

[0050] Figure 9A–9B provides information on serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination (i.e., day 28). Figure 9A ) and IgG2b titer ( Figure 9B The second vaccination was administered as follows: placebo (i.e., AlOH + CpG adjuvant; group A); or SEQ ID NO:132 (AlOH + CpG adjuvant; group D); administered according to the vaccination protocol described elsewhere in this document (Experiment 2), as determined by standard ELISA.

[0051] Figures 10A-10B Provides serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination (i.e., day 28). Figure 10A ) and IgG2b titer ( Figure 10B The figure shows that the second vaccination was administered as: placebo (i.e., AlOH + CpG adjuvant; group A); or SEQ ID NO:133 (AlOH + CpG adjuvant; group E); by standard ELISA assay, according to the vaccination protocol described elsewhere in this document (Experiment 2).

[0052] Figure 11A-11B Provides serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination (i.e., day 28). Figure 11A ) and IgG2b titer ( Figure 11B The second vaccination was administered as follows: placebo (i.e., AlOH + CpG adjuvant; group A); or SEQ ID NO:135 (AlOH + CpG adjuvant; group F); administered according to the vaccination protocol described elsewhere in this document (Experiment 2), as determined by standard ELISA.

[0053] Figure 12 A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, 218-219, 132-133, and 135) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using HLA-DR4 mouse serum approximately 28 days after the initial vaccination. The initial vaccination was performed as: placebo (i.e., AlOH + CpG adjuvant; Group A); or SEQ ID NO: 132 (AlOH + CpG adjuvant; Group D); according to the vaccination protocol described elsewhere in this document (Experiment 2). For each type of coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the figure, where the left and right bars represent the data of groups A and D in Experiment 2, respectively.

[0054] Figures 13A-13C Provides a bar graph showing the production of Th1 / Th17 / pro-inflammatory cytokine IL-17A after restimulation of spleen cells from HLA-DR4 mice immunized with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135. Figure 13A ), IL-2 Figure 13B ) and IL-6 ( Figure 13C The immunization regimens were: placebo (i.e., AlOH + CpG adjuvant; group A); SEQ ID NO:127 (AlOH + CpG adjuvant; group B); SEQ ID NO:128 (AlOH + CpG adjuvant; group C); SEQ ID NO:132 (AlOH + CpG adjuvant; group D); SEQ ID NO:133 (AlOH + CpG adjuvant; group E); or SEQ ID NO:135 (AlOH + CpG adjuvant; group F); assays were performed using a cell measurement bead array (CBA) (BD Biosciences).

[0055] Figures 14A-14B Provides a bar graph showing the production of Th1 / pro-inflammatory cytokines: TNF-α (…) after restimulation of spleen cells from HLA-DR4 mice immunized with the following protocols using SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135. Figure 14A ) and IFN-γ Figure 14B The immunization regimens were: placebo (i.e., AlOH + CpG adjuvant; group A); SEQ ID NO:132 (AlOH + CpG adjuvant; group D); SEQ ID NO:133 (AlOH + CpG adjuvant; group E); or SEQ ID NO:135 (AlOH + CpG adjuvant; group F); and assays were performed using a cell measurement bead array (CBA) (BD Biosciences).

[0056] Figures 15A-15B Provides a bar graph showing the production of the Th2 / anti-inflammatory cytokine IL-4 after restimulation of spleen cells from HLA-DR4 mice immunized with the following protocols using SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135. Figure 15A ) and IL-10 ( Figure 15BThe immunization regimens were: placebo (i.e., AlOH + CpG adjuvant; group A); SEQ ID NO:132 (AlOH + CpG adjuvant; group D); SEQ ID NO:133 (AlOH + CpG adjuvant; group E); or SEQ ID NO:135 (AlOH + CpG adjuvant; group F); and assays were performed using a cell measurement bead array (CBA) (BD Biosciences).

[0057] Figure 16A –16B provides serum IgG1 titers in HLA-DR4 mice over time (days) following inoculation with SEQ ID NO:132 (AddaS03™ + CpG adjuvant). Figure 16A ) and IgG2b titer ( Figure 16B The figure was obtained by standard ELISA assay according to the vaccination protocol described in this article (Experiment 3).

[0058] Figure 17A –17C provides serum (days) showing changes in HLA-DR4 mice over time after inoculation with SEQ ID NO:132 (AddaS03™ + CpG adjuvant). Figure 17A ) and urine ( Figure 17B-17C IgA( Figures 17A-17B ) and IgG(H)( Figure 17C The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 3).

[0059] Figure 18 A bar graph is provided showing the peptide ELISA results, where wells were coated with certain peptides (i.e., SEQ ID NO:17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, 218-219, 132-133, and 135) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using HLA-DR4 mouse serum approximately 21 and 41 days post-vaccination with SEQ ID NO:132 (AddaS03™ + CpG adjuvant); following the vaccination protocol described elsewhere in this document (Experiment 3). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the graph, with the left and right bars representing data from samples collected on day 21 and day 41 post-vaccination, respectively.

[0060] Figures 19A-19B Provides serum IgG1 titers in HLA-DR4 mice approximately 14 days after the second vaccination ( Figure 19A ) and IgG2b titer ( Figure 19B The second vaccination was administered as follows: placebo (i.e., AlOH adjuvant; group A); or SEQ ID NO:132 (AlOH adjuvant; group B); administered according to the vaccination protocol described herein (Experiment 8) as determined by standard ELISA.

[0061] Figures 20A-20C Provides serum from HLA-DR4 mice approximately 14 days after the second vaccination ( Figure 20A ) and urine ( Figure 20B-20C IgA( Figures 20A-20B ) and IgG(H)( Figure 20C The second vaccination was performed using either a placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO:132 (AlOH adjuvant; group B), as determined by standard ELISA, according to the vaccination protocol described herein (Experiment 8).

[0062] Figures 21A-21B Provides serum IgG1 levels in HLA-DR4 mice approximately 14 days after the second vaccination ( Figure 21A ) and IgG2b ( Figure 21B The titer plot, where the second vaccination was administered with: placebo (i.e., AlOH adjuvant; group A); or SEQ ID NO:135 (AlOH adjuvant; group C); as determined by standard ELISA, according to the vaccination protocol described herein (Experiment 8).

[0063] Figures 22A-22C Provides serum from HLA-DR4 mice approximately 14 days after the second vaccination ( Figure 22A ) and urine ( Figure 22B-22C IgA( Figures 22A-22B ) and IgG(H)( Figure 22C The second vaccination was performed using either a placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO:135 (AlOH adjuvant; group C), as determined by standard ELISA, according to the vaccination protocol described herein (Experiment 8).

[0064] Figure 23A bar graph is provided showing the peptide ELISA results, where wells were coated with certain peptides (i.e., SEQ ID NO:17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, 218-219, 132-133, and 135) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using HLA-DR4 mouse serum approximately 14 days after the second vaccination, where the second vaccination was: placebo (i.e., AlOH adjuvant; group A); or SEQ ID NO:132 (AlOH adjuvant; group B); according to the vaccination protocol described elsewhere in this document (Experiment 8). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the graph, with the left and right bars representing the data for groups A and B of Experiment 8, respectively.

[0065] Figure 24 A bar graph is provided showing the peptide ELISA results, where wells were coated with certain peptides (i.e., SEQ ID NO:17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, 218-219, 132-133, and 135) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using CD1 mouse serum approximately 14 days after the second vaccination, which was administered as: placebo (i.e., AlOH adjuvant; group A); or SEQ ID NO:135 (AlOH adjuvant; group C); according to the vaccination protocol described elsewhere in this document (Experiment 8). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the graph, with the left and right bars representing the data for groups A and C in Experiment 8, respectively.

[0066] Figures 25A-25C Provides a bar graph showing the production of Th1 / Th17 / pro-inflammatory cytokine IL-17A after restimulation of spleen cells from CD1 mice immunized with SEQ ID NO:132 or SEQ ID NO:135. Figure 25A ), IL-2 Figure 25B ) and IL-6 ( Figure 25C The immunization regimens were: placebo (i.e., AlOH adjuvant; group A); SEQ ID NO:132 (AlOH adjuvant; group B); or SEQ ID NO:135 (AlOH adjuvant; group C); and assays were performed using a cell measurement bead array (CBA) (BDBiosciences).

[0067] Figures 26A-26BProvided are bar graphs showing the production of Th1 / pro-inflammatory cytokine TNF-α (TNF-α) by spleen cells of CD1 mice immunized with placebo (i.e., AlOH adjuvant; group A); SEQ ID NO:132 (AlOH adjuvant; group B); or SEQ ID NO:135 (AlOH adjuvant; group C) after restimulation with SEQ ID NO:132 or SEQ ID NO:135. Figure 26A ) and IFN-γ Figure 26B The assay was performed using a cell measurement bead array (CBA) (BD Biosciences).

[0068] Figures 27A-27B Provided is a bar chart showing the production of Th2 / anti-inflammatory cytokine IL-4 (L / A) by CD1 spleen cells immunized with placebo (i.e., AlOH adjuvant; group A) SEQ ID NO:132 (AlOH adjuvant; group B); or SEQ ID NO:135 (AlOH adjuvant; group C) after restimulation with SEQ ID NO:132 or SEQ ID NO:135. Figure 27A ) and IL-10 ( Figure 27B The assay was performed using a cell measurement bead array (CBA) (BD Biosciences).

[0069] Figures 28A-28E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:136 (AddaS03™+CpG adjuvant) or placebo. Figure 28A ), IgG2b ( Figure 28B ) and IgA( Figure 28C ) titer or level and urinary IgA ( Figure 28D ) and IgG(H)( Figure 28E The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 4).

[0070] Figures 29A-29E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:137 (AddaS03™+CpG adjuvant) or placebo. Figure 29A ), IgG2b ( Figure 29B ) and IgA( Figure 29C ) titer or level and urinary IgA ( Figure 29D ) and IgG(H)( Figure 29E The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 4).

[0071] Figures 30A-30EProvides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:138 (AddaS03™+CpG adjuvant) or placebo. Figure 30A ), IgG2b ( Figure 30B ) and IgA( Figure 30C ) titer or level and urinary IgA ( Figure 30D ) and IgG(H)( Figure 30E The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 4).

[0072] Figures 31A-31E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:139 (AddaS03™+CpG adjuvant) or placebo. Figure 31A ), IgG2b ( Figure 31B ) and IgA( Figure 31C ) titer or level and urinary IgA ( Figure 31D ) and IgG(H)( Figure 31E The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 4).

[0073] Figures 32A-32E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:140 (AddaS03™+CpG adjuvant) or placebo. Figure 32A ), IgG2b ( Figure 32B ) and IgA( Figure 32C ) titer or level and urinary IgA ( Figure 32D ) and IgG(H)( Figure 32E The levels were plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 4).

[0074] Figures 33A-33C A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 136-140) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using serum from HLA-DR4 mice approximately 14 days after the second vaccination, wherein the second vaccination was: placebo (group A); SEQ ID NO: 136 (group B) (…). Figure 33A ); SEQ ID NO:138 (Group D) Figure 33B); or SEQ ID NO:140(Group F)( Figure 33C ); according to the inoculation protocol described elsewhere in this document (Experiment 4). For each coating shown on the x-axis, two bars are given in the figure (e.g., SEQ ID NO:17), where the left and right bars represent groups A and B of Experiment 4, respectively. Figure 33A Group A and Group D Figure 33B ); and Groups A and F ( Figure 33C (Data).

[0075] Figures 34A-34F A bar chart is provided showing the results of splenocytes from HLA-DR4 mice immunized with placebo (AddaS03™ + CpG; Group A); SEQ ID NO:136 (AddaS03™ + CpG; Group B); SEQ ID NO:137 (AddaS03™ + CpG; Group C); SEQ ID NO:138 (AddaS03™ + CpG; Group D); SEQ ID NO:139 (AddaS03™ + CpG; Group E); and SEQ ID NO:140 (AddaS03™ + CpG; Group F) (Experiment 4) using SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140 (AddaS03™ + CpG; Group F) (Experiment 4). NO:140 produces Th1 / Th17 / pro-inflammatory cytokines or Th2 / anti-inflammatory cytokines after restimulation or no stimulation: IFN-γ ( Figure 34A ), IL-4 Figure 34B ), IL-6 Figure 34C ), IL-22 Figure 34D ), IL-10 Figure 34E ) and IL-17A ( Figure 34F ); measured by cell metering bead array (CBA) (BD Biosciences).

[0076] Figures 35A-35CA bar chart is provided depicting the activation induction markers (AIM) OX40 and PD-L1 in spleen cells approximately 49 days after initial vaccination and approximately 3 days after challenge with UPEC25 in placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:137 (Group C); SEQ ID NO:138 (Group D); SEQ ID NO:139 (Group E); and SEQ ID NO:140 (Group F), following restimulation or no stimulation with SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:139 and SEQ ID NO:140. Figure 35A CD69 and PD-L1 Figure 35B ) and CD25 and PD-L1 ( Figure 35C The percentage of ); according to the vaccination protocol described elsewhere in this document (Experiment 4).

[0077] Figures 36A-36E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:136 (Group B); SEQ ID NO:136 (Group E); or placebo (Group A). Figure 36A ), IgG2b ( Figure 36B ) and IgA( Figure 36C ) titer or level and urinary IgA ( Figure 36D ) and IgG(H)( Figure 36E The level graph was obtained by standard ELISA assay, following the vaccination protocol described in this article (Experiment 5).

[0078] Figures 37A-37E Provides serum IgG1 levels showing changes over time (days) in HLA-DR4 mice after vaccination with SEQ ID NO:138 (Group C); SEQ ID NO:138 (Group F); or placebo (Group D). Figure 37A ), IgG2b ( Figure 37B ) and IgA( Figure 37C ) titer or level and urinary IgA ( Figure 37D ) and IgG(H)( Figure 37E The level was plotted; measured by standard ELISA, according to the vaccination protocol described in this article (Experiment 5).

[0079] Figures 38A-38BProvided bar charts showing peptide ELISA results, where wells are coated with certain peptides (i.e., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 136-140) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated wells) using serum from HLA-DR4 mice approximately 14 days after the second vaccination, wherein the second vaccination was performed using SEQ ID NO: 136 (group B) or SEQ ID NO: 138 (group C). Figure 38A ) and SEQ ID NO:136 (Group E) or SEQ ID NO:138 (Group F) Figure 38B ); according to the inoculation protocol described elsewhere in this document (Experiment 5). For each coating shown on the x-axis, two bars are given in the figure (e.g., SEQ ID NO: 17), where the left and right bars represent groups B and C of Experiment 5, respectively. Figure 38A ); and Groups E and F ( Figure 38B (Data).

[0080] Figures 39A-39F Provided is a bar graph showing the production of Th1 / Th17 / pro-inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ ( ) after restimulation or no stimulation of spleen cells from HLA-DR4 mice immunized with each of the following regimens approximately 33 days after the initial vaccination, using SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140. Figure 39A ), IL-4 Figure 39B ), IL-6 Figure 39C ), IL-22 Figure 39D ), IL-10 Figure 39E ) and IL-17A ( Figure 39F The immunization regimens were: placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:138 (Group C); placebo (Group D); SEQ ID NO:136 (Group E); and SEQ ID NO:138 (Group F) (Experiment 5); assays were performed using a cell bead array (CBA) (BD Biosciences).

[0081] Figure 40A-40CProvide a bar chart depicting the percentage of activation-inducible markers (AIMs) of spleen cells after restimulation with or without stimulation using SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and SEQ ID NO:139 approximately 53 days after initial vaccination and approximately 7 days after UPEC25 challenge with placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:138 (Group C); placebo (Group D); SEQ ID NO:136 (Group E); and SEQ ID NO:138 (Group F), approximately 53 days after initial vaccination and approximately 7 days after UPEC25 challenge: OX40 vs. PD-L1. Figure 40A CD69 and PD-L1 Figure 40B ) and CD69 and CD86 ( Figure 40C );; follow the vaccination protocol described elsewhere in this article (Experiment 5).

[0082] Figures 41A-41E Provides serum IgG1 levels in HLA-DR4 mice approximately 34 days post-vaccination. Figure 41A ), IgG2b ( Figure 41B ) and IgA( Figure 41C ) titer or level and urinary IgA ( Figure 41D ) and IgG(H)( Figure 41E The levels of the inoculation were as follows: SEQ ID NO:141 (Group B); SEQ ID NO:142 (Group C); SEQ ID NO:143 (Group D); SEQ ID NO:144 (Group E); and SEQ ID NO:145 (Group F); compared with placebo (Group A); determined by standard ELISA according to the inoculation protocol described herein (Experiment 6).

[0083] Figures 42A-42D A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 141-145) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using serum from HLA-DR4 mice approximately 34 days after the initial vaccination, with the initial vaccination being: placebo (group A) and SEQ ID NO: 141 (group B) (…). Figure 42A Placebo (Group A) vs. SEQ ID NO:142 (Group C) Figure 42B Placebo (Group A) vs. SEQ ID NO:143 (Group D) Figure 42C Placebo (Group A) vs. SEQ ID NO:144 (Group E) Figure 42D ); according to the inoculation protocol described elsewhere in this document (Experiment 6). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the figure, where the left and right bars represent groups A and B of Experiment 6, respectively. Figure 42A Group A and Group C Figure 42B Group A and Group D Figure 42C Group A and Group E Figure 42D (Data).

[0084] Figures 43A-43F Provided are bar graphs showing the production of Th1 / Th17 / pro-inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ ( ) after restimulation of spleen cells from HLA-DR4 mice immunized with each of the following regimens with SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:137 approximately 47 days after initial vaccination and approximately 7 days after UPEC25 challenge. Figure 43A ), IL-4 Figure 43B ), IL-6 Figure 43C ), IL-22 Figure 43D ), IL-10 Figure 43E ) and IL-17A ( Figure 43F The immunization regimens were: placebo (Group A); SEQ ID NO:141 (Group B); SEQ ID NO:142 (Group C); SEQ ID NO:143 (Group D); SEQ ID NO:144 (Group E); and SEQ ID NO:145 (Group F), as described elsewhere in this document (Experiment 6); assays were performed using a Cell Measurement Bead Array (CBA) Magpix®.

[0085] Figures 44A-44C Provide a bar chart depicting the percentage of activation-inducible markers (AIMs) of spleen cells after restimulation with or without stimulation using SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, and SEQ ID NO:145 approximately 47 days after initial vaccination and approximately 7 days after UPEC25 challenge with placebo (Group A); SEQ ID NO:141 (Group B); SEQ ID NO:142 (Group C); SEQ ID NO:143 (Group D); SEQ ID NO:144 (Group E); and SEQ ID NO:145 (Group F), ... with placebo (Group A); SEQ ID NO:141 (Group B); SEQ ID NO:142 (Group C); SEQ ID NO:143 (Group D); SEQ ID NO:144 (Group E); and SEQ ID NO:145 (Group F): OX40 vs PD-L1 (Group F). Figure 44A CD69 and PD-L1 Figure 44B ) and CD69 and CD86 ( Figure 44C ); according to the vaccination protocol described elsewhere in this article (Experiment 6).

[0086] Figures 45A-45E Provides serum IgG1 levels in HLA-DR4 mice approximately 35 days post-vaccination. Figure 45A ), IgG2b ( Figure 45B ) and IgA ( Figure 45C ) titer or level, and urinary IgA ( Figure 45D ) and IgG(H)( Figure 45E The levels of the vaccine were as follows: placebo (Group A); SEQ ID NO:138(1) (Group B); SEQ ID NO:138(2) (Group C); placebo (Group D); SEQ ID NO:138(2) (Group E); placebo (Group F); or SEQ ID NO:138(2) (Group G); administered by standard ELISA according to the vaccination protocol described herein (Experiment 7).

[0087] Figure 46 A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 141-145) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using HLA-DR4 mouse serum approximately 35 days after the initial vaccination, with the initial vaccination being: placebo (Group A); SEQ ID NO: 138 (Group B); or SEQ ID NO: 138 (Group C); according to the vaccination protocol described elsewhere in this document (Experiment 7). For each type of coating shown on the x-axis (e.g., SEQ ID NO:17), three bars are given in the figure, where the left, middle, and right bars represent the data of groups A, B, and C in Experiment 7, respectively.

[0088] Figures 47A-47F Provide a bar chart showing the production of Th1 / Th17 / pro-inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ ( ) after restimulation of spleen cells from HLA-DR4 mice immunized with the following regimens with SEQ ID NO:138(1), SEQ ID NO:138(2), or SEQ ID NO:139 approximately 55 days after the initial vaccination and approximately 7 days after UPEC25 challenge. Figure 47A ), IL-4 Figure 47B ), IL-6 Figure 47C ), IL-22 Figure 47D ), IL-10 Figure 47E) and IL-17A ( Figure 47F The immunization regimens were: placebo (Group A); SEQ ID NO:138(1) (Group B); SEQ ID NO:138(2) (Group C); placebo (Group D); SEQ ID NO:138(2) (Group E); placebo (Group F); or SEQ ID NO:138(2) (Group G); as described elsewhere in this document (Experiment 7); assays were performed using a Cell Measurement Bead Array (CBA) Magpix®.

[0089] Figures 48A-48F Provided is a bar chart showing the production of Th1 / Th17 / pro-inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ, after bladder cells from HLA-DR4 mice immunized with the following regimens were restimulated with SEQ ID NO:138(1) or SEQ ID NO:138(2) approximately 55 days after the initial vaccination and approximately 7 days after UPEC25 challenge. Figure 48A ), IL-4 Figure 48B ), IL-6 Figure 48C ), IL-22 Figure 48D ), IL-10 Figure 48E ) and IL-17A ( Figure 48F The immunization regimens were: placebo (Group A); SEQ ID NO:138(1) (Group B); SEQ ID NO:138(2) (Group C); placebo (Group D); SEQ ID NO:138(2) (Group E); placebo (Group F); or SEQ ID NO:138(2) (Group G); as described elsewhere in this document (Experiment 7); assays were performed using a Cell Measurement Bead Array (CBA) Magpix®.

[0090] Figures 49A-49C Provides a bar chart depicting the percentage of activation-inducible markers (AIMs) in spleen cells of HLA-DR4 mice approximately 55 days after initial vaccination and approximately 7 days after UPEC25 challenge, following restimulation with or without stimulation using SEQ ID NO:138(1), SEQ ID NO:138(2), and SEQ ID NO:139: OX40 vs PD-L1 ( Figure 49A CD69 and PD-L1 Figure 49B ) and CD69 and CD86 ( Figure 49C For the first dose, the following regimens were used: placebo (Group A); SEQ ID NO:138(1) (Group B); SEQ ID NO:138(2) (Group C); placebo (Group D); SEQ ID NO:138(2) (Group E); placebo (Group F); or SEQ ID NO:138(2) (Group G); according to the vaccination protocol described elsewhere in this document (Experiment 7).

[0091] Figures 50A-50B Provides a bar chart depicting the percentage of activation-inducible markers (AIMs) in HLA-DR4 mouse bladder cells after restimulation with SEQ ID NO:138(1), SEQ ID NO:138(2) or without stimulation approximately 55 days after initial vaccination and approximately 7 days after UPEC25 challenge: OX40 vs PD-L1 ( Figure 50A ) and CD69 and PD-L1 ( Figure 50B For the first dose, the following regimens were used: placebo (Group A); SEQ ID NO:138(1) (Group B); SEQ ID NO:138(2) (Group C); placebo (Group D); SEQ ID NO:138(2) (Group E); placebo (Group F); or SEQ ID NO:138(2) (Group G); according to the vaccination protocol described elsewhere in this document (Experiment 7).

[0092] Figures 51A-51E Provides serum IgG1 levels in C57BL / 6 mice approximately 34 days post-vaccination. Figure 51A ), IgG2b ( Figure 51B ) and IgA ( Figure 51C ) titer or level, and urinary IgA ( Figure 51D ) and IgG(H) ( Figure 51E The levels of the vaccine, wherein the vaccine was administered using: placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:137 (Group C); SEQ ID NO:138 (Group D); and SEQ ID NO:139 (Group E); as determined by standard ELISA, according to the vaccination protocol described herein (Experiment 9).

[0093] Figures 52A-52B A bar graph is provided showing the peptide ELISA results, where wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 136-139) and controls (i.e., tetanus toxin (TTX), serum albumin (SA), and uncoated), using serum from C57BL / 6 mice approximately 34 days after the initial vaccination, with the initial vaccination being: placebo (group A), SEQ ID NO: 136 (group B), or SEQ ID NO: 138 (group D). Figure 52A ); and placebo (Group A), SEQ ID NO:137 (Group C) ( Figure 52B) or SEQ ID NO:139 (Group E); according to the inoculation protocol described elsewhere in this document (Experiment 9). For each coating shown on the x-axis (e.g., SEQ ID NO:17), three bars are given in the figure, where the left, middle, and right bars represent Groups A, B, and D of Experiment 9, respectively. Figure 52A ) and Groups A, C, and E ( Figure 52B (Data).

[0094] Figures 53A-53F Provide a bar graph showing that approximately 52 days after the first vaccination and approximately 7 days after UPEC25 challenge, spleen cells from C57BL / 6 mice immunized according to the following regimens were used with SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:138. Figure 52B ), SEQ ID NO:139, Th1 / Th17 / pro-inflammatory cytokine or Th2 / anti-inflammatory cytokine produced after restimulation or no stimulation: IL-17A ( Figure 53A ), IL-6 Figure 53B ), TNF-α Figure 53C ), IFN-γ Figure 53D ), IL-4 Figure 53E ) and IL-10 Figure 53F The immunization regimen is: placebo (Group A); SEQ ID NO:136 (Group B); or SEQ ID NO:138 (Group D) Figure 52A ); and placebo (Group A); SEQ ID NO:137 (Group C) ( Figure 52B ); or SEQ ID NO:139 (Group E); as described elsewhere in this document (Experiment 9); measured using a Cell Measurement Bead Array (CBA) Magpix®.

[0095] Figures 54A-54B Provides a bar chart depicting the percentage of activation-inducible markers (AIMs) in spleen cells of C57BL / 6 mice after restimulation with or without stimulation using SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and SEQ ID NO:139 approximately 52 days after initial vaccination and approximately 7 days after UPEC25 challenge: OX40 vs. PD-L1 ( Figure 54A ) and CD86 and CD69 ( Figure 54B The initial doses were administered as follows: placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:137 (Group C); SEQ ID NO:138 (Group D); and SEQ ID NO:139 (Group E); according to the vaccination protocol described elsewhere in this document (Experiment 9).

[0096] Figure 55 Provided are bar graphs depicting the percentages of activation-inducing markers (AIM) OX40 and PD-L1 in bladder cells of C57BL / 6 mice after restimulation with SEQ ID NO:136, SEQ ID NO:138, or no stimulation, approximately 52 days after the initial vaccination and approximately 7 days after UPEC25 challenge, wherein the initial vaccination was performed with: placebo (Group A); SEQ ID NO:136 (Group B); SEQ ID NO:137 (Group C); SEQ ID NO:138 (Group D); and SEQ ID NO:139 (Group E); according to the vaccination protocol described elsewhere in this document (Experiment 9).

[0097] Figures 56A-56E Provides serum IgG1 levels in C3H / HeN mice approximately 33 days after the first vaccination ( Figure 56A ), IgG2a ( Figure 56B ) and IgA ( Figure 56C ) titer, and urinary IgA ( Figure 56D ) and IgG(H) levels ( Figure 56E The graph shows the levels of the initial vaccinations, where the initial vaccinations were: placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D); administered via standard ELISA according to the vaccination protocol described herein (Experiment 12).

[0098] Figures 57A-57B A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219 and 225) and controls (i.e. tetanus toxin (TTX), serum albumin (SA) and uncoated), and C3H / HeN mouse serum was used approximately 33 days after the initial vaccination, in which the initial vaccination was: placebo (Group A; Figure 57A ); SEQ ID NO:225 (Group B; Figure 57A ); placebo (Group C; Figure 57B); and SEQ ID NO:225 (Group D; Figure 57B ); according to the inoculation protocol described elsewhere in this document (Experiment 12). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the figure, where the left and right bars represent groups A and B of Experiment 12, respectively. Figure 57A ) and Groups C and D ( Figure 57B (Data).

[0099] Figure 58A bar graph is provided showing the production of IFN-γ in the spleen, lymph nodes, and bladder of C3H / HeN mice immunized with the following regimens approximately 33 days after the first vaccination: restimulation with SEQ ID NO:225 or no stimulation (i.e., Unstim); the immunization regimens being: placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D); as described elsewhere in this document (Experiment 12); measurements were taken using a Cell Measurement Bead Array (CBA) Magpix®.

[0100] Figures 59A-59E Provides serum IgG1 levels in C57BL / 6 mice approximately 33 days after the first vaccination. Figure 59A ), IgG2b ( Figure 59B ) and IgA ( Figure 59C ) titer or level, and urinary IgA ( Figure 59D ) and IgG(H)( Figure 59E The graph shows the levels of the following vaccines: for the first dose, placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D); administered via standard ELISA according to the vaccination protocol described herein (Experiment 10).

[0101] Figures 60A-60B A bar graph is provided showing the peptide ELISA results, in which wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219 and 225) and controls (i.e. tetanus toxin (TTX), serum albumin (SA) and uncoated), and serum from C57BL / 6 mice approximately 33 days after the initial vaccination was used, with the initial vaccination being: placebo (Group A; Figure 60A ); SEQ ID NO:225 (Group B; Figure 60A ); placebo (Group C; Fig. 60B); and SEQ ID NO:225 (Group D; Fig. 60B); according to the vaccination protocol described elsewhere in this document (Experiment 10). For each coating shown on the x-axis (e.g., SEQ ID NO:17), two bars are given in the figure, with the left and right bars corresponding to Groups A and B of Experiment 10, respectively. Figure 60A ) and Groups C and D ( Figure 60B (Data).

[0102] Figure 61Provided bar graphs showing IFN-γ production in spleen, lymph nodes, and bladder of C57BL / 6 mice immunized approximately 33 days after initial vaccination, following restimulation with SEQ ID NO:225 or no stimulation (i.e., Unstim); the immunization regimens being: placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D); as described elsewhere herein (Experiment 10); measured using a Cell Measurement Bead Array (CBA) Magpix®.

[0103] Figures 62A-62E Provides serum IgG1 levels in C57BL / 6 mice approximately 33 days after the first vaccination (approximately 12 days after the second vaccination). Figure 62A ), IgG2b ( Figure 62B ) and IgA ( Figure 62C ) titer or level, urine IgA ( Figure 62D ) and IgG(H)( Figure 62E The levels of the vaccine were plotted, with the first dose being either SEQ ID NO:225 (groups A, C, E, G, I) or placebo (groups B, D, F, H); measured by standard ELISA, according to the vaccination protocol described herein (Experiment 11).

[0104] Figures 63A-63E Provided bar charts showing peptide ELISA results, where wells were coated with certain peptides (i.e., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219 and 225) and controls (i.e. tetanus toxin (TTX), serum albumin (SA) and uncoated), and C57BL / 6 mouse serum was used approximately 33 days after the first vaccination (approximately 12 days after the second vaccination), with the first vaccination using: SEQ ID NO: 225 (Group A; Figure 63A Placebo (Group B); Figure 63B ); SEQ ID NO:225 (Group C; Figure 63B Placebo (Group D); Figure 63C ); SEQ ID NO:225 (Group E; Figure 63C Placebo (Group F); Figure 63D ); SEQ ID NO:225 (Group G; Figure 63D Placebo (Group H); Figure 63E ) and SEQ ID NO:225 (Group I; Figure 63E ); according to the vaccination protocol described in this article (Experiment 11). For Figures 63B-63EFor each coating shown on the x-axis (e.g., SEQ ID NO: 17), two bars are given in the figure, where the left and right bars represent groups B and C of Experiment 11, respectively. Figure 63B Groups D and E Figure 63C Groups F and G Figure 63D ) and groups H and I ( Figure 63E (Data).

[0105] Figure 64 A bar chart is provided depicting the percentages of activation-inducing markers (AIM) OX40 and PDL1 in spleen cells obtained from C57BL / 6 mice approximately 45 days after the initial vaccination, wherein the initial vaccination was performed using: SEQ ID NO:225 (Group A); placebo (Group B); SEQ ID NO:225 (Group C); placebo (Group D); SEQ ID NO:225 (Group E); placebo (Group F); SEQ ID NO:225 (Group G); placebo (Group H); and SEQ ID NO:225 (Group I); according to the vaccination protocol described herein (Experiment 11). Spleen cells derived from the designated treatment group or placebo group (i.e., AI group) were restimulated (+) or not stimulated (-) with SEQ ID NO:225.

[0106] Figure 65 A bar graph is provided showing the production of IFN-γ after spleens from C57BL / 6 mice immunized with the following protocols were restimulated with SEQ ID NO:225 or unstimulated (i.e., Unstim) approximately 12 days after the second vaccination; the immunization protocols are: SEQ ID NO:225 (Group A); placebo (Group B); SEQ ID NO:225 (Group C); placebo (Group D); SEQ ID NO:225 (Group E); placebo (Group F); SEQ ID NO:225 (Group G); placebo (Group H); and SEQ ID NO:225 (Group I) (Experiment 11); measured using a Cell Measurement Bead Array (CBA) Magpix® according to the vaccination protocol described herein (Experiment 11). Detailed Implementation

[0107] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0108] In this document, values ​​expressed in range format should be interpreted flexibly, including not only the numerical values ​​explicitly indicated as range limits, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly stated. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise stated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0109] In this document, unless the context clearly specifies otherwise, the terms “a,” “an,” or “the” are used to include one or more. Unless otherwise stated, the term “or” is used to refer to a non-exclusive “or.” The expressions “at least one of A and B” or “at least one of A or B” have the same meaning as “A, B, or A and B.” Furthermore, it should be understood that the wording or terminology used herein (unless otherwise defined) is descriptive and not restrictive. Section headings are used to aid reading and should not be construed as limiting; information relating to a section heading may appear within or outside that particular section. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety as if individually cited.

[0110] In this document, the term "each" is used to refer to each of two or more members of a group, and also to a single element of a group that has only one member. For example, when group 1 consists of A, B, and C, the term "each" applied to group 1 refers to each of A, B, and C. Similarly, when group 2 consists of A, the term "each" applied to group 2 refers to A.

[0111] In the methods described herein, actions may be performed in any order unless the timing or sequence of operations is explicitly stated. Furthermore, specific actions may be performed simultaneously unless the explicit language of the claims states that they may be performed individually. For example, the claimed action of doing X and the claimed action of doing Y may be performed simultaneously in a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0112] definition The term “about” as used herein may allow for a degree of variability in the value or range, for example, within 10%, 5%, or 1% of the said limits of the value or range, and includes the exact value or range.

[0113] As used herein, the term "adjuvant" refers to a substance that increases and / or modulates the immune response to a vaccine. In some embodiments, when administered in combination with one or more antigens, adjuvants can accelerate, prolong, and / or enhance the antigen-specific immune response in a subject.

[0114] An infection, disease, or disorder will “improve” if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both decrease.

[0115] The term "anionic lipid" refers to any lipid that carries a negative charge at a physiological pH (e.g., approximately 7.0). These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine salt, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoylphosphatidylglycerol (POPG), and other anionic modifying groups that bind to neutral lipids.

[0116] As used herein, the term "antigen" refers to a substance capable of inducing an immune response, such as a major histocompatibility complex (MHC) cell surface protein or an antigen-binding region capable of binding immunoglobulin molecules. T-cell-mediated immune responses are achieved through the presentation of antigens. The term "antigen" as used herein includes, but is not limited to, antigenic determinants, haptens, and immunogens, which can be peptides, small molecules, carbohydrates, lipids, nucleic acids, or combinations thereof. Skilled immunologists will recognize that when discussing antigens processed for presentation to T cells, the term "antigen" refers to a portion of the antigen, namely a T-cell epitope (e.g., a peptide fragment) presented by the MHC to the T-cell receptor. When used in the form of antibodies specific to the "antigen" in B-cell-mediated immune responses, the portion of the antigen containing the complementary determinant region (i.e., the binding portion) of the antibody-binding variable domains (i.e., the light and heavy chains) can be a linear or three-dimensional epitope.

[0117] As used in this article, the term "antigenic peptide" refers to a portion of a polypeptide antigen that is specifically recognized by B cells or T cells. B cells respond to foreign antigenic determinants via antibodies, while T lymphocytes mediate cellular immunity. Therefore, an antigenic peptide is a portion of an antigen that, in the case of MHC, is recognized by antibodies or T cell receptors.

[0118] As used herein, the term "bacterial infection" refers to any infection caused directly or indirectly by one or more Gram-negative, Gram-positive, or atypical bacteria. This term is not limited to infections caused solely by bacteria. Non-limiting examples of bacterial infections considered within the scope of this disclosure include urinary tract infections (UTIs), sepsis (such as neonatal sepsis), and pneumonia.

[0119] The term "cationic lipid" refers to any of a variety of lipid substances that carry a net positive charge at a selected pH value (such as physiological pH, e.g., pH approximately 7.0). It has been found that cationic lipids comprising an alkyl chain having multiple unsaturated sites (e.g., at least two or three unsaturated sites) are particularly useful for forming lipid particles with increased membrane fluidity. Certain cationic lipids and related analogues also used in this disclosure have been described in U.S. Patent Publications 20060083780 and 20060240554; U.S. Patent Publications 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992; and PCT Publication WO 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids include protonable tertiary amine head groups (such as pH-titrile amines), C 18 Alkyl chains, head groups, and ether bonds between alkyl chains, as well as 0 to 3 double bonds. Examples of this type of lipid include, for instance, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0120] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one polypeptide or compound that can be used in this invention with a pharmaceutically acceptable carrier. This pharmaceutical composition facilitates the administration of the polypeptide or compound to a patient or subject. Various techniques for administering compounds exist in the art, including but not limited to intravenous, subcutaneous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0121] As used herein, the term "conjugated lipid" refers to a lipid conjugated to one or more polymer groups that inhibits the aggregation of lipid particles. Such lipid conjugations include, but are not limited to, polyamide oligomers (e.g., ATTA lipid conjugations), PEG lipid conjugations such as PEG coupled to dialkoxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG coupled to ceramides (e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to lipids or linked to lipids via a linker portion. Any linker portion suitable for coupling PEG to lipids can be used, including, for example, ester-free and ester-containing linker portions. In a preferred embodiment, an ester-free linker portion is used.

[0122] The “effective amount” or “therapeutic effective amount” of a compound refers to the amount of the compound sufficient to produce a beneficial effect on a subject administering the peptide or compound. The “effective amount” of a delivery medium refers to the amount sufficient to effectively bind or deliver the compound.

[0123] In particular, in the case of mRNA, the “effective amount” or “therapeutic effective amount” of the therapeutic nucleic acid associated with mRNA is an amount sufficient to produce the desired effect, for example, the amount of targeted mRNA expression of a polypeptide or protein that elicits the desired biological effect in an organism expressing a polypeptide or protein. Suitable assays for measuring mRNA or protein expression include, but are not limited to, dot blot, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function assays, and phenotypic assays known to those skilled in the art.

[0124] As used herein, the term "encoded" refers to a product (e.g., protein and RNA) specified by a given nucleotide sequence in a nucleic acid (i.e., DNA and / or RNA) during transcription or translation of DNA or RNA, respectively. In some embodiments, the term "encoded" refers to an RNA sequence specified by transcription of a DNA sequence. In some embodiments, the term "encoded" refers to an amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In some embodiments, the term "encoded" refers to an amino acid sequence specified by DNA transcribed into mRNA and subsequently translated into mRNA encoded by a DNA sequence. In some embodiments, the encoded product may include a direct transcription or translation product. In some embodiments, the encoded product may include post-translational modifications understood or reasonably expected by those skilled in the art.

[0125] As used herein, the term “fetus” refers to any prenatal organism that develops normally within the uterus, from conception to birth (e.g., a fertilized egg and an embryo). This definition also includes prenatal organisms that are first conceived in vitro and then implanted in the uterus. After birth, and for approximately 28 days thereafter, the fetus is referred to herein as a “newborn” (e.g., “its newborn”).

[0126] The term "complete encapsulation" means that the active agent or therapeutic agent in the lipid particles will not significantly degrade upon exposure to serum or nuclease or protease assays, which significantly degrade free DNA, RNA, or protein. In a completely encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particles degrades in treatments that typically degrade 100% of the free active agent or therapeutic agent, more preferably less than about 10%, and most preferably less than about 5% of the active agent and therapeutic agent in the particles degrades. In the context of nucleic acid therapeutic agents, complete encapsulation can be determined by the OLIGREEN® assay. OLIGREEN® is a highly sensitive fluorescent nucleic acid staining agent for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif). "Complete encapsulation" also indicates that the lipid particles are serum stable, meaning that they do not rapidly degrade into their constituent parts after administration in vivo.

[0127] As used herein, the term "helper lipid" refers to lipids that enhance the effectiveness of delivering lipid-based particles (such as cationic lipid-based particles) to a target site, preferably to cells. Helper lipids can be neutral, positively charged, or negatively charged. In some embodiments, the helper lipid is neutral or negatively charged. Non-limiting examples of helper lipids include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC).

[0128] The term "immune cells" as used in this article refers to any cell involved in initiating an immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (such as dendritic cells and macrophages), monocytes, neutrophils, eosinophils, basophils, etc.

[0129] The term "immunogenic fragment" as used in this article refers to a portion of a polypeptide sequence that specifically binds to or is specifically bound to by antibodies produced in an immune response.

[0130] Unless the context clearly indicates otherwise, the term "independently selected" as used herein refers to a group of references that are the same, different, or a mixture thereof. Therefore, according to this definition, the phrase "X" 1 X 2 and X 3 "Independently selected from inert gases" will include the following cases: for example, X 1 X 2 X 3 They are all the same, among which X1 X 2 and X 3 They are all different, among which X 1 and X 2 Same but X 3 Different, and other similar arrangements.

[0131] As used herein, the term "ionizable lipid" refers to a lipid (e.g., a cationic lipid) having at least one protonable or deprotonable group, such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH. Those skilled in the art will understand that the addition or removal of protons with pH changes is an equilibrium process, and references to charged or neutral lipids refer to the properties of the dominant species and do not require all lipids to be present in a charged or neutral form. Typically, the pKa of the protonable group in ionizable lipids is in the range of about 4 to about 7. "Isolated" means altered or removed from its natural state. Isolated nucleic acids can exist in a substantially purified form or in a non-natural environment, such as a host cell. "Isolated" nucleic acids include segments or fragments of nucleic acids separated from sequences flanking the naturally occurring state, such as DNA fragments removed from sequences typically adjacent to fragments in a naturally occurring genome. The term also applies to nucleic acids substantially purified from other components naturally accompanying nucleic acids (e.g., RNA or DNA or proteins, which are naturally associated with nucleic acids in cells). Thus, the term includes, for example, mRNA or recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or prokaryotic or eukaryotic genomic DNA, or existing as separate molecules independent of other sequences (e.g., as cDNA or genomic or cDNA fragments produced by PCR or restriction enzyme digestion). Isolation does not require absolute purity and can include at least 50% isolated proteins, peptides, nucleic acids, or viral molecules, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0132] The term “lipid” refers to a group of organic compounds, including but not limited to fatty acid esters, characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, including fats, oils and waxes; (2) “complex lipids”, including phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0133] As used herein, "encapsulated lipid" can refer to lipid particles that provide active agents or therapeutic agents, such as nucleic acids (e.g., mRNA cargo), and have complete or partial encapsulation. In a preferred embodiment, the nucleic acid is completely encapsulated in the lipid particles.

[0134] The term "lipid nanoparticles" refers to particles having at least one dimension on the nanometer scale (e.g., 1-1000 nm), which include one or more lipids and / or additional reagents.

[0135] The term "lipid particle" is used herein to refer to lipid formulations that can be used to deliver active agents or therapeutic agents (such as nucleic acids (e.g., mRNA)) to target sites of interest. In the lipid particles of this disclosure, they are typically formed from one or more cationic or ionizable lipids, one or more non-cationic lipids (e.g., helper lipids and / or cholesterol), and one or more conjugated lipids that prevent particle aggregation. The active agent or therapeutic agent may be encapsulated within the lipids, thereby protecting the reagent from enzymatic degradation.

[0136] As used herein, the term "mRNA" or "messenger RNA" refers to a ribonucleic acid sequence encoding a peptide or protein. In some embodiments, the mRNA may comprise a "transcription" that is produced using a DNA template and encodes a peptide or protein. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA can be produced from a DNA template by in vitro transcription. Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transfer kits are commercially available. According to the invention, in addition to the modifications according to the invention, the mRNA can also be modified by further stabilization modifications and cap formation.

[0137] The term "neutral amino acid" refers to any of a variety of amino acids having a side chain containing a substituent (e.g., H, methyl, isopropyl, isobutyl, hydroxyl, and thiol, etc.) that is electrically neutral at physiologically relevant pH values. Non-limiting examples of neutral amino acids and / or amino acids having neutral side chains include glycine, alanine, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, proline, glutamine, phenylalanine, tyrosine, tryptophan, asparagine, and glutamine.

[0138] The term “newborn” as used in this article refers to an infant aged 0 to approximately 28 days (e.g., a human infant).

[0139] The term "neutral lipids" refers to any of a variety of lipids that exist as either uncharged or neutral zwitterions at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0140] The term "non-cationic lipid" refers to any amphiphilic lipid as well as any other neutral or anionic lipid.

[0141] As used herein, the term "nucleic acid" refers to a polymer in single-stranded or double-stranded form containing at least two deoxyribonucleotides or ribonucleotides, including DNA and RNA. DNA can be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, and viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or links that are synthetic, naturally occurring, or non-naturally occurring, and have binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphate thioesters, phosphoramide esters, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless specifically limited, the term includes nucleic acids containing known natural nucleotide analogs that have binding properties similar to a reference nucleic acid. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)).

[0142] As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, wherein a fragment containing up to 60 nucleotides is generally called an oligonucleotide, and a longer fragment is called a polynucleotide. In certain embodiments, the oligonucleotides of this disclosure are about 15 to about 60 nucleotides in length. Nucleic acids can be administered alone in the lipid particles of the present invention, or can be administered in combination (e.g., co-administered) with lipid particles of the present invention comprising peptides, polypeptides, or small molecules (such as conventional pharmaceuticals). In other embodiments, nucleic acids can be administered in a viral vector.

[0143] A “nucleotide” comprises a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. A “base” includes purines and pyrimidines, as well as natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.,260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0144] As used herein, the term "oil-in-water emulsion adjuvant" refers to a biocompatible formulation comprising fine droplets (e.g., microdroplets and / or nanodroplets) of oil (e.g., squalene) dispersed in an aqueous phase, stabilized by a surfactant or emulsifier (e.g., polysorbate 80). In some embodiments, the oil phase comprises a metabolizable oil (e.g., squalene and / or α-tocopherol). In some embodiments, the aqueous phase comprises water or a buffered saline solution (e.g., phosphate-buffered saline). In some embodiments, the surfactant comprises a nonionic, cationic, or anionic agent suitable for stabilizing the emulsion and ensuring uniform distribution of the oil droplets. Non-limiting examples of oil-in-water emulsion adjuvants used in this invention include AddaS03™ and AS03®. In some embodiments, AddaS03™ and / or AS03® comprise nanoemulsions of DL-α-tocopherol (5% v / v) (i.e., racemic α-tocopherol) in squalene oil (5% v / v) and Tween® 80 (1.8% v / v) in phosphate-buffered saline (PBS) (pH 6.8). In some embodiments, the nanoemulsions are produced using a microfluidic apparatus and filtered through a 0.22 µm filter to substantially reduce or remove large droplets in the final product, sterilize the final product, and / or substantially reduce or eliminate endotoxins present in the final product.

[0145] The term "operable linkage" or "operational linkage" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, allowing them to function in a desired manner (e.g., leading to the expression of the latter). This term includes the location of the regulatory region and the sequence to be transcribed in the nucleic acid to influence the transcription or translation of that sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of a polypeptide translation reading frame is typically located 1 to approximately 50 nucleotides downstream of the promoter. However, the promoter can be located up to approximately 5000 nucleotides upstream of the translation initiation site or approximately 2000 nucleotides upstream of the transcription initiation site.

[0146] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, as described herein. In some non-limiting embodiments, the patient, subject, or individual is a human.

[0147] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, which are commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and to long chains, which are commonly referred to in the art as proteins, of which there are many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0148] As used herein, the term “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting with any component of the contained composition in a harmful manner.

[0149] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, relating to the delivery or transport of compounds useful in this invention into or to a patient so that they can perform their intended function. Typically, such a construct delivers or transports from one organ or part of the body to another organ or another part of the body. Each carrier must be "acceptable," meaning compatible with other components of the formulation, including peptides or compounds useful in this invention, and harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical formulations. As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coatings, antimicrobial and antifungal agents, and absorption delay agents that are compatible with the activity of the polypeptides or compounds used in this invention and are physiologically acceptable to patients. Additional active compounds may also be incorporated into the composition. "Pharmaceutically acceptable carriers" may also include pharmaceutically acceptable salts of peptides or compounds that can be used in the present invention. Other additional ingredients that may be included in pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0150] As used in this article, the term "pneumonia" refers to inflammation of the lungs that primarily affects the small air sacs called alveoli. Symptoms typically include a combination of some of these, such as a sputumy or dry cough, chest pain, fever, and difficulty breathing. Pneumonia is usually caused by a bacterial or viral infection. Non-limiting exemplary bacteria that are commonly isolated from subjects with pneumonia include Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae Haemophilus influenzae ( ) Haemophilus influenzae Chlamydia pneumoniae ( Chlamydophila pneumoniae Mycoplasma pneumoniae ( ) Mycoplasma pneumoniae Staphylococcus aureus ( Staphylococcus aureus), Moraxella catarrhalis ( Staphylococcus aureus ) and Legionella pneumophila ( Legionella pneumophila ).

[0151] The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide compound, DSPE-PEG-carboxyl-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, etc.

[0152] As used herein, the terms “prevent,” “preventing,” or “prevention” refer to the avoidance or delay of the onset or recurrence of symptoms associated with an infection, disease, or condition in a subject who did not experience these symptoms at the start of administration of the drug or compound. Infection, disease, condition, and / or disorder may be used interchangeably herein.

[0153] As used herein, the term "sepsis" refers to a life-threatening condition that occurs when the body's response to infection results in damage to its own tissues and organs. Common symptoms include fever, rapid heart rate, increased respiratory rate, confusion, cough, and painful urination. Infections that cause sepsis typically include bacterial infections, although fungal, parasitic, and / or viral infections can also cause sepsis. In some embodiments, bacterial infections that cause and / or contribute to the onset of sepsis may include Gram-positive or Gram-negative bacteria. Non-limiting exemplary bacteria that may cause and / or contribute to the onset of sepsis include Staphylococcus spp. Staphylococci species), Klebsiella genus ( Klebsiella species), Streptococcus pyogenes ( Klebsiella species), Escherichia coli ( Escherichia coli ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa (etc.) The term “neonatal sepsis” used in this article specifically refers to the occurrence of sepsis in newborn infants (i.e., newborns).

[0154] In the context of amino acid sequences, the terms “sequence homology,” “identity percentage (%),” “sequence identity,” or “identity percentage” refer to a quantitative measurement of the similarity between two amino acid sequences in the alignment region of two amino acid sequences.

[0155] The term “specifically bind” or “specifically binds” as used in this article means that a first molecule (e.g., an antibody) preferentially binds to a second molecule (e.g., an antigen and / or an immunogenic fragment), but not necessarily only to that second molecule.

[0156] As used herein, the term “substantially” means most or a majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. As used herein, the term “substantially free” can mean free from or containing a small amount of material such that the amount present does not affect the material properties of the composition comprising the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free" can mean having a small amount of the material, such that the composition is about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01 or about 0.001 wt% or less, or about 0 wt%.

[0157] As used in this article, the terms “treat,” “treating,” and “treatment” refer to the reduction of the frequency or severity of symptoms of a disease or condition experienced by a subject through the administration of a drug (such as a peptide or compound).

[0158] The term “UPEC” or “UPEC25” used in this article refers to “urinary tract pathogenic Escherichia coli” with a diameter of approximately 25µm to approximately 250µm, which is a common pathological type of Escherichia coli that causes urinary tract infections (UTIs).

[0159] As used herein, the term "urinary tract infection" or "UTI" refers to a bacterial infection affecting a part of the body that produces and / or carries urine (i.e., the urinary tract), including the kidneys, ureters, bladder, and / or urethra. When it affects the lower urinary tract, it is also called a bladder infection (cystitis), and when it affects the upper urinary tract, it is also called a kidney infection (pyelonephritis). Symptoms of a lower UTI may include painful urination, urinary frequency, and a feeling of needing to urinate even when the bladder is empty, while symptoms of a kidney infection typically include fever and back pain, often combined with lower UTI symptoms. UTIs can also lead to life-threatening invasive Escherichia coli infections (such as bacteremia, sepsis, or urosepticemia). The most common cause of UTIs is Escherichia coli. However, UTIs can also be caused by other Gram-negative bacteria (such as Klebsiella pneumoniae). Klebsiella pneumonia ) and Proteus mirabilis ( Proteus mirabilis Risk factors include female anatomy, sexual intercourse, diabetes, obesity, and family history. UTI is more common in women than men, and often occurs between the ages of 16 and 35. UTI also frequently occurs in older men and women.

[0160] A “vector” is a composition of substances containing isolated nucleic acids and / or polypeptides that can be used to deliver isolated nucleic acids and / or polypeptides into the cell. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses. The term is also interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, etc. An “expression vector” is a vector containing a polynucleotide having an expression control sequence operatively linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other expression elements may be provided by a host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as granules, plasmids (e.g., naked plasmids or plasmids contained in liposomes), phage particles, BACs, YACs, and viral vectors (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0161] describe As discussed elsewhere in this article, bacterial infections, including urinary tract infections (UTIs), sepsis (such as neonatal sepsis), and pneumonia, pose a significant public health risk, infecting millions of people each year. There is a lack of treatments for these infections (typically caused by E. coli). E . coli Klebsiella pneumoniae ( K. pneumonia ) and / or Proteus mirabilis ( P. mirabilisThere is an unmet need for compositions and / or methods for the treatment, prevention and / or improvement of bacterial infections (e.g., urinary tract infections (UTI), sepsis and / or neonatal sepsis and pneumonia) and / or the broad production of immunity against one or more pathogenic bacterial infections.

[0162] Therefore, in one aspect, the present invention relates to compositions and / or methods for treating, preventing, and / or improving bacterial infections in subjects and / or generating immunity against one or more bacterial pathogens. In another aspect, the present invention relates to vaccine compositions and methods of use thereof to meet this unmet need.

[0163] In some embodiments, the compositions and / or methods described herein are suitable for maternal immunization (i.e., use in pregnant subjects). In some embodiments, the compositions and / or methods described herein can be used to treat, prevent, and / or improve bacterial infections in subjects and / or fetuses or their corresponding newborns and / or generate immunity against one or more bacterial pathogens. In one aspect, maternal immunization using the compositions and / or methods described herein can be used to reduce infant morbidity and / or mortality associated with bacterial infections (e.g., neonatal sepsis).

[0164] Vaccination has been widely used as a method of preventing viral infections (e.g., SARS-CoV-2 and COVID-19 infection), but only to a limited extent for preventing bacterial infections (e.g., Mycobacterium tuberculosis). Mycobacterium tuberculosis (Bacterial and tuberculosis infections). However, in both cases, vaccination is performed in a similar manner.

[0165] In short, vaccination involves exposing a subject to one or more antigens that are normally present on the surface of foreign particles, such as bacteria, thereby stimulating an immune response. The subject's first exposure to one or more of these antigens triggers a primary immune response, in which B cells produce antibodies specific to the antigen, ultimately leading to the destruction of the foreign particle by the host's immune system, such as T cells. Furthermore, B cells produce memory cells, which help to produce a more rapid response upon repeated exposure to the same antigen or antigen.

[0166] The surface of *E. coli* contains certain proteins that can be detected by the host's immune system, including iron receptor proteins, flagellated proteins, and non-flagellated proteins (such as pili, curli, and / or fimbriae). These proteins may contribute to immune responses and the elimination of infecting bacteria. Non-limiting examples of proteins detectable by the host's immune system include AfaD, Afa / Dr, Ag43, BmaE, CfaE, CFA / I, ChuA, Cnf1, CsgA, dmLT, EatA, ECOK1_3385, EibD, EstA, F17G, FdeC, FimH, FliC, FmlH, FyuA, GspK, Hia, HlyA, HRA-1, IatA, IatB, etc. IatC, IatD, Iha, Pertactin, IroN, IutA, MrpH, NaIP, OmpA, OmpT, OmpX, PapG, PapC, Pertactin, PNAG, SfaS, SsIE, S fimbriae, TolC, TosA, UpaB, UpaC, YadC, Yad fimbriae, YeeJ, YghA, YghJ, YgiL, Ygi fimbriae, and YncE.

[0167] Therefore, in one aspect, the present invention relates to compositions comprising bacterial surface proteins and / or immunogenic fragments thereof, suitable for inducing an immune response in a subject to generate immunity against infection by one or more pathogenic bacteria, and / or to treat, prevent, and / or improve a bacterial infection in a subject (e.g., urinary tract infection, sepsis, or pneumonia). In some embodiments, the present invention relates to maternal immunity. In other embodiments, the present invention relates to compositions encoding bacterial surface proteins and / or immunogenic fragments thereof, which, upon delivery and translation, are suitable for inducing an immune response in a subject to generate immunity against infection by one or more pathogenic bacteria, and / or to treat, prevent, and / or improve a bacterial infection in a subject (e.g., urinary tract infection).

[0168] lipids Ionizable and / or cationic lipids The terms "cationic lipid" and "ionizable lipid" are used interchangeably herein. Non-limiting examples of ionizable and / or cationic lipids contemplated for use in the lipid nanoparticles of this disclosure include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinyloxy-N,N-dimethylaminopropane (DLenDMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, "XTC2") ), 2,2-diaminopropyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-diaminopropyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-diaminopropyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-diaminopropyl-4- N-Methylpiperazinyl-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylaminoacetoxy)propane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-di DODMA (dimethylaminopropane), DSDMA (1,2-distearyloxy-N,N-dimethylaminopropane), DOTMA (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-Cholesterol (3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol) ol), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoxy-N-[2-(spermine-formamido)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylamide)glycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA) 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxaproloxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In some embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof.

[0169] The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as other cationic lipids, is described in U.S. Provisional Application No. 61 / 104,212, filed October 9, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLin-DAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as other cationic lipids, is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA and other cationic lipids is described in U.S. Patent Publication No. 20060240554, the entire contents of which are incorporated herein by reference for all purposes.

[0170] The range of ionizable and / or cationic lipids intended for use in the lipid nanoparticles of this disclosure is not limited to the species described herein, and may include any ionizable and / or cationic lipids known to those skilled in the art.

[0171] non-cationic lipids In the lipid nanoparticles disclosed herein, non-cationic lipids may include, for example, one or more anionic lipids, accessory lipids, and / or neutral lipids. In some embodiments, non-cationic lipids include one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) phospholipids; or (3) a mixture of phospholipids and cholesterol or a derivative thereof.

[0172] Examples of cholesterol derivatives include, but are not limited to, cholesterol alcohols, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and mixtures thereof. The synthesis of cholesterol-2'-hydroxyethyl ether is known to those skilled in the art and is described in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 1,114,1378, all of which are incorporated herein by reference in their entirety for all purposes.

[0173] Non-limiting examples of non-cationic lipids or accessory lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, hexadecyl phosphate, distearate, dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), and palmitoylphosphatidylcholine (POP). C) Palmitoleoylphosphatidylethanolamine (POPE), palmitoleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearateoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, ditransoleoylphosphatidylethanolamine (DEPE), stearoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0174] Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids can be, for example, derived from substances with a C... 10 -C 24 The acyl group of the fatty acid in the carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Other examples of non-cationic lipids include sterols, such as cholesterol and its derivatives, such as cholesterolanol, cholesterolanone, cholesterolenone, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and mixtures thereof. In some embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0175] Conjugated lipids In the lipid nanoparticles disclosed herein, the conjugated lipids that inhibit particle aggregation may include, for example, one or more of the following: polyethylene glycol (PEG) lipid conjugates, polyamide (ATTA) lipid conjugates, cationic polymer lipid conjugates (CPL), or mixtures thereof. In some embodiments, the nucleic acid lipid particles comprise PEG lipid conjugates or ATTA lipid conjugates.

[0176] PEG is a linear, water-soluble polymer of ethylene-PEG repeating units having two terminal hydroxyl groups. PEG is classified according to its molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2000 Daltons, and PEG 5000 has an average molecular weight of approximately 5000 Daltons. PEG is commercially available from Sigma Chemical Co. and other companies, including substances such as monomethoxy polyethylene glycol (MePEGOH), monomethoxy polyethylene glycol succinate (MePEGS), monomethoxy polyethylene glycol succinimide succinate (MePEG-S-NHS), monomethoxy polyethylene glycol amine (MePEG-NH2), monomethoxy polyethylene glycol trifluoroethanesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol imidazocarboxyl ester (MePEG-IM). Other PEGs, such as those described in U.S. Patents 6774180 and 7053150 (e.g., mPEG (20 kDa) amine), can also be used to prepare the PEG lipid conjugates of this disclosure. For all purposes, the disclosures of these patents are incorporated herein by reference in their entirety. Furthermore, monomethoxy polyethylene glycol acetic acid (MePEG-CH2COOH) is particularly suitable for the preparation of PEG lipid conjugates, including PEG-DAA conjugates.

[0177] In some embodiments, PEG lipid conjugates or ATTA lipid conjugates are used in conjunction with CPL. Conjugated lipids that inhibit particle aggregation may include PEG lipids, including, for example, PEG diacylglycerol (DAG), PEG dialkyloxypropyl (DAA), PEG phospholipids, PEG ceramide (Cer), or mixtures thereof. PEGDAA conjugates may be PEG dilauryloxypropyl (C... 12 ), PEG dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), PEG distearate oxypropyl (C 18 (or mixtures thereof).

[0178] Other PEG lipid conjugates applicable to this disclosure include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. However, other PEG lipid conjugates applicable to this disclosure include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propoxy)-carboxamido-3',6'-dioxooctadecyl]carbamoylmethyl polyethylene glycol (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, which is incorporated herein by reference in its entirety for all purposes.

[0179] The PEG moiety of the PEG lipid conjugate described herein may contain an average molecular weight of about 550 Daltons to about 10,000 Daltons. In some cases, the average molecular weight of the PEG moiety is about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, about 750 Daltons, and about 2,000 Daltons, etc.). In some embodiments, the average molecular weight of the PEG moiety is about 2,000 Daltons or about 750 Daltons.

[0180] In addition to the foregoing, those skilled in the art will readily understand that other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used to replace PEG include, but are not limited to, polyvinylpyrrolidone, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid and derived celluloses such as hydroxymethyl cellulose or hydroxyethyl cellulose.

[0181] In addition to the components described above, the particles of this disclosure (e.g., LNP) may also include cationic polyethylene glycol (PEG) lipids or CPLs (e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). For example, suitable SPLPs and SPLP-CPLs for use in this disclosure, as well as methods for manufacturing and using SPLs and SPLP-CPLs, are disclosed in U.S. Patent No. 6,852,334 and PCT Publication No. WO 00 / 62813, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0182] In some cases, the conjugated lipids that inhibit particle aggregation (e.g., PEG lipid conjugates) may comprise about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2%, about 1 mol% to about 2 mm, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8%, about 1.2 mol% to about 1.7%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any part or range thereof) of the total lipids present in the particles.

[0183] In the lipid nanoparticles of this disclosure, the active agent or therapeutic agent can be completely encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation. In some embodiments, nucleic acid lipid particles containing nucleic acids such as messenger RNA (i.e., mRNA) are completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In some cases, the nucleic acid in the nucleic acid lipid particles remains substantially undegraded after exposure to nucleases at 37°C for at least about 20, 30, 45, or 60 minutes. In some other cases, the nucleic acid in the nucleic acid lipid particles remains substantially undegraded after incubation in serum at 37°C for at least about 30, 45, or 60 minutes, or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agent or therapeutic agent (e.g., nucleic acid such as siRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulation disclosed herein is that the lipid particle composition is essentially non-toxic to mammals such as humans.

[0184] Lipid nanoparticles (LNP) In another aspect, the present invention provides a lipid nanoparticle (LNP) composition comprising the isolated nucleic acid of the present invention. In some embodiments, the isolated nucleic acid of the present invention is isolated mRNA. In some embodiments, the isolated nucleic acid of the present invention is isolated DNA. In some embodiments, the ratio of lipids to isolated mRNA in the LNP is about 5:1 to about 25:1. In some embodiments, the isolated mRNA is at least partially encapsulated in the LNP. In some embodiments, the ratio of lipids to isolated DNA in the LNP is about 5:1 to about 25:1. In some embodiments, the isolated DNA is at least partially encapsulated in the LNP.

[0185] In some embodiments, the isolated polynucleotide is at least partially encapsulated in an LNP. In some embodiments, the isolated polynucleotide is completely encapsulated in an LNP.

[0186] In another aspect, the present invention provides a lipid nanoparticle (LNP) composition comprising the polynucleotides isolated according to the present invention. In some embodiments, the ratio of lipids to isolated polynucleotides in the LNP is about 5:1 to about 25:1. In some embodiments, isolated mRNA is completely encapsulated in the LNP. In some embodiments, isolated DNA is completely encapsulated in the LNP.

[0187] In some implementations, the LNP includes: (a) At least one ionizable lipid; (b) at least one accessory lipid; (c) Cholesterol or its modified derivatives, and any combination thereof; and (d) At least one conjugated lipid.

[0188] In some embodiments, the ionizable lipid is selected from at least one of the following: DLinDMA, DLenDMA, DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DLin-KDMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, DLin-EG-DMA, DODAC, DODMA, DSDMA, DOTMA, DDAB, DOTAP, DC-Chol, DMRIE, DOSPA, DOGS, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, and DLincarbDAP.

[0189] In some embodiments, the at least one ionizable lipid accounts for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or approximately 99 mol%.

[0190] In some embodiments, the at least one ionizable lipid constitutes less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or approximately 99 mol%.

[0191] In some embodiments, the at least one ionizable lipid constitutes more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 of the LNP. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or approximately 99 mol%.

[0192] In some embodiments, the auxiliary lipid is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC).

[0193] In some embodiments, the at least one auxiliary lipid accounts for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25 mol of LNP.

[0194] In some embodiments, the at least one auxiliary lipid accounts for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25 mol of LNP.

[0195] In some embodiments, the at least one auxiliary lipid constitutes more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25 mol% of the LNP.

[0196] In some embodiments, cholesterol accounts for approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or approximately 60 mol of LNP.

[0197] In some embodiments, cholesterol accounts for less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol% of LNP.

[0198] In some embodiments, cholesterol constitutes a percentage of LNP greater than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or about 60 mol%.

[0199] In some embodiments, the conjugated lipid is selected from at least one of the following: 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (PEG-DMG), 1,2-distearate-sn-glycerol-methoxy polyethylene glycol (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol-methoxy polyethylene glycol (PEG-DPG), mPEG-OH, mPEG-AA (mPEG-CM), mPEG-CH2CH2CH2-NH2, mPEG-DMG, mPEG-N,N-bis(tetradecyl)acetamide (ALC-0159), mPEG-DSPE, and mPEG-DPPE.

[0200] In some embodiments, the at least one conjugated lipid accounts for about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0 mol% of LNP.

[0201] In some embodiments, the at least one conjugated lipid constitutes less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or about 5.0 mol% of the LNP.

[0202] In some embodiments, the at least one conjugated lipid constitutes a fraction of LNP greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0 mol%.

[0203] Nucleic acid Messenger RNA (mRNA) In one aspect, the present invention provides an isolated messenger ribonucleic acid (mRNA) encoding the polypeptide of the present invention. In some embodiments, the mRNA is codon-optimized for expression in mammals. In some embodiments, the mammal is a human. In some embodiments, the mRNA is codon-optimized for expression in prokaryotes. In some embodiments, the prokaryote is *Escherichia coli*.

[0204] In another aspect, the present invention provides an isolated polynucleotide encoding the mRNA of the present invention, wherein the polynucleotide comprises one or more promoters and / or polyadenylation signals operatively linked to a sequence encoding the mRNA.

[0205] In some embodiments, the disclosed nucleic acid is or includes ribonucleic acid (RNA). The non-restrictive ribonucleic acid is messenger RNA (mRNA). The term messenger RNA (mRNA) can refer to any ribonucleic acid that directly encodes a polypeptide of interest. Therefore, the disclosed mRNA can be translated to produce one or more coding polypeptides of interest. In some non-restrictive embodiments, the mRNA is produced by in vitro transcription.

[0206] mRNA can be of any suitable length. For example, the length can vary depending on the size of the encoded polypeptide. The length of an mRNA molecule is typically between 200 and 10,000 nucleotides. In some non-limiting embodiments, the mRNA comprises about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,8 ... 0, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000 nucleotides, with or without a poly(A) tail, 5'UTR and / or 3'UTR.

[0207] mRNA can be codon-optimized. For example, mRNA can be codon-optimized for expression in eukaryotic or prokaryotic cells (such as *E. coli*). Eukaryotic cells can be cells derived from or originate from a specific organism, such as plants or mammals, including but not limited to human or non-human eukaryotic or animal or mammalian organisms such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. Codon optimization describes a genetic engineering approach that uses changes from rare codons to synonymous codons, which are used more frequently in the cell type of interest, with the aim of increasing protein yield. Generally, codon optimization involves modifying a nucleic acid sequence by replacing at least one codon of the native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon that is more frequently or most frequently used in the gene of the host cell, to enhance expression in the host cell of interest while maintaining the native amino acid sequence. Various species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon usage between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of a selected tRNA in a cell often reflects the most frequently used codons in peptide synthesis. Therefore, genes can be tailored based on codon optimization to achieve optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" at www.kazusa.orjp / codon / , and these tables can be modified in various ways. See, for example, Nakamura, Y. et al., Nucl. Acids Res., 28:292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some non-limiting embodiments, one or more codons in the mRNA (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) correspond to the most commonly used codon for a particular amino acid.

[0208] Typically, the disclosed isolated messenger ribonucleic acid (mRNA) comprises a 5' untranslated region (UTR), a 3' UTR, and an open reading frame (also known as a coding region). In some non-limiting embodiments, the mRNA further includes a 5' cap or its analogue, a poly(A) tail, one or more modified nucleotides, or combinations thereof. In some embodiments, the mRNA comprises at least a 5' cap or its analogue, a 5' UTR, a 3' UTR, one or more open reading frames, and a poly(A) tail. In some embodiments, the mRNA comprises at least a 5' cap or its analogue, a 5' UTR, a 3' UTR, one or more open reading frames, a poly(A) tail, and one or more modified nucleotides.

[0209] mRNA can include different caps or cap analogues (e.g., ARCA). The body of the mRNA can use modified nucleosides. One or more coding sequences or open reading frames can include various elements such as signal peptides, localization signals (e.g., NLS), inteins, etc. The structure of mRNA can be engineered to optimize GC motifs, folding, circularization signals, and / or structured UTR elements.

[0210] 5' cap Typically, the 5' cap of mRNA participates in nuclear export, increasing mRNA stability and binding to the mRNA cap-binding protein (CBP). The CBP is responsible for mRNA stability and translational capacity within the cell; it binds to poly(A)-binding proteins to form mature circular mRNA species. Endogenous mRNA molecules can be capped at the 5' end, creating a 5'-ppp-5'-triphosphate link between the terminal guanosine cap residue and the sense nucleotide transcribed at the 5' end. This 5'-guanosine cap can then be methylated, producing an N7-methyl-guanosine residue. In some non-limiting embodiments, the mRNA contains a non-hydrolyzable cap that prevents or inhibits decapping, thereby increasing the mRNA's half-life. Since cap hydrolysis requires cleavage of the 5'-ppp-5' phosphodiester linker, the 5' cap may contain modified nucleotides to prevent such hydrolysis.

[0211] The 5' cap can be a single nucleotide or a series of nucleotides. For example, the cap can include 1 to 10 nucleotides in length, such as 2-9, 3-8, 4-7, 1-5, 5-10, or at least 1 or 2, or 10 or fewer nucleotides. In some non-limiting embodiments, there is no cap.

[0212] Cap analogs differ chemically from natural (e.g., endogenous, wild-type, or physiological) 5'-caps while retaining cap function. Cap analogs can be chemically (e.g., non-enzymatically) or enzymatically synthesized and / or linked to nucleic acid molecules. For example, the cap of an inverse cap analog (ARCA) contains two guanines linked by 5'-5'-triphosphate groups, one of which contains an N7 methyl and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m... 7 G-3'mppp-G; can be equivalently specified as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of another unmodified guanine is linked to the 5' terminal nucleotide of a capped nucleic acid molecule (such as mRNA). N7 and 3'-O-methylguanine provide the terminal portion of the capped nucleic acid molecule. Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m...). 7 Gm-ppp-G).

[0213] In some non-limiting embodiments, the 5' cap may include an endogenous cap or a cap analogue. For example, the 5' cap may contain a guanine analogue. Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-dezo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0214] Suitable 5' caps or analogues that can be included in mRNA are known in the art, including but not limited to 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), ARCA, β-S-ARCA, m7G, mCAP, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, trimethylguanosine (TMG), nicotinamide adenine dinucleotide (NAD), cap AG, cap AU, cap GG, and 2-azido-guanosine.

[0215] Deoxyribonucleic acid (DNA) In one aspect, the present invention provides an isolated deoxyribonucleic acid (DNA) encoding the polypeptide of the present invention. In some embodiments, the DNA is a codon optimized for mammalian expression. In some embodiments, the mammal is a human.

[0216] DNA can be of any suitable length. For example, the length can vary depending on the size of the encoded polypeptide. The length of a DNA molecule is typically between 200 and 10,000 nucleotides. In some non-limiting embodiments, the DNA comprises about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800 nucleotides. 0, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and 100,000 nucleotides, with or without a poly(A) tail, 5'UTR and / or 3'UTR.

[0217] DNA can be codon-optimized. For example, DNA can be codon-optimized for expression in prokaryotic cells. In some embodiments, the prokaryotic cell is *Escherichia coli*. In other embodiments, DNA can be codon-optimized for expression in eukaryotic cells. Eukaryotic cells can be cells derived from or originate from a specific organism, such as plants or mammals, including but not limited to human or non-human eukaryotes or animals or mammals, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates.

[0218] In some embodiments, DNA (e.g., codon-optimized DNA for mammalian expression) may be administered to mammals in a vector, wherein expression may occur in mammals (e.g., humans) to confer a therapeutic effect. In other embodiments, DNA (e.g., codon-optimized DNA for prokaryotic expression) may be administered and / or delivered to prokaryotic cells (e.g., E. coli) to produce and isolate their protein products.

[0219] Non-translated area The untranslated region (UTR) is a region of a gene that is transcribed but not translated. Typically, the 5' UTR begins at the transcription start site and continues until the start codon, but not to the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. The 5' UTR may contain specific regions, such as Kozak sequences, which are involved in the initiation of ribosomal translation. The 5' UTR is also known to form secondary structures involved in elongation factor binding. UTRs can play important regulatory roles in the associated mRNA, such as affecting mRNA stability and / or translation. Typically, the translation efficiency of mRNA (including the activation or repression of translation) can be controlled by the UTR. In some non-limiting embodiments, regulatory features of the UTR may be incorporated into the disclosed mRNA to enhance molecular stability. In some non-limiting embodiments, the mRNA is engineered to contain UTRs found in highly expressed genes to enhance mRNA stability and protein yield. For example, introducing the 5'UTR of liver-expressed mRNAs, such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII, can be used to enhance mRNA expression. Similarly, using the 5'UTR of other tissue-specific mRNAs to improve expression in that tissue is possible for muscle (MyoD, myosin, myoglobin, myocyte-derived protein, herculin), endothelial cells (Tie-1, CD36), myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0220] Polymer A-tail During RNA processing, a long chain of adenine nucleotides, called poly(A) tails, can be added to polynucleotides (such as mRNA) to improve stability. After transcription, the 3' end of the transcript may be immediately cleaved, releasing a 3' hydroxyl group. Poly(A) polymerase then adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly(A) tail, which can be, for example, between approximately 100 and 250 residues in length.

[0221] In some non-limiting embodiments, the poly(A) tail includes about 10-100, about 100-300, about 100-250, or about 100-200 adenines. In some non-limiting embodiments, the poly(A) tail comprises about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides.

[0222] open reading frame (ORF) encoded peptides mRNA contains a sequence encoding a polypeptide of interest. For example, mRNA may contain one or more open reading frames, each encoding one or more polypeptides. Typically, open reading frames encode antigens (such as proteins or peptides) from pathogenic microorganisms (such as bacteria, fungi, protozoa, or viruses). In some non-limiting embodiments, open reading frames encode one or more proteins from viruses, or immune-inducing fragments or variants thereof.

[0223] Suitable variants may include at least one point mutation or substitution (e.g., 1, 2, 3, 4, 5 or more mutations or substitutions) at any amino acid residue relative to a reference. In some non-limiting embodiments, amino acid substitutions include conserved amino acid substitutions, but non-conserved substitutions may also be used. Examples of conserved amino acid substitutions include those in which the substitution is within one of the following five groups: 1) small aliphatic, nonpolar or micropolar residues (Ala, Ser, Thr, Pro, Gly); 2) negatively charged polar residues and their amides (Asp, Asn, Glu, Gln); positively charged polar residues (His, Arg, Lys); large aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and large aromatic residues (Phe, Tyr, Trp). Examples of nonconservative amino acid substitutions include: 1) hydrophilic residues, such as serine or threonine, replacing (or being replaced by) hydrophobic residues, such as leucine, isoleucine, phenylalanine, valine, or alanine; 2) cysteine ​​or proline replacing (or being replaced by) any other residue; 3) residues with positively charged side chains, such as lysine, arginine, or histidine, replacing (or being replaced by) negatively charged residues, such as glutamine or aspartic acid; or 4) residues with bulky side chains, such as phenylalanine, replacing (or being replaced by) residues without side chains, such as glycine.

[0224] Other polynucleotides The polynucleotides disclosed herein may also include functional polynucleotide regions (e.g., non-coding polynucleotides). The polynucleotides may include one or more promoters and / or polyadenylation signals operatively linked to a sequence encoding mRNA. In some non-limiting embodiments, the polynucleotide is a plasmid or contained within a plasmid. In some non-limiting embodiments, the polynucleotide is a vector or contained within a vector, such as an expression vector.

[0225] Expression vectors include all vectors known in the art, such as visceral particles, plasmids (e.g., naked plasmids or plasmids contained in liposomes), phage particles, artificial chromosomes (e.g., BAC, YAC), and viral vectors incorporating polynucleotides (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0226] In some non-limiting embodiments, a polynucleotide (e.g., the portion encoding mRNA therein) is operatively linked to a control element, such as a transcriptional control element, like a promoter. Transcriptional control elements can function in eukaryotic cells, such as mammalian cells, or prokaryotic cells (e.g., bacterial or archaea cells). In some non-limiting embodiments, a polynucleotide (e.g., the portion encoding its mRNA) is operatively linked to multiple control elements that allow the expression of the polynucleotide sequence encoding mRNA in prokaryotic or eukaryotic cells. Depending on the host / vector system used, any of certain suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc., can be used in the expression vector (e.g., U6 promoter, HI promoter, CMV promoter, T7 promoter, SV40 promoter, bGH-poly(A) signal, SV40-poly(A) signal, etc.).

[0227] Many vectors and expression systems are commercially available from vendors including Addgene, Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Suitable expression vectors include, but are not limited to, viral vectors, such as those based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and human immunodeficiency virus; retroviral vectors (such as murine leukemia virus, spleen necrosis virus); and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentiviruses, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Viral vectors can be derived from DNA viruses (e.g., dsDNA or ssDNA viruses) or RNA viruses (e.g., ssRNA viruses).

[0228] Those skilled in the art are aware of many suitable expression vectors, many of which are commercially available, including pET29 (Novagen), pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pCDNA 3.1, and pSVLSV40 (Pharmacia). However, any other vector may be used, provided it is compatible with the host cell.

[0229] Based on the above, any cell can be used. In some non-limiting embodiments, the cell is a prokaryotic cell (e.g., an archaea or bacterial cell). In some non-limiting embodiments, the cell is *Escherichia coli*. In other forms, the cell is a eukaryotic cell. For example, the cell can be a cell of a single-celled eukaryote, a plant cell, an algal cell, or a fungal cell (such as a yeast cell). The cell can be a mammalian cell. The mammalian cell can be a human or non-human mammal, such as primate, cow, sheep, pig, dog, rodent, monkey, rat, or mouse cell.

[0230] The production of polynucleotides can be achieved using any suitable genetic engineering techniques known in the art, including but not limited to standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)).

[0231] carrier In another aspect, this disclosure provides a vector comprising the isolated nucleic acid of this disclosure. In some embodiments, the isolated nucleic acid comprises RNA. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is adeno-associated virus (AAV). In some embodiments, the AAV is AAV9.

[0232] In another aspect, this disclosure provides a vector comprising the polynucleotides isolated by this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is adeno-associated virus (AAV). In some embodiments, the AAV is AAV9.

[0233] In some non-limiting embodiments, the vector encoding vaccine antigens (e.g., mRNA and / or peptides) is a viral vector. In some non-limiting embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, to facilitate and expedite regulatory approval, an AAV2-derived ITR sequence or its deleted form (ΔITR) is used. However, ITRs from other AAV sources can be selected. If the ITR is derived from AAV2 and the AAV capsid is derived from another AAV source, the resulting vector may be referred to as a pseudoform.

[0234] AAV is a nonpathogenic single-stranded DNA virus that has been actively used for many years to deliver therapeutic genes in vitro and in vivo (Choi, et al., Curr. Gene Ther., 5:299-310, (2005)). AAV belongs to the parvovirus family and relies on co-infection with other viruses (mainly adenoviruses) for replication. Each end of the single-stranded DNA genome contains an inverted terminal repeat (ITR), which is the only cis-acting element required for genome replication and packaging. The single-stranded AAV genome contains three genes: Rep (replication), Cap (capsid), and aap (assembly). These three genes produce at least nine gene products through the use of three promoters, alternative translation initiation sites, and differential splicing. These coding sequences are flanked by ITRs. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), while Cap expression produces viral capsid proteins (VP; VP1 / VP2 / VP3), which form a protective outer shell for the viral genome and actively participate in cell binding and internalization. It is estimated that the viral capsid is composed of 60 proteins arranged in an icosahedral structure, with a molar ratio of capsid proteins of 1:1:10 (VP1:VP2:VP3).

[0235] Recombinant AAV vectors lacking the Rep and / or Cap genes can be non-integrative. In the absence of the Rep protein, the ITR flanking transgene encoded within the rAAV can form circular polymorphs, existing as free isolates in the nucleus of transduced cells. Because the recombinant free DNA does not integrate into the host genome, it eventually becomes diluted over time as the cell undergoes repeated replication. This ultimately leads to the loss of the transgene and its expression.

[0236] The sequence placed between ITRs typically includes a promoter, the gene of interest (e.g., encoding the disclosed mRNA), and a terminator. Promoters can be naturally occurring or non-natural. In many cases, a strong, constitutively active promoter is required to express the gene of interest at high levels. Examples of promoters include, but are not limited to, viral, plant, and mammalian promoters. Commonly used promoters include the CMV (cytomegalovirus) promoter / enhancer, EF1a (elongation factor 1a), SV40 (simian virus 40), chicken β-actin, and CAG (CMV, chicken β-actin, rabbit β-globin) and their variants. All of these promoters provide constitutively active, high levels of gene expression in most cell types. Some of these promoters may undergo silencing in certain cell types, so this consideration can be evaluated for each application.

[0237] Examples of terminators include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit β-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences and any variants thereof.

[0238] Viral vectors (such as AAV vectors) may also have one or more restriction sites located near the promoter sequence to provide an insertion of a nucleic acid sequence encoding the mRNA / protein of interest.

[0239] The AAV vectors used in the disclosed compositions and methods can be naturally occurring AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, artificial variants such as AAV.rh1O, AAV.rh32 / 33, AAV.rh43, AAV.rh64R1, rAAV2-retro, AAV-DJ, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, or other engineered versions of AAV. In one particular form, the AAV vector is AAV9. These serotypes differ in their tropism or the cell types they infect, making AAV a very useful system for transducing specific cell types in some embodiments. Typically, the packaging size of the AAV vector is limited to ~4.7 kb. AAV itself may be immunogenic and can be used as an adjuvant in certain cases.

[0240] An AAV vector expression cassette typically includes an AAV 5'-ITR, a coding sequence, and an arbitrary control sequence, as well as an AAV 3'-ITR. However, other arrangements of these elements may be suitable. A shortened version of the 5'-ITR, called ΔITR, is described, lacking the D sequence and terminal dissociation sites (trs). In other embodiments, full-length AAV 5'-ITRs and 3'-ITRs are used.

[0241] For example, the expression cassette typically contains a promoter sequence as part of the expression control sequence located between the selected 5'-ITR sequence and the coding sequence.

[0242] In addition to the promoter, the expression cassette and / or vector also includes one or more other suitable transcription initiation, termination, enhancer sequences, and effective RNA processing signals such as splicing and polyadenylation (polyA) signals. It may contain sequences that stabilize the mRNA; sequences that improve translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance the secretion of the encoded product.

[0243] Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA.

[0244] Examples of suitable enhancers include, for example, the alpha-fetoprotein enhancer, the TTR minimal promoter / enhancer, and the LSP (TH-binding globulin promoter / alpha-microglobulin / biskunisin enhancer). In some embodiments, the expression cassette includes one or more expression enhancers. In some embodiments, the expression cassette contains two or more expression enhancers. These enhancers may be the same as or different from each other. Enhancers may be present in two adjacent copies. Optionally, the two copies of the enhancer may be separated by one or more sequences. In another embodiment, the expression cassette also includes introns, such as the Promega intron. Other suitable introns include those known in the art, such as those described in International Patent Application No. WO 2011 / 126808, the entire contents of which are incorporated herein by reference for all purposes.

[0245] Recombinant AAV viral vectors are well-suited for delivering the coding sequences described herein. Such AAV vectors are ITRs derived from the same AAV source as the capsid. Alternatively, AAV ITRs may be derived from a different AAV source than the one providing the capsid.

[0246] However, other promoters can be selected, including tissue-specific promoters. Methods for preparing and isolating AAV viral vectors suitable for delivery to subjects are known in the art. For example, U.S. Patent Application Publication No. US2007 / 0036760 (February 15, 2007), U.S. Patents 7,790,449, 7,282,199, and 7,588,772; and International Publications WO2003 / 042397, WO2005 / 033321, and WO2006 / 11689; all of these documents are incorporated herein by reference in their entirety for all purposes. The sequence of AAV8 and methods for preparing vectors based on the AAV8 capsid are described in U.S. Patents 7,282,199, 7,790,449, and 8,318,480; all of these patents are incorporated herein by reference in their entirety. In some embodiments, the vector is based on an AAV9 capsid.

[0247] In some embodiments, the vector is a bacterial expression vector. In some embodiments, the bacterial expression vector comprises *Escherichia coli*. In some embodiments, the bacterial expression vector includes the pET *E. coli* T7 expression vector. In some embodiments, the pET *E. coli* T7 expression vector comprises pET29b (Novagen).

[0248] Composition In one aspect, this disclosure provides a polypeptide comprising o Individual B instances p One T instance, q There are L instances, where: Each occurrence of B (if present) independently contains an immunogenic fragment of a bacterial surface protein. Each instance of B has a C-terminus and an N-terminus; Each occurrence of T (if present) independently contains an immunogenic fragment of the iron receptor protein. Each instance of T has a C-terminus and an N-terminus; Each occurrence of L independently comprises a polypeptide of 1 to 40 amino acids. Each instance of L has a C-terminus and an N-terminus; Each instance of B is covalently linked to one or two independent instances of L via a covalent peptide bond between the C-terminus of B and the N-terminus of L and / or between the N-terminus of B and the C-terminus of L; Each instance of T is covalently linked to one or two independent instances of L via a covalent peptide bond between the C-terminus of T and the N-terminus of L and / or between the N-terminus of T and the C-terminus of L; o It is an integer between 0 and 15; p It is an integer between 0 and 15; q It is an integer between 5 and 30; and in o + p ≥6.

[0249] In some embodiments, the C-terminus of the polypeptide may contain one or more amino acids (e.g., a polyHis tag or HHHHHH) relevant to polypeptide synthesis and / or purification. Those skilled in the art will understand that the design of polyHis tags (i.e., the terminal position and a relatively short length of 6-10 residues) does not confer any biological activity (e.g., immunogenicity) to the polypeptides to which they are covalently linked. Those skilled in the art will further understand that the design of polyHis tags minimizes and / or eliminates interference with the biological activity of the polypeptides to which they are covalently linked. Those skilled in the art will recognize, particularly in consideration of this disclosure, that this disclosure is not limited to the exemplary polypeptides containing polyHis tags described herein (e.g., SEQ ID NO: 127-145), but also includes their analogues lacking polyHis functionalization (e.g., SEQ ID NO: 223-241).

[0250] In some embodiments, the N-terminus of the polypeptides of this disclosure may contain one or more amino acids (e.g., methionine) relevant to polypeptide synthesis and / or purification. In some embodiments, certain exemplary polypeptides of this disclosure contain N-terminal methionine residues. As will be understood by those skilled in the art, the N-terminal methionine residues present in certain exemplary polypeptides are products of methods for synthesizing polypeptides in which the AUG start codon (i.e., adenine-uracil-guanine) of the mRNA transcript encodes methionine. Therefore, those skilled in the art will recognize, particularly in consideration of this disclosure, that this disclosure is not limited to the exemplary polypeptides described herein that contain N-terminal methionine residues (e.g., SEQ ID NO: 127-145 and SEQ ID NO: 223-241), but also includes analogs lacking N-terminal methionine residues (e.g., SEQ ID NO: 242-260). Furthermore, those skilled in the art understand methods for post-translational removal of N-terminal methionine residues (i.e., N-terminal methionine excision or cleavage). In some implementations, the N-terminal methionine cleavage and / or cutting is catalyzed by methionine aminopeptidase (MetAP).

[0251] In some embodiments, each occurrence of L independently comprises a polypeptide of 1 to 10 amino acids. In some embodiments, each occurrence of L independently comprises a polypeptide of 3 to 10 amino acids. In some embodiments, each occurrence of L independently comprises a polypeptide of 3 to 25 amino acids. In some embodiments, multiple occurrences of L may be positioned adjacent to each other.

[0252] In some implementations, each occurrence of L independently includes a number selected from SEQ ID NO:124 (GSGS), SEQ ID NO:125 (GPGP), SEQ ID NO:126 (LLSVGG), SEQ ID NO:146-155 (SGSG). 1-10 The amino acid sequences of SEQ ID NO:156 (SSSS), SEQ ID NO:157 (GGGS), SEQ ID NO:158 (GGC), SEQ ID NO:159 (GGS), SEQ ID NO:160 ((GGC)8), SEQ ID NO:161 ((GGGGS)3), and SEQ ID NO:162 (GGAAY). In some embodiments, each occurrence of L can be cleaved by a protease.

[0253] In some implementations, B does not exist. In some implementations, 1 to 15 instances of B are represented as B0. 1 B 2 B 3 B4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 and B 15 In some implementations, each instance of B is independently represented as B. 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 Or B 15 .

[0254] In some implementations, T does not exist. In some implementations, 1 to 15 instances of T are represented as Ti. 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 and T 15 In some implementations, each instance of T is independently represented as T. 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 or T 15 .

[0255] In some implementations, 5 to 30 instances of L are respectively represented as L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 L 24 L 25 L 26 L 27 L 28 L 29 and L 30 In some implementations, each instance of L is independently represented as L. 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 L 24 L 25 L 26 L 27 L 28 L 29 or L 30 .

[0256] In some embodiments, the polypeptide has B 1 -L1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 -L 26 -B 14 -L 27 -T 14 -L 28 -B 15 -L 29 -T 15 The structure.

[0257] In some embodiments, the polypeptide may include an L instance (i.e., a terminal L) that is not adjacent to two B instances, two T instances, or one instance of each of B and T, as shown in the following non-limiting embodiments: B 1 -L 1 -T1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 -L 26 -B 14 -L 27 -T 14 -L 28 -B 15 -L 29 -T 15 -L 30 .

[0258] This disclosure includes any arrangement of any instance of B and any instance of T.

[0259] In some embodiments, the polypeptide may be initiated by an instance of T (i.e., the N-terminus of the polypeptide), as shown in the following non-limiting embodiments: T 1-L 1 -B 1 -L 2 -T 2 -L 3 -B 2 -L 4 -T 3 -L 5 -B 3 -L 6 -T 4 -L 7 -B 4 -L 8 -T 5 -L 9 -B 5 -L 10 -T 6 -L 11 -B 6 -L 12 -T 7 -L 13 -B 7 -L 14 -T 8 -L 15 -B 8 -L 16 -T 9 -L 17 -B 9 -L 18 -T 10 -L 19 -B 10 -L 20 -T 11 -L 21 -B 11 -L 22 -T 12 -L 23 -B 12 -L 24 -T 13 -L 25 -B 13 -L 26 -T 14 -L 27 -B 14 -L 28 -T 15 -L 29 -B 15 .

[0260] In some implementations, two B instances and / or two T instances can be separated by L instances (e.g., B 1 -L 1 -B 2 and / or T 1 -L2 -T 2 As shown in the following non-limiting embodiments: B 1 -L 1 -B 2 -L 2 -T 1 -L 3 -T 2 -L 4 -B 3 -L 5 -B 4 -L 6 -T 3 -L 7 -T 4 -L 8 -B 5 -L 9 -B 6 -L 10 -T 5 -L 11 -T 6 -L 12 -B 7 -L 13 -B 8 -L 14 -T 7 -L 15 -T 8 -L 16 -B 9 -L 17 -B 10 -L 18 -T 9 -L 19 -T 10 -L 20 -B 11 -L 21 -B 12 -L 22 -T 11 -L 23 -T 12 -L 24 -B 13 -L 25 -B 14 -L 26 -T 13 -L 27 -T 14 -L 28 -B 15 -L 29 -T 15 B 1 -L 1 -B 2 -L 2 -B 3-L 3 -B 4 -L 4 -B 5 -L 5 -B 6 -L 6 -B 7 -L 7 -B 8 -L 8 -B 9 -L 9 -B 10 -L 10 -B 11 -L 11 -B 12 -L 12 -B 13 -L 13 -B 14 -L 14 -B 15 -L 15 -T 1 -L 16 -T 2 -L 17 -T 3 -L 18 -T 4 -L 19 -T 5 -L 20 -T 6 -L 21 -T 7 -L 22 -T 8 -L 23 -T 9 -L 24 -T 10 -L 25 -T 11 -L 26 -T 12 -L 27 -T 13 -L 28 -T 14 -L 29 -T 15 ; or many other implementations in which the arrangements of instances of B and T are interchanged, all of which are considered herein.

[0261] In some implementations, B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8B 9 B 10 B 11 B 12 B 13 B 14 B 15 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 T 15 L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 L 20 L 21 L 22 L 23 L 24 L 25 L 26 L 27 L 28 L 29 or L 30 Any one of them may or may not exist, such that there are at least 5 L instances and a total of 6 B and / or T instances, as shown in the following non-limiting implementation: B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -B 5 -L 5 -B 6;T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 ;B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -B 5 -L 5 -T 1 ;T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -B 1 ;B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -T 1 -L 5 -T 2 ;T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -B 1 -L 5 -B 2 ;B 10 -L 11 -B 11 -L 12 -B 12 -L 13 -B 13 -L 14 -B 14 -L 15 -T 25; or many other embodiments of truncated polypeptides, in which there are at least 5 L instances and a total of 6 instances in B and T, all of which are considered herein.

[0262] In some implementations, B is defined. 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 B 15 T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 and T 15 The identity of each of them can replace any two identical or different instances of B (e.g., Bi). 1 and B 2 ) and / or any two identical or different instances of T (e.g., T) 1 and T 2 The position of ) is shown in the following non-limiting embodiments: B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T[[ID=4^2]] 11 -L 22 -B 12 -L 23 -T 12 and B 2 -L 1 -T 1 -L 2 -B 1 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 [[ID=^9]]-L 10 -B 6 -L 11 -T[[ID=9 ^]] 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11-L 21 -T 11 -L 22 -B 12 -L 23 -T 12 (e.g. B) 1 and B 2 The position was replaced); and B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 and B 1 -L 1 -T 2 -L 2 -B 2 -L 3 -T 1 -L 4 -B 3 -L 5 -T3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 (e.g. T) 1 and T 2 The position was replaced.

[0263] In some embodiments, the polypeptide has the structure B. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 Polypeptides.

[0264] In some embodiments, the polypeptide has the structure B. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 Polypeptides.

[0265] In some embodiments, the polypeptide has a structure of T. 1 -L 1 -T 2-L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 Polypeptides.

[0266] In some embodiments, the polypeptide has a structure of T. 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 Polypeptides.

[0267] In some embodiments, the polypeptide has the structure B. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 Polypeptides.

[0268] In some embodiments, the polypeptide has a structure of T. 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 -L 12 -T 13 Polypeptides.

[0269] In some embodiments, the polypeptide has the structure B. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4-B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 Polypeptides.

[0270] In some embodiments, the polypeptide has the structure B. 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -T 1 -L 5 -T 2 -L 6 -T 3 -L 7 -T 4 -L 8 -B 5 -L 9 -B 6 -L 10 -B 7 -L 11 -B8 -L 12 -T 5 -L 13 -T 6 -L 14 -T 7 -L 15 -T 8 -L 16 -B 9 -L 17 -B 10 -L 18 -B 11 -L 19 -B 12 -L 20 -T 9 -L 21 -T 10 -L 22 -T 11 -L 23 -T 12 Polypeptides.

[0271] In some embodiments, the polypeptide has a structure of T. 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -B 1 -L 5 -B 2 -L 6 -B 3 -L 7 -B 4 -L 8 -T 5 -L 9 -T 6 -L 10 -T 7 -L 11 -T 8 -L 12 -B 5 -L 13 -B 6 -L 14 -B 7 -L 15 -B 8 -L 16 -T 9 -L 17 -T 10 -L 18 -T 11 -L 19 -T 12-L 20 -B 9 -L 21 -B 10 -L 22 -B 11 -L 23 -B 12 Polypeptides.

[0272] Those skilled in the art will understand that this disclosure is not limited to the embodiments explicitly disclosed herein.

[0273] In some embodiments, the polypeptide comprises a polypeptide of formula (I) or a salt or solvation thereof: (I), in: B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5 L 6 T 1 T 2 and T 3 Each occurrence of can either exist or not exist; B 1 B 2 and B 3 Each occurrence of (if present) independently contains an immunogenic fragment of a bacterial surface protein; T 1 T 2 and T 3 Each occurrence (if present) independently contains an immunogenic fragment of the iron receptor protein. Among them, T 1 T 2 and T 3 At least two exist, or T 1 T 2 and T 3 At least one of them exists, and n is at least 2; L 1 L 2 L 3 L 4 L 5 and L 6 Each occurrence of (if present) is an independent polypeptide consisting of 1-10 amino acids. Where L 1 L 2 L 3 L 4 L5 and L 6 At least one of them exists, and Among them B 1 B 2 B 3 T 1 T 2 and T 3 Every time L appeared, it was 1 L 2 L 3 L 4 L 5 and L 6 At least one of them is separated from the other; and n is an integer selected from 1, 2, 3, 4, and 5.

[0274] In some embodiments, the polypeptide of formula (I) comprises n One (i.e., 1, 2, 3, 4, or 5) of independently occurring In some non-limiting embodiments, wherein n It is 2, the first time it appears independently. Covalently linked to the second independently occurring [product / product] via an amide (peptide) bond. In some implementations, covalent bonding includes each occurrence of... A linear arrangement. For example, in some implementations, the first occurrence... terminal B 1 B 2 B 3 T 1 T 2 or T 3 Covalently linked to the second occurrence The end L 1 L 2 L 3 L 4 L 5 or L 6 For example, in a non-limiting embodiment, wherein n For 2, B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5 L 6 T 1 T 2 and T 3 Each of them exists in two occurrences. In this context, the polypeptide of formula (I) has the following structure (for example, where the terminal L appears for the first time). 6 Covalently bonded to the second occurrence of terminal B 1 ): .

[0275] In other words, B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5 L 6 T 1 T 2 or T 3 Does any of them exist in a given occurrence? (its frequency of occurrence is determined by) n (Definition) and whether each group exists in any other occurrence It is irrelevant to the middle. For example, in a non-limiting embodiment, wherein n The value is 2, the first time it appears. It can include only B 1 L 1 T 1 and L 2 ( (The second time) It can include only T 1 L 5 and T 3 ( In the above non-limiting embodiments, the polypeptide of formula (I) has the following structure: Therefore, in the above non-limiting embodiment, B 1 L 1 and L 2 Only exists in the first appearance In the middle, L 5 and T 3 It only exists in the second occurrence In the middle, T 1 Existing in the first and second occurrences middle.

[0276] In addition, in each occurrence In (its frequency of occurrence is defined by n), B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5L 6 T 1 T 2 and T 3 The identity of each of them is independent of each other. For example, in the above non-limiting embodiment, wherein n For 2, T 1 Existing in two occurrences In the middle, in the first appearance T in 1 It may contain an immunogenic fragment of the iron receptor protein as defined in SEQ ID NO:10, in the second occurrence. T in 1 It may contain immunogenic fragments of the iron receptor protein as defined in SEQ ID NO:12.

[0277] In the polypeptide of formula (I), B 1 B 2 and B 3 Each occurrence of B (if present) independently contains an immunogenic fragment of a bacterial surface protein. In some embodiments, B 1 B 2 and / or B 3 Each occurrence of B exists or does not exist independently. In some implementations, B 1 B 2 and B 3 Each occurrence of B may contain the same immunogenic fragment of a bacterial surface protein. In some embodiments, B 1 B 2 and B 3 Each occurrence of B can contain different immunogenic fragments of bacterial surface proteins. Therefore, in some non-limiting embodiments, B is present. 1 and B 2 Each of the elements appears once, and B 1 and B 2 All bacterial surface proteins can correspond to SEQ ID NO:1, B 1 Immunogenic fragments of bacterial surface proteins may include SEQ ID NO:16, B 2 Immunogenic fragments of bacterial surface proteins may include SEQ ID NO:17. Furthermore, the identity of bacterial surface proteins in B... 1 B 2 and B 3 The occurrence of B does not need to be the same in each instance. In some implementations, B 1 B 2 and B 3 Each occurrence of [a specific protein] may contain immunogenic fragments of different bacterial surface proteins. For example, in some non-limiting embodiments, B is present.1 and B 2 Each of the single occurrences in B 1 Bacterial surface proteins may include SEQ ID NO:1, B 2 The bacterial surface proteins may contain SEQ ID NO:2. Similarly, in some non-limiting embodiments, the polypeptide of formula (I) contains B. 1 B appeared twice. 1 The first appearance of this may include bacterial surface protein SEQ ID NO:1, B 1 The second occurrence may include the bacterial surface protein SEQ ID NO:2. Optionally, in some non-limiting embodiments, the polypeptide of formula (I) comprises B. 1 B appeared twice. 1 Both the first and second occurrences of this can contain bacterial surface protein SEQ ID NO:1.

[0278] In the polypeptide of formula (I), T 1 T 2 and T 3 Each occurrence of T (if present) independently contains an immunogenic fragment of the iron receptor protein. In some embodiments, T 1 T 2 and / or T 3 Each occurrence of T exists or does not exist independently. In some implementations, T 1 T 2 and T 3 Each occurrence of T may contain the same immunogenic fragment of the iron receptor protein. In some embodiments, T 1 T 2 and T 3 Each occurrence of T may contain different immunogenic fragments of the iron receptor protein. Therefore, in some non-limiting embodiments, T is present. 1 and T 2 Each of the elements appears once, and T 1 and T 2 All iron receptor proteins can correspond to SEQ ID NO:10, T 1 The immunogenic fragment of the iron receptor protein in the protein may include SEQ ID NO:42, T 2 Immunogenic fragments of iron receptor proteins in T cells may include SEQ ID NO:43. Furthermore, the identity of iron receptor proteins in T cells... 1 T 2 and T 3 The occurrence of T does not need to be the same in each instance. In some implementations, T 1 T 2 and T 3Each occurrence of may contain immunogenic fragments of different iron receptor proteins. For example, in some non-limiting embodiments, T is present. 1 and T 2 Each of the single occurrences in T 1 Iron receptor proteins may include SEQ ID NO:10, T 2 The iron receptor protein may contain SEQ ID NO: 12. Similarly, in some non-limiting embodiments, the polypeptide of formula (I) contains T twice. 1 T 1 The first appearance may include the iron receptor protein SEQ ID NO:10, T 1 The second occurrence may include the iron receptor protein SEQ ID NO:12. Optionally, in some non-limiting embodiments, the polypeptide of formula (I) comprises T appearing twice. 1 T 1 Both the first and second occurrences can contain the iron receptor protein SEQ ID NO:10.

[0279] In the polypeptide of formula (I), L 1 L 2 L 3 L 4 L 5 and L 6 Each occurrence of L (if present) independently comprises a polypeptide of 1-10 amino acids. In some embodiments, L 1 L 2 L 3 L 4 L 5 and / or L 6 Each occurrence of L exists independently or does not exist independently. In some implementations, L 1 L 2 L 3 L 4 L 5 and / or L 6 Each occurrence of L can contain a different polypeptide of 1-10 amino acids. Therefore, in some non-limiting embodiments, L is present. 1 and L 2 Each of the single occurrences in L 1 It may contain SEQ ID NO:124, L 2 It may include SEQ ID NO: 125. Optionally, in some non-limiting embodiments, L is present. 1 and L 2 Each of the single occurrences in L 1 and L 2All can contain SEQ ID NO:124. Similarly, in some non-limiting embodiments, wherein n The polypeptide of formula (I) contains L twice, which is 2. 1 L 1 The first occurrence of can include SEQ ID NO:124, L 1 The second occurrence may include SEQ ID NO:125. Optionally, in some non-limiting embodiments, wherein n The polypeptide of formula (I) contains L twice, which is 2. 1 L 1 Both the first and second occurrences of this can contain SEQ ID NO:124.

[0280] In some implementations, there is B 1 B 2 and B 3 At least one of them.

[0281] In some embodiments, each bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0282] In some embodiments, each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

[0283] In some embodiments, each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:40.

[0284] In some implementations, B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 and B 15Each of the following (if present) has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with the polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

[0285] In some embodiments, each iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0286] In some embodiments, each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the following: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:6 ... NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ IDNO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123.

[0287] In some embodiments, each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:85, and SEQ ID NO:88.

[0288] In some implementations, T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 and T 15each of which (if present) is independently selected from SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ IDThe polypeptides of SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

[0289] In some implementations, L 1 L 2 L 3 L 4 L 5 and L 6 Each of them (if present) independently contains a polypeptide, wherein each amino acid residue side chain contains a neutral (i.e., uncharged) substituent.

[0290] In some implementations, L 1 L 2 L 3 L 4 L 5 and L 6 Each of these (if present) independently comprises a polypeptide of 4 amino acids. In some embodiments, L 1 L 2 L 3 L 4 L 5 and L 6 Each of the following (if present) is an independent polypeptide comprising 5 amino acids. In some embodiments, L 1 L 2 L 3 L 4 L 5 and L 6 Each of them (if present) independently contains a polypeptide of 6 amino acids.

[0291] In some implementations, L 1 L 2 L 3 L 4 L 5 and L 6 Each of these (if it exists) is independently selected from GSGS (SEQ ID NO:124), GPGP (SEQ ID NO:125), LLSVGG (SEQ ID NO:126), and (SGSG). 1-2(SEQ ID NO:146-147), SSSS (SEQ ID NO:156), GGGS (SEQ ID NO:157), GGC (SEQ ID NO:158), GGS (SEQ ID NO:159), (GGC)8 (SEQ ID NO:160), (GGGGS)3 (SEQ ID NO:161), and GGAAY (SEQ ID NO:162).

[0292] In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3. In some implementations, n is 4. In some implementations, n is 5.

[0293] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides selected from SEQ ID NO:130 and SEQ ID NO:129.

[0294] In some embodiments, the polypeptide is selected from SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 ... The polypeptides of SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259 and SEQ ID NO:260 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

[0295] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:135.

[0296] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0297] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0298] In some embodiments, the polypeptide includes a polypeptide selected from the following, or a salt or solvation thereof: (a)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 ; (b) T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 ; (c)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 ; (d)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8-T 9 ; (e)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 1 -L 10 -B 6 -L 11 -T 2 -L 12 -B 7 -L 13 -T 3 -L 14 -B 8 -L 15 -T 4 -L 16 -B 9 -L 17 -T 1 -L 18 -B 10 -L 19 -T 2 -L 20 -B 11 -L 21 -T 3 -L 22 -B 12 -L 23 -T 4 ; (f)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 ; (g)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 -L 12 -T 13 ; (h)B 1 -L 1 -T 1 -L 2 -B 2-L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 ; (i)B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -T 1 -L 5 -T 2 -L 6 -T 3 -L 7 -T 4 -L 8 -B 5 -L 9 -B 6 -L 10 -B 7 -L11 -B 8 -L 12 -T 5 -L 13 -T 6 -L 14 -T 7 -L 15 -T 8 -L 16 -B 9 -L 17 -B 10 -L 18 -B 11 -L 19 -B 12 -L 20 -T 9 -L 21 -T 10 -L 22 -T 11 -L 23 -T 12 ; and (j)T 1 -L 1 -T 2 -L 2 -T 3 [[ID=__63]]-L 3 -T 4 -L 4 -B 1 -L 5 -B 2 -L 6 -B 3 -L 7 -B 4 -L 8 -T 5 -L 9 -T 6 -L 10 -T 7 -L 11 -T 8 -L 12 -B 5 -L 13 -B 6 -L 14 -B 7 -L 15 -B 8 -L 16 -T 9 -L 17 -T 10 -L 18 -T 11 -L 19 -T 12 -L 20-B 9 -L 21 -B 10 -L 22 -B 11 -L 23 -B 12 ; in: Each occurrence of B (if present) independently contains an immunogenic fragment of a bacterial surface protein. Each instance of B has a C-terminus and an N-terminus; Each occurrence of T independently contains an immunogenic fragment of the iron receptor protein. Each instance of T has a C-terminus and an N-terminus; Each occurrence of L independently comprises a polypeptide of 1-10 amino acids. Each instance of L has a C-terminus and an N-terminus; Each instance of B is covalently linked to one or two independent instances of L via a covalent peptide bond between the C-terminus of B and the N-terminus of L and / or between the N-terminus of B and the C-terminus of L; and Each instance of T is covalently linked to one or two independent instances of L via a covalent peptide bond between the C-terminus of T and the N-terminus of L and / or between the N-terminus of T and the C-terminus of L.

[0299] In some embodiments, each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

[0300] In some embodiments, each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:40.

[0301] In some embodiments, each iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0302] In some embodiments, each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the following: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:6 ... NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ IDNO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123.

[0303] In some embodiments, each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:85, and SEQ ID NO:88.

[0304] In some implementations, L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each of them (if present) independently contains a polypeptide, wherein each amino acid residue side chain contains a neutral (i.e., uncharged) substituent.

[0305] In some implementations, L 1 L 2 L 3 L 4 L 5L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each of them (if present) independently contains 4 to 6 amino acids of polypeptide.

[0306] In some implementations, L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each of these (if it exists) is independently selected from GSGS (SEQ ID NO:124), GPGP (SEQ ID NO:125), LLSVGG (SEQ ID NO:126), and (SGSG). 1-2(SEQ ID NO:146-147), SSSS (SEQ ID NO:156), GGGS (SEQ ID NO:157), GGC (SEQ ID NO:158), GGS (SEQ ID NO:159), (GGC)8 (SEQ ID NO:160), (GGGGS)3 (SEQ ID NO:161) and GGAAY (SEQ ID NO:162).

[0307] In some embodiments, the polypeptide is selected from SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:23 ... NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:253, SEQ ID NO:254, SEQID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259 and SEQ ID The polypeptide of NO:260 has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

[0308] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0309] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0310] method In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one polypeptide of this disclosure to the subject.

[0311] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one isolated mRNA of this disclosure to the subject.

[0312] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one isolated DNA of this disclosure to the subject.

[0313] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering to the subject at least one isolated polynucleotide of this disclosure.

[0314] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one carrier of this disclosure to the subject.

[0315] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one LNP of this disclosure to the subject.

[0316] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one pharmaceutical composition of this disclosure to the subject.

[0317] In another aspect, this disclosure provides a method for treating, preventing, and / or improving bacterial infections in a subject in need. In some embodiments, the method includes administering at least one vaccine composition of this disclosure to the subject.

[0318] In some embodiments, the bacterial infection is a urinary tract infection. In some embodiments, the bacterial infection includes both a urinary tract infection and sepsis. In some embodiments, the bacterial infection is sepsis. In some embodiments, the sepsis is neonatal sepsis. In some embodiments, the bacterial infection is pneumonia.

[0319] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering at least one polypeptide of this disclosure to the subject.

[0320] In another aspect, this disclosure provides a method for inducing immunity in a subject against infection with one or more pathogenic bacteria. In some embodiments, the method includes administering to the subject at least one isolated mRNA of this disclosure. In some embodiments, the method includes administering to the subject at least one isolated DNA of this disclosure.

[0321] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering to the subject at least one isolated polynucleotide of this disclosure.

[0322] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering at least one carrier of this disclosure to the subject.

[0323] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering at least one LNP of this disclosure to the subject.

[0324] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering at least one pharmaceutical composition of this disclosure to the subject.

[0325] In another aspect, this disclosure provides a method for inducing immunity against infection with one or more pathogenic bacteria in a subject. In some embodiments, the method includes administering at least one vaccine composition of this disclosure to the subject.

[0326] In some embodiments, the one or more pathogenic bacteria include at least one selected from the following: Escherichia coli (Escherichia coli) Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ), Proteus mirabilis ( Proteus mirabilis ), Shigella dysenteriae ( Shigella dysenteriae Salmonella enterica ( Salmonella enterica Streptococcus pneumoniae () Salmonella enterica Haemophilus influenzae ( ) Haemophilus influenzae Chlamydia pneumoniae ( Chlamydophila pneumoniae Mycoplasma pneumoniae ( ) Mycoplasma pneumoniae Staphylococcus aureus ( Staphylococcus aureus ), Moraxella catarrhalis ( Moraxella catarrhalis ) and Legionella pneumophila ( Legionella pneumophila Streptococcus pyogenes ( Legionella pneumophila ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Salmonella Bongo ( Salmonella bongori ).

[0327] In some implementations, immunity can prevent bacterial infections.

[0328] In some embodiments, the bacterial infection is a urinary tract infection (UTI). In some embodiments, the bacterial infection includes both urinary tract infection and sepsis. In some embodiments, the bacterial infection is sepsis. In some embodiments, the sepsis is neonatal sepsis. In some embodiments, the bacterial infection is pneumonia.

[0329] In some embodiments, after the initial administration, the subject is given one or more additional doses of the disclosed polypeptide. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the disclosed polypeptide.

[0330] In some embodiments, after the initial administration, the subject is given one or more additional doses of the isolated mRNA of this disclosure. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the isolated mRNA of this disclosure.

[0331] In some embodiments, after the initial administration, the subject is given one or more additional doses of the isolated polynucleotide of this disclosure. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the isolated polynucleotide of this disclosure.

[0332] In some embodiments, after the initial administration, the subject is given one or more additional doses of the carrier of this disclosure. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the carrier of this disclosure.

[0333] In some embodiments, after the initial administration, the subject is given one or more additional doses of the LNP. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the LNP of this disclosure.

[0334] In some embodiments, after the initial administration, the subject is given one or more additional doses of the pharmaceutical composition of this disclosure. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the pharmaceutical composition of this disclosure.

[0335] In some embodiments, after the initial administration, the subject is given one or more additional doses of the vaccine composition. In some embodiments, after the initial administration, the subject is given a second, third, or fourth dose of the vaccine composition of this disclosure.

[0336] In some implementations, each additional dose is administered at intervals of about 1 to about 365 days. In some embodiments, the interval between dose administrations is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 20 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 31 5, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, and 365 days.

[0337] In some implementations, the subject is a mammal. In some implementations, the mammal is a human.

[0338] In some implementations, administration includes maternal immunization.

[0339] In some implementations, the subject becomes pregnant.

[0340] In some embodiments, a bacterial infection is treated, prevented, and / or improved in at least one of a subject and a fetus or newborn. In some embodiments, a bacterial infection is treated, prevented, and / or improved in both a subject and a fetus or newborn. In some embodiments, a bacterial infection is treated, prevented, and / or improved in a newborn or infant after birth.

[0341] In some embodiments, immunity against one or more pathogenic bacteria is generated in at least one of the subject and the fetus or newborn. In some embodiments, immunity against one or more pathogenic bacteria is generated in both the subject and the fetus or newborn. In some embodiments, immunity against one or more pathogenic bacteria is generated in the newborn or infant after birth.

[0342] Pharmaceutical compositions and formulations In another aspect, the present invention provides a vaccine composition comprising the polypeptide of the present disclosure and at least one pharmaceutically acceptable excipient.

[0343] In another aspect, the present invention provides a vaccine composition comprising at least one LNP disclosed herein and a pharmaceutically acceptable excipient.

[0344] In some embodiments, the polypeptide is selected from SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:23 ... NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:253, SEQ ID NO:254, SEQID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259 and SEQ ID The polypeptide of NO:260 has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

[0345] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0346] In some embodiments, the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0347] In some embodiments, the vaccine composition further comprises an adjuvant. In some embodiments, the adjuvant is at least one selected from the following: aluminum hydroxide (AlOH), double-mutant heat-labile toxin (dmLt), CpG, AddaSO3™, AddaVax. TM MF59®, W 20 5EC and / or W 80 5EC (NanoBio®), Matrix-M TM Quil-A®, monophosphoryl lipid A (MPLA; e.g., PHAD® (synthetic) or E. coli-derived), monophosphoryl lipid A Minnesota Salmonella ( Salmonella Minnesota (MPLA-SM; for example, MPLA-SM VacciGrade) TM Alhydrogel®, aluminum phosphate (ALPO4; e.g., Impject® alum adjuvant), chitosan, Class A CpG adjuvants (e.g., OND 1585, ODN 2216, and ODN 2336), Class B CpG adjuvants (e.g., ODN 1018, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, ODN D-SL01, and ODN 7909), Class C CpG adjuvants (e.g., ODN 2395, ODN M362, and ODN D-SL03), cCpG, compositions comprising one or more cationic liposomes and / or one or more immunomodulators (e.g., CAF01), AS01b, gardisil adjuvant, AS02, AS03®, AS04, GLA-SE (e.g., gluconopyranoside lipid adjuvant, synthetic TLR4 agonist), IC31®, Montanide TM ISA-51 VG, Montamide TMISA-70 VG, Immunostimulatory Complexes (ISCOM) (e.g., H5N1), Cholera Toxin (CT), Cholera Toxin B Subunit (CTB), Heat-Insensitive Enterotoxin B Subunit (LTB), Imiquimod, Resiquimod (R848), Double-Stranded RNA (dsRNA) Hairpins (e.g., Polyinosinic-Polycytidine (i.e., PolyI:C)), Riboxxim®, Rintatolimod (Ampligen®), PolyICLC (e.g., Hiltonol®), AF03, Polyphosphazene (PCEP and / or PCPP), Cyclic Guanosyl Monophosphate (c-bis-GMP or CDG), CAF01, CAF05, CAF09, Trehalose Disorbate (TDB), Furfurman, Curdlan, Retinoic Acid, Protollin, Proteasome (ID Biomedical), Endocine TM (L3B), N30A, N30ASq, NanoVax (NanoBio®), inulin, delta inulin (e.g., Advax) TM Advax-CpG55.2, polyacrylamide and / or α-polyacrylamide, mannan peptide, β-glucosylceramide, α-galactosylceramide, cholesterol, IFN-α, flagellin, MALT chemokines (e.g., CCL25, CCL27 and CCL28), glucopyranoyl lipid adjuvant, ADP ribosyl toxin, trehalose and hydroxypropyl methacrylamide (HPMA) copolymer.

[0348] In some embodiments, the adjuvant comprises aluminum hydroxide. In some embodiments, the adjuvant comprises dmLT. In some embodiments, the adjuvant comprises CpG. In some embodiments, the adjuvant comprises AddaSO3™. In some embodiments, the adjuvant comprises ASSO3®.

[0349] In some embodiments, the adjuvant includes AlOH and CpG. In some embodiments, the adjuvant includes AddaSO3™ and / or ASO3® and CpG. In some embodiments, the adjuvant includes AddaSO3™ and CpG. In some embodiments, the adjuvant includes ASO3® and CpG.

[0350] In some embodiments, the adjuvants in the compositions of this disclosure suitable for treating and / or preventing urinary tract infections in subjects include AddaS03™ and / or ASO3® and CpG. In some embodiments, the adjuvants in the compositions of this disclosure suitable for treating and / or preventing urinary tract infections in subjects include AddaS03™ and CpG. In some embodiments, the adjuvants in the compositions of this disclosure suitable for treating and / or preventing urinary tract infections in subjects include ASO3® and CpG.

[0351] In some embodiments, the adjuvant in the presently disclosed compositions suitable for treating and / or preventing neonatal sepsis in a subject includes AlOH. In some embodiments, the adjuvant in the presently disclosed compositions suitable for treating and / or preventing neonatal sepsis in a subject includes AlOH and CpG.

[0352] In some embodiments, the compositions of this disclosure comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or approximately 100µg of polypeptide.

[0353] In some embodiments, the compositions of this disclosure comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or about 500 µg of polypeptide.

[0354] In some embodiments, the peptide concentration of the vaccine composition is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or about 5.0 µg / µL.

[0355] In some embodiments, the vaccine composition is administered in a volume selected from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 µL.

[0356] In some embodiments, the compositions of this disclosure comprise about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 300 0, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 or approximately 6000 µg CpG.

[0357] In some embodiments, the compositions disclosed herein comprise about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or about 750 µg of AlOH.

[0358] In some embodiments, the compositions of this disclosure comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25 µg dmLT.

[0359] In some embodiments, the mass ratio of peptide to CpG in the pharmaceutical or vaccine composition is about 10:6000, 15:6000, 20:6000, 25:6000, 30:6000, 35:6000, 40:6000, 45:6000, 50:6000, 55:6000, 60:6000, 65:6000, 70:6000, 75:6000, 80:6000, 85:6000, 90:6000, 95:6000, 100:6000, 10:5750, 15:5750, 20:5750, 25:5750, 30:5750, 35:5750, 40:5750, 45:5750, 5 0:5750, 55:5750, 60:5750, 65:5750, 70:5750, 75:5750, 80:5750, 85:5750, 90:5750, 95:5750, 100:5750, 10:5500, 15:5500, 20:5500, 25:5500, 30:5 500, 35:5500, 40:5500, 45:5500, 50:5500, 55:5500, 60:5500, 65:5500, 70:5500, 75:5500, 80:5500, 85:5500, 90:5500, 95:5500, 100:5500, 10:5250 15:5250, 20:5250, 25:5250, 30:5250, 35:5250, 40:5250, 45:5250, 50:5250, 55:5250, 60:5250, 65:5250, 70:5250, 75:5250, 80:5250, 85:5250, 90:5 250, 95:5250, 100:5250, 10:5000, 15:5000, 20:5000, 25:5000, 30:5000, 35:5000, 40:5000, 45:5000, 50:5000, 55:5000, 60:5000, 65:5000, 70:5000 75:5000, 80:5000, 85:5000, 90:5000, 95:5000, 100:5000, 10:4750, 15:4750, 20:4750, 25:4750, 30:4750, 35:4750, 40:4750, 45:4750, 50:4750, 55:4 750, 60:4750, 65:4750, 70:4750, 75:4750, 80:4750, 85:4750, 90:4750, 95:4750, 100:4750, 10:4500, 15:4500, 20:4500, 25:4500, 30:4500, 35:450040:4500、45:4500、50:4500、55:4500、60:4500、65:4500、70:4500、75:4500、80:4500、85:4500、90:4500、95:4500、100:4500、10:4250、15:4250、20:4250、25:4250、30:4250、35:4250、40:4250、45:4250、50:4250、55:4250、60:4250、65:4250、70:4250、75:4250、80:4250、85:4250、90:4250、95:4250、100:4250、10:4000、15:4000、20:4000、25:4000、30:4000、35:4000、40:4000、45:4000、50:4000、55:4000、60:4000、65:4000、70:4000、75:4000、80:4000、85:4000、90:4000、95:4000、100:4000、10:3750、15:3750、20:3750、25:3750、30:3750、35:3750、40:3750、45:3750、50:3750、55:3750、60:3750、65:3750、70:3750、75:3750、80:3750、85:3750、90:3750、95:3750、100:3750、10:3500、15:3500、20:3500、25:3500、30:3500、35:3500、40:3500、45:3500、50:3500、55:3500、60:3500、65:3500、70:3500、75:3500、80:3500、85:3500、90:3500、95:3500、100:3500、10:3250、15:3250、20:3250、25:3250、30:3250、35:3250、40:3250、45:3250、50:3250、55:3250、60:3250、65:3250、70:3250、75:3250、80:3250、85:3250、90:3250、95:3250、100:3250、10:3000、15:3000、20:3000、25:3000、30:3000、35:3000、40:3000、45:3000、50:3000、55:3000、60:3000、65:3000、70:3000、75:3000、80:3000、85:3000、90:3000、95:3000、100:3000、10:2750、15:2750、20:2750、25:2750、30:2750、35:2750、40:2750、45:2750、50:2750、55:2750、60:2750、65:2750、70:2750、75:2750、80:2750、85:2750、90:2750、95:2750、100:2750、10:2500、15:2500、20:2500、25:2500、30:2500、35:2500、40:2500、45:2500、50:2500、55:2500、60:2500、65:2500、70:2500、75:2500、80:2500、85:2500、90:2500、95:2500、100:2500、10:2250、15:2250、20:2250、25:2250、30:2250、35:2250、40:2250、45:2250、50:2250、55:2250、60:2250、65:2250、70:2250、75:2250、80:2250、85:2250、90:2250、95:2250、100:2250、10:2000、15:2000、20:2000、25:2000、30:2000、35:2000、40:2000、45:2000、50:2000、55:2000、60:2000、65:2000、70:2000、75:2000、80:2000、85:2000、90:2000、95:2000、100:2000、10:1750、15:1750、20:1750、25:1750、30:1750、35:1750、40:1750、45:1750、50:1750、55:1750、60:1750、65:1750、70:1750、75:1750、80:1750、85:1750、90:1750、95:1750、100:1750、10:1500、15:1500、20:1500、25:1500、30:1500、35:1500、40:1500、45:1500、50:1500、55:1500、60:1500、65:1500、70:1500、75:1500、80:1500、85:1500、90:1500、95:1500、100:1500、10:1250、15:1250、20:1250、25:1250、30:1250、35:1250、40:1250、45:1250, 50:1250, 55:1250, 60:1250, 65:1250, 70:1250, 75:1250, 80:1250, 85:1250, 90:1250, 95:1250, 100:1250, 10:1000, 15:1000, 20:1000, 25:1000, 30:1000, 35:1000, 40:1000, 45:1000, 50:1000, 55:1000, 60:1000, 65:1000, 70:10 00, 75:1000, 80:1000, 85:1000, 90:1000, 95:1000, 100:1000, 10:750, 15:750, 20:750, 25:750, 30:750, 35:750, 40:750, 45:750, 50:750, 55:750, 60:750, 65:750, 70:750, 75:750, 80:750, 85:750, 90:750, 95:750, 100:750, 10:500, 15:500 20:500, 25:500, 30:500, 35:500, 40:500, 45:500, 50:500, 55:500, 60:500, 65:500, 70:500, 75:500, 80:500, 85:500, 90:500, 95:500, 100:500, 10:250, 15:250, 20:250, 25:250, 30:250, 35:250, 40:250, 45:250, 50:250, 55:250, 60:250, 6 5:250, 70:250, 75:250, 80:250, 85:250, 90:250, 95:250, 100:250, 10:100, 15:100, 20:100, 25:100, 30:100, 35:100, 40:100, 45:100, 50:100, 55:100, 60:100, 65:100, 70:100, 75:100, 80:100, 85:100, 90:100, 95:100, or approximately 100:100 (peptide: CpG).

[0360] In some embodiments, the pharmaceutical composition contains about 0.4% AlOH (w / w%). In some embodiments, the mass ratio of the polypeptide to AlOH in the pharmaceutical or vaccine composition is about 10:750, 15:750, 20:750, 25:750, 30:750, 35:750, 40:750, 45:750, 50:750, 55:750, 60:750, 65:750, 70:750, 75:750, 80:750, 85:750, 90:750, 95:750, 100:750, 10:700, 15:700, 20:700, 25:700, 30:700, 35:700, 40:700, 45:700, 50:700, 55:700, 60:7 00, 65:700, 70:700, 75:700, 80:700, 85:700, 90:700, 95:700, 100:700, 10:650, 15:650, 20:650, 25:650, 30:650, 35:650, 40:650, 45:650, 50:650, 55:650, 60:650, 65:650, 70:650, 75:650, 80:650, 85:650, 90:650, 95:650, 100:650, 10:600, 15:600, 20:600, 25:600, 30:600, 35:600, 40:60 0, 45:600, 50:600, 55:600, 60:600, 65:600, 70:600, 75:600, 80:600, 85:600, 90:600, 95:600, 100:600, 10:550, 15:550, 20:550, 25:550, 30:550, 35:550, 40:550, 45:550, 50:550, 55:550, 60:550, 65:550, 70:550, 75:550, 80:550, 85:550, 90:550, 95:550, 100:550, 10:500, 15:500, 20:50 0, 25:500, 30:500, 35:500, 40:500, 45:500, 50:500, 55:500, 60:500, 65:500, 70:500, 75:500, 80:500, 85:500, 90:500, 95:500, 100:500, 10:450, 15:450, 20:450, 25:450, 30:450, 35:450, 40:450, 45:450, 50:450, 55:450, 60:450, 65:450, 70:450, 75:450, 80:450, 85:450, 90:450, 95:450100:450, 10:400, 15:400, 20:400, 25:400, 30:400, 35:400, 40:400, 45:400, 50:400, 55:400, 60:400, 65:400, 70:400, 75:400, 80:400, 85:400, 90:400, 95:400, 100: 400, 10:350, 15:350, 20:350, 25:350, 30:350, 35:350, 40:350, 45:350, 50:350, 55:350, 60:350, 65:350, 70:350, 75:350, 80:350, 85:350, 90:350, 95:350, 100:350, 1 0:300, 15:300, 20:300, 25:300, 30:300, 35:300, 40:300, 45:300, 50:300, 55:300, 60:300, 65:300, 70:300, 75:300, 80:300, 85:300, 90:300, 95:300, 100:300, 10:25 0, 15:250, 20:250, 25:250, 30:250, 35:250, 40:250, 45:250, 50:250, 55:250, 60:250, 65:250, 70:250, 75:250, 80:250, 85:250, 90:250, 95:250, or approximately 100:250 (peptide: AlOH).

[0361] In some embodiments, the pharmaceutical composition contains about 50% AddaS03™.

[0362] In some embodiments, the mass ratio of peptide to dmLT in the pharmaceutical or vaccine composition is about 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 10:2, 15:2, 20:2, 25:2, 30:2, 35:2, 40:2, 45:2, 50:2, 55:2, 60:2, 65:2, 70:2, 75:2, 80:2, 85:2, 90:2, 95:2, 100:2, 10:3, 15:3, 20:3, 25:3, 3 0:3, 35:3, 40:3, 45:3, 50:3, 55:3, 60:3, 65:3, 70:3, 75:3, 80:3, 85:3, 90:3, 95:3, 100:3, 10:4, 15:4, 20:4, 25:4, 30:4, 35:4, 40:4, 45:4, 50:4, 55: 4, 60:4, 65:4, 70:4, 75:4, 80:4, 85:4, 90:4, 95:4, 100:4, 10:5, 15:5, 20:5, 25:5, 30:5, 35:5, 40:5, 45:5, 50:5, 55:5, 60:5, 65:5, 70:5, 75:5, 80:5 85:5, 90:5, 95:5, 100:5, 10:10, 15:10, 20:10, 25:10, 30:10, 35:10, 40:10, 45:10, 50:10, 55:10, 60:10, 65:10, 70:10, 75:10, 80:10, 85:10, 90:10 95:10, 100:10, 10:15, 15:15, 20:15, 25:15, 30:15, 35:15, 40:15, 45:15, 50:15, 55:15, 60:15, 65:15, 70:15, 75:15, 80:15, 85:15, 90:15, 95:15, 1 00:15, 10:20, 15:20, 20:20, 25:20, 30:20, 35:20, 40:20, 45:20, 50:20, 55:20, 60:20, 65:20, 70:20, 75:20, 80:20, 85:20, 90:20, 95:20, 100:20, 10:25, 15:25, 20:25, 25:25, 30:25, 35:25, 40:25, 45:25, 50:25, 55:25, 60:25, 65:25, 70:25, 75:25, 80:25, 85:25, 90:25, 95:25 or approximately 100:25 (peptide: dmLT).

[0363] In some embodiments, the oil-in-water emulsion adjuvant comprises about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10.0 mg of polysorbate 80. In some embodiments, the oil-in-water emulsion adjuvant comprises about 2.43 mg of polysorbate 80. In some embodiments, the oil-in-water emulsion adjuvant comprises about 4.86 mg of polysorbate 80. In some embodiments, the oil-in-water emulsion adjuvant comprises about 9.72 mg of polysorbate 80.

[0364] In some embodiments, the oil-in-water emulsion adjuvant comprises about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, or 13. 0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, or about 25.0 mg of squalene. In some embodiments, the oil-in-water emulsion adjuvant contains about 5.35 mg of squalene. In some embodiments, the oil-in-water emulsion adjuvant contains about 10.69 mg of squalene. In some embodiments, the oil-in-water emulsion adjuvant contains about 21.38 mg of squalene.

[0365] In some embodiments, the oil-in-water emulsion adjuvant comprises about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5. 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, or about 25.0 mg of α-tocopherol (e.g., racemic α-tocopherol). In some embodiments, the oil-in-water emulsion adjuvant contains about 5.93 mg of α-tocopherol. In some embodiments, the oil-in-water emulsion adjuvant contains about 11.86 mg of α-tocopherol. In some embodiments, the oil-in-water emulsion adjuvant contains about 23.72 mg of α-tocopherol.

[0366] In some embodiments, the oil-in-water emulsion adjuvant comprises about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or about 75% (v / v) of the vaccine composition disclosed herein.

[0367] In some embodiments, this disclosure provides a vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:225, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide; (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) Squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, wherein the polysorbate 80 comprises about 0.9% (v / v) of the vaccine composition; The polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0368] In some embodiments, the present invention provides a vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:225, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide; (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) Squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80, wherein the polysorbate 80 comprises about 0.9% (v / v) of the vaccine composition; (e) CpG, wherein the vaccine contains about 100 µg to about 6000 µg of CpG; The polypeptide, α-tocopherol, squalene, polysorbate and CpG are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0369] In some embodiments, this disclosure provides a vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:226, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide; (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) Squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, wherein the polysorbate 80% comprises about 0.9% (v / v) of the vaccine composition; The polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0370] In some embodiments, this disclosure provides a vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:226, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide; (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) Squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80, wherein the polysorbate 80 comprises about 0.9% (v / v) of the vaccine composition; (e) CpG, wherein the vaccine contains about 100 µg to about 6000 µg of CpG; The polypeptide, α-tocopherol, squalene, polysorbate and CpG are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0371] In another aspect, the present invention provides a pharmaceutical composition comprising at least one LNP disclosed herein and a pharmaceutically acceptable excipient.

[0372] This invention provides pharmaceutical compositions comprising at least one polypeptide or compound of the present invention, or a salt or solvation thereof, for use in carrying out the methods of the present invention. Such pharmaceutical compositions may consist of at least one polypeptide or compound of the present invention, or a salt or solvation thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one peptide or compound of the present invention, or a salt or solvation thereof, and one or more pharmaceutically acceptable carriers, one or more other components, or any combination thereof. At least one polypeptide or compound of the present invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, for example, in combination with physiologically acceptable cations or anions, as known in the art.

[0373] In some embodiments, the pharmaceutical composition used to carry out the method of the present invention may be administered to deliver a dose of 1 ng / kg / day to 1 mg / kg / day. In other embodiments, the pharmaceutical composition used to carry out the present invention may be administered to deliver a dose of 100 ng / kg / day to 1 mg / kg / day.

[0374] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any other components in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the patient being treated and the route of administration of the composition. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0375] Pharmaceutical compositions that can be used in the methods of the present invention can be suitably developed for use via nasal, inhalation, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, oral, ocular, epidural, intrathecal, intravenous, or other routes of administration. Compositions that can be used in the methods of the present invention can be directly applied to the brain, brainstem, or any other part of the central nervous system of mammals or birds. Other formulations considered include projected nanoparticles, microspheres, liposome formulations, coated particles, polymer conjugates, resealed red blood cells containing the active ingredient, and immunologically based formulations.

[0376] In some embodiments, the compositions of the present invention are part of a pharmaceutical matrix that allows for the manipulation of insoluble substances and improvement of their bioavailability, the development of controlled-release or sustained-release products, and the generation of homogeneous compositions. For example, pharmaceutical matrices can be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction techniques, molecular complexes (such as cyclodextrins), microparticles, and particle and formulation coating processes. Amorphous or crystalline phases can be used in such processes.

[0377] The route of administration will be obvious to those skilled in the art and will depend on many factors, including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, etc.

[0378] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or subsequently developed in the fields of pharmacology and pharmaceutics. Generally, such preparation methods involve the step of combining the active ingredient with a carrier or one or more other excipients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose units.

[0379] As used herein, “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient administered to a subject or a convenient portion of such dose, such as half or one-third of such dose.

[0380] Although the description of the pharmaceutical compositions provided herein is primarily directed toward pharmaceutical compositions suitable for ethical human administration, those skilled in the art will understand that such compositions are generally suitable for administration to a wide variety of animals. Modifications to pharmaceutical compositions suitable for human administration are well known to be made to make the compositions suitable for a wide range of animals, and such modifications can generally be designed and carried out by a skilled veterinary pharmacologist through routine (if any) experimentation. The intended recipients of the pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, and rodents.

[0381] In some embodiments, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one polypeptide or compound of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and recombinant human albumin (such as RECOMBUMIN). ® ), soluble gelatin (such as GELOFUSINE) ® Other pharmaceutically acceptable carriers, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0382] The carrier can be a solvent or dispersion medium, comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), recombinant human albumin, soluble gelatin, suitable mixtures thereof, and vegetable oils. For example, appropriate flowability can be maintained by using coatings such as lecithin, maintaining the desired particle size in the dispersed state, and using surfactants. Antimicrobial activity can be achieved using various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, sodium chloride, or polyols such as mannitol and sorbitol, are included in the composition. The absorption time of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate or gelatin.

[0383] The formulations may be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carriers, suitable for parenteral, intravenous, subcutaneous, transcutaneous, or any other suitable route of administration known in the art. Pharmaceutical formulations may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure buffers, colorants, flavoring agents, and / or fragrance-importing substances. Where desired, they may also be combined with other active agents, such as other analgesics, anxiolytics, or hypnotics. As used herein, “other ingredients” includes, but is not limited to, one or more ingredients that can be used as a drug carrier.

[0384] The compositions of the present invention may contain a preservative comprising about 0.005% to 2.0% by weight of the total composition. The preservative is used to prevent deterioration upon exposure to contaminants in the environment. Examples of preservatives that can be used in the present invention include, but are not limited to, those selected from benzyl alcohol, sorbic acid, parabens, imidurea, and any combination thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

[0385] The composition may include antioxidants and chelating agents that inhibit the degradation of peptides or compounds. Some antioxidants for peptides or compounds are BHT, BHA, α-tocopherol, and ascorbic acid, with exemplary ranges from about 0.01% to 0.3% of the total weight of the composition, or 0.03% to 0.1% of BHT. The amount of chelating agent may be from 0.01% to 0.5% of the total weight of the composition. Exemplary chelating agents include edetate (e.g., disodium edetate) and citric acid, with weight ranges from about 0.01% to 0.20%, or 0.02% to 0.10% of the total weight of the composition. Chelating agents can be used to chelate metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidants and chelating agents for certain peptides or compounds, other suitable and equivalent antioxidants and chelating agents can be substituted, as those skilled in the art will recognize.

[0386] Liquid suspensions can be prepared using conventional methods to suspend active ingredients in aqueous or oily mediators. Aqueous mediators include, for example, water and isotonic saline. Oily mediators include, for example, vegetable oils such as almond oil, oily esters, ethanol, peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may also contain one or more other components, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, modifiers, preservatives, buffers, salts, flavorings, colorings, and sweeteners. Oily suspensions may also contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, gum arabic, and cellulose derivatives such as sodium carboxymethyl cellulose, methylcellulose, and hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, naturally occurring phospholipids such as lecithin, and condensation products of oxidized alkenes with fatty acids, long-chain fatty alcohols, and partial esters derived from fatty acids and hexanols or from fatty acids and hexanol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitan monooleate, and polyoxyethylene dehydrated sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin, gum arabic, and ionic or nonionic surfactants. Known preservatives include, but are not limited to, methylparaben, ethylparaben, or n-propylparaben, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin.

[0387] Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, an "oily" liquid refers to a liquid containing carbon-containing liquid molecules and exhibiting a lower polarity than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described with respect to liquid suspensions, and it should be understood that suspending agents do not necessarily contribute to the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0388] The powder and particle formulations of the pharmaceutical preparations of this invention can be prepared using known methods. Such formulations can be administered directly to a subject, for example, for forming tablets, filling capsules, or for preparing aqueous or oily suspensions or solutions by adding an aqueous or oily medium. Each of these formulations may also contain one or more of a dispersant or wetting agent, a suspending agent, an ionic or nonionic surfactant, and a preservative. Other excipients, such as fillers and sweeteners, flavoring agents, or coloring agents, may also be present in these formulations.

[0389] The pharmaceutical compositions of the present invention can also be prepared, packaged, or marketed as oil-in-water emulsions or water-in-oil emulsions. The oil phase can be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or a combination thereof. Such compositions may also contain one or more emulsifiers, such as naturally occurring gums, such as gum arabic or tragacanth, naturally occurring phospholipids, such as soybean or lecithin phospholipids, esters or fractional esters derived from combinations of fatty acids and hexanyl anhydrides, such as sorbitan monooleate, and condensation products of such fractional esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain other ingredients, such as sweeteners or flavorings.

[0390] Methods of impregnating or coating materials with chemical compositions are known in the art, including but not limited to methods of depositing or incorporating chemical compositions onto surfaces, methods of incorporating chemical compositions into the structure of materials during material synthesis (i.e., with physiologically degradable materials), and methods of absorbing aqueous or oily solutions or suspensions into absorbent materials, whether or not subsequent drying is performed. Methods of mixing components include physical milling, the use of granules in solid and suspension formulations, and mixing in transdermal patches, as known to those skilled in the art.

[0391] Application / Dosage In a clinical setting, the delivery system of the compositions described herein can be introduced into a subject by any of a variety of methods, each of which is well known in the art. For example, the pharmaceutical formulation of the compositions can be administered via intravenous injection.

[0392] The administration regimen can affect the composition of the effective dose. Therapeutic agents can be administered to subjects before or after the onset of symptoms associated with the disease or condition (i.e., to prevent, treat, and / or improve the subject's infection or symptoms, and / or prevent recurrence of the subject's infection or symptoms).

[0393] The administration of the compositions of the present invention to subjects, preferably mammals, more preferably humans and / or laboratory mammals (e.g., rodents), can be performed using known procedures at doses and time periods to effectively treat, prevent, and / or improve a subject's disease or condition. The effective amount of the composition required to achieve a therapeutic effect can vary depending on factors such as: the time of administration; the duration of administration; other drugs, peptides, compounds, or materials used in combination with the composition; the state of the disease or disorder; the age, sex, weight, condition, general health status, and medical history of the subject being treated; and similar factors well known in the medical field. Dosing regimens can be adjusted to provide an optimal therapeutic response. For example, the dosage may be annual or semi-annually, or it may be proportionally reduced depending on the urgency of the treatment situation. Those skilled in the art will be able to investigate the relevant factors and determine the effective amount of the composition without excessive experimentation. The formulation can be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carriers, suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable route of administration known in the art. Pharmaceutical preparations can be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure buffers, colorants, flavoring agents, and / or aromatic substances. They can also be combined with other active agents, such as other analgesics, when needed.

[0394] The routes of administration for any composition of the present invention include oral, nasal, rectal, intravaginal, parenteral, oral, sublingual, or topical. The polypeptides used in the present invention can be formulated for administration via any suitable route, such as oral or parenteral, transdermal, transmucosal (e.g., sublingual, tongue, oral cavity, urethra, vagina (e.g., vaginal and perivasal), nasal (intranasal) and rectal (intrarectal), intrabladder, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical application.

[0395] In some preferred embodiments, the composition of the present invention is administered intramuscularly.

[0396] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troche, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, granules, emulsions, lozenges, creams, pastes, plasters, lotions, discs, suppositories, nasal or oral liquid sprays, inhaled dry powder or nebulized formulations, and compositions and formulations for intravesical administration. It should be understood that the formulations and compositions that can be used in this invention are not limited to the specific formulations and compositions described herein.

[0397] Oral administration For oral applications, tablets, lozenges, liquids, drops, suppositories or capsules, capsules, and gel caps are particularly suitable. Compositions for oral administration can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from inert, non-toxic pharmaceutical excipients suitable for tablet manufacturing. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate. Tablets may be uncoated or coated using known techniques to improve aesthetics or delay the release of the active ingredient. Oral formulations may also be presented in the form of hard gelatin capsules, wherein the active ingredient is mixed with an inert diluent.

[0398] For oral administration, the polypeptides or compounds of the present invention may be in the form of tablets or capsules, prepared by conventional methods using pharmaceutically acceptable excipients such as: binders (e.g., polyvinylpyrrolidone, hydroxypropyl cellulose, or hydroxypropyl methylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium glycolate); or wetting agents (e.g., sodium dodecyl sulfate). If desired, the tablets may be coated using suitable methods and coating materials, such as the OPADRY™ film coating system (e.g., OPADRY® OY, OYC, organic enteric OY-P, aqueous enteric OY-A, OY-PM, and OPADRY™ White, 32K18400) available from Colorcon, West Point, Pa. Liquid formulations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid formulations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous mediators (e.g., almond oil, oily esters, or ethanol); and preservatives (e.g., methylparaben, propylparaben, or sorbic acid).

[0399] External application For parenteral administration, the polypeptides or compounds of the present invention can be formulated for injection or infusion, such as intravenous, intramuscular, or subcutaneous injection or infusion, or for single-dose and / or continuous infusion administration. Suspensions, solutions, or emulsions in oily or aqueous media can be used, optionally containing other formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0400] Controlled-release formulations and drug delivery systems In some embodiments, the formulations of the present invention may be, but are not limited to, short-term formulations, rapid-offset formulations, and controlled formulations, such as sustained-release, delayed-release, and pulsatile-release formulations.

[0401] The term sustained-release, in its traditional sense, refers to a pharmaceutical formulation that provides a gradual release of the drug over a prolonged period of time and may (though not necessarily) result in a substantially constant blood level of the drug over that extended period. This period may be as long as a month or longer and should be a longer release than the same amount of drug administered in a single dose.

[0402] For sustained release, the peptide or compound can be formulated with a suitable polymer or hydrophobic material to provide sustained-release properties. Therefore, the peptide or compound used in the methods of the present invention can be administered in particulate form, for example by injection, or by implantation in the form of a disc or disk.

[0403] In some embodiments, a sustained-release formulation is used to administer the polypeptide or compound of the present invention, alone or in combination with another pharmaceutical agent, to a patient.

[0404] As used in this article, the term delayed release, in its conventional sense, refers to a pharmaceutical formulation that provides an initial release of the drug after a delay following drug administration, which may include a delay of approximately 10 minutes to approximately 12 hours.

[0405] The term pulse release is used in its conventional sense in this document, referring to a pharmaceutical formulation that delivers drug release in a manner that produces a pulsed plasma spectrum of the drug after administration.

[0406] The term immediate release, in its conventional sense, refers to a pharmaceutical preparation that releases the drug immediately after administration.

[0407] As used herein, "short term" means any time period of time, up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any or all of its increments.

[0408] As used herein, rapid resolution means any time period after drug administration, up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any or all of its increments.

[0409] Dosage The therapeutically effective amount or dosage of the peptides, compounds, and / or compositions of the present invention depends on the patient's age, sex, and weight, the patient's current medical condition, and the progression of the disease or disorder anticipated herein in the patient being treated. Those skilled in the art can determine the appropriate dosage based on these and other factors.

[0410] Suitable dosages of the peptides, compounds, and / or compositions of the present invention can range from about 1 ng to about 1 mg per dose, for example, from about 100 ng to about 1 mg, or from about 100 ng to about 500 ng. In some embodiments, suitable dosages of the peptides, compounds, and / or compositions of the present invention can range from about 10 µg to about 100 µg. This dosage can be administered as a single dose or in multiple doses, for example, one to four or more times. When multiple doses are used, the amount of each dose can be the same or different.

[0411] In some embodiments, the composition may be administered to the subject in one to four doses. In some embodiments, a single dose is administered to the subject. In some embodiments, two doses are administered to the subject. In some embodiments, three doses are administered to the subject. In some embodiments, four doses are administered to the subject. In some embodiments, more than four doses are administered to the subject.

[0412] In some embodiments, the interval between doses is approximately one week. In some embodiments, the interval between doses is approximately one week or less. In some embodiments, the interval between doses is approximately one week or longer. In some embodiments, the interval between doses is approximately two weeks. In some embodiments, the interval between doses is approximately two weeks or less. In some embodiments, the interval between doses is approximately two weeks or longer. In some embodiments, the interval between doses is approximately three weeks. In some embodiments, the interval between doses is approximately three weeks or less. In some embodiments, the interval between doses is approximately three weeks or longer. In some embodiments, the interval between doses is approximately four weeks. In some embodiments, the interval between doses is approximately four weeks or less. In some embodiments, the interval between doses is approximately four weeks or longer.

[0413] In some embodiments, the subject is given two doses, with each dose administered approximately 2 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 3 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 4 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 5 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 6 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 7 weeks apart. In some embodiments, the subject is given two doses, with each dose administered approximately 8 weeks apart.

[0414] In some embodiments, the subject is given three doses, with each dose administered approximately 2 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 3 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 4 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 5 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 6 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 7 weeks apart. In some embodiments, the subject is given three doses, with each dose administered approximately 8 weeks apart.

[0415] In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately two weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately three weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately four weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately five weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately six weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately seven weeks between doses. In some embodiments, the subject is given four doses, with each dose administered at an interval of approximately eight weeks between doses.

[0416] In some preferred, non-limiting embodiments, the subject is given four doses, with an interval of approximately two weeks between doses (i.e., the first dose is on day 0, the second dose is on approximately day 14, the third dose is on approximately day 28, and the fourth dose is on approximately day 42).

[0417] In some preferred, non-limiting embodiments, the subject is given two doses with an interval of approximately 3 weeks between doses (i.e., the first dose is on day 0, and the second dose is on approximately day 21). In some preferred, non-limiting embodiments, the subject is given two doses with an interval of approximately 4 weeks between doses (i.e., the first dose is on day 0, and the second dose is on day 28). In some preferred, non-limiting embodiments, the subject is given two doses with an interval of approximately 8 weeks between doses (i.e., the first dose is on day 0, and the second dose is on day 56).

[0418] The actual dose level of cells in the pharmaceutical formulation of the present invention can be varied to obtain a composition amount that effectively achieves the desired therapeutic response for a specific subject, composition, and administration method without toxicity to the subject.

[0419] The toxicity and efficacy of this treatment regimen can optionally be determined in cell cultures or laboratory animals, including but not limited to LD50. 50 (The dose that would be fatal to 50% of the population) and ED 50 The determination of the dose (the therapeutically effective dose in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, expressed as LD50. 50 and ED 50 The ratio between. Data obtained from cell culture experiments and animal studies may optionally be used to formulate dosage ranges for human use. The dosage of such peptides, compounds, and / or compositions is preferably within the range of ED, which has minimal toxicity. 50 Within the cyclic concentration range. The dose may optionally vary within this range, depending on the dosage form and route of administration used.

[0420] Example Various embodiments of this application can be better understood by referring to the following examples, which are provided by way of example. The scope of this application is not limited to the embodiments given herein.

[0421] Preparation of antigens In some embodiments, the polypeptides described herein are prepared using one or more bacterial expression vectors (e.g., Escherichia coli) according to methods known to those skilled in the art. Front. Microbiol (2014, 5:172). In one aspect, the construction of such a recombinant DNA molecule includes the following steps: (a) generating a single-stranded DNA copy (cDNA) of purified messenger RNA (mRNA) as a template for the desired protein; (b) converting the cDNA into double-stranded DNA; (c) binding the DNA to a suitable site in a suitable cloning vector to form a recombinant DNA molecule; and (d) transforming a suitable host with this recombinant DNA molecule. This transformation enables the host to produce the desired protein.

[0422] Expression vectors possess any characteristics that a vector might have, such as origin of replication, selection markers, and suitable sites for gene insertion, such as multiple cloning sites. Cloned genes can be transferred from specialized cloning vectors to expression vectors, although they can also be cloned directly into expression vectors. The cloning process can occur in *E. coli*. Vectors used for protein production in organisms other than *E. coli*, in addition to having suitable origins of replication for propagation in *E. coli*, may also have elements that allow them to be maintained in another organism (i.e., shuttle vectors).

[0423] HLA-DR4 mice The HLA-DR4 allele is associated with the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. To provide a mouse model of these diseases, a hybrid MHC class II molecule between the peptide-binding domain of human HLA-DRA and HLA-DRB*0401 and the proximal membrane domain of mouse IE (H2-E) was engineered and co-injected into C57BL / 6 zygotes. The transgenic offspring were bred into mice that could not express other MHC class II molecules (Abb knockout in the B6 background). By retaining the α2 and β2 domains of mouse MHC class II, the interaction with the CD4 co-receptor on T cells was preserved. The mouse was healthy and reproduced normally. Immunization with peptides of proteins known to bind to HLA-DR4 elicits a strong T-cell proliferative response, leading to CNS white matter inflammatory damage and symptoms of experimental allergic encephalomyelitis (Ito K, et al. J. Exp. Med. 1996, 183(6):2635-2644).

[0424] Immunization in HLA-DR4 mice HLA-DR4 transgenic mice were used in the experiments described herein, with all mice being within one week of each other in age. HLA-DR4 mice were randomly assigned to treatment and control groups (e.g., blank control and / or placebo). On designated days, mice were immunized twice with a total volume of 50 µL or 100 µL of a vaccine composition comprising 100 µg of antigen (e.g., SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 135) and an adjuvant (e.g., AlOH, dmLT, CpG, and / or AddaSO3™) diluted to the total volume with phosphate-buffered saline (PBS). In some embodiments, HLA-DR4 mice were immunized as described in Tables 1–7 (i.e., Experiments 1–7). Serum and urine samples were collected from the mice at designated time points, as described elsewhere herein.

[0425] Table 1. HLA-DR4 mice a Immunity (Experiment 1)

[0426] a n = 3 mice per group, total volume 100 μL, PBS diluent, 100 μg antigen, administered subcutaneously twice (with an interval of approximately 3 weeks between inoculations); b Blank control group; c Aluminum hydroxide; d Double-mutant heat-labile toxin (dmLT); e CpG ODN 2395.

[0427] Table 2. HLA-DR4 mice a Immunity (Experiment 2)

[0428] a n = 5 mice per group, total volume 100 μL, PBS diluent, 100 μg antigen, administered subcutaneously twice, mice aged 9-10 months at the time of first vaccination (with an interval of about 2 weeks between vaccinations); b Placebo control group; c Aluminum hydroxide; d CpG ODN2395.

[0429] Table 3. HLA-DR4 mice a Immunity (Experiment 3)

[0430] a n = 5 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 8-10 weeks at the time of first vaccination (with an interval of about 3 weeks between vaccinations); b CpG ODN 2395.

[0431] Table 4. HLA-DR4 mice a Immunity (Experiment 4)

[0432] a n=(A) 5 mice per group, (BF) 4 mice per group, total volume 50μL, PBS diluent, 100μg antigen, intramuscular administration twice, mice aged 6-7 weeks at the time of first vaccination (interval between vaccinations is about 3 weeks). b Placebo control group; c CpG ODN 2395.

[0433] Table 5. HLA-DR4 mice a Immunity (Experiment 5)

[0434] a n = 5 mice per group, total volume 50 μL, PBS diluent, 100 μg antigen, administered intramuscularly twice, mice aged 9-10 weeks at the time of first vaccination (with an interval of about 3 weeks between vaccinations); b Placebo control group; c Aluminum hydroxide; d CpG ODN2395.

[0435] Table 6. HLA-DR4 mice a Immunity (Experiment 6)

[0436] a n = 3 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 6-8 weeks at the time of first vaccination (with an interval of about 3 weeks between vaccinations); b Placebo control group; c CpG ODN 2395.

[0437] Table 7. HLA-DR4 mice a Immunity (Experiment 7)

[0438] a n = (AB and DG) 3 mice per group, (C) 4 mice per group, total volume 50 μL, PBS diluent, 100 μg antigen, intramuscular administration twice (with an interval of about 3 weeks between vaccinations), mice aged 7-9 weeks at the time of the first vaccination, all mice were challenged once with UPEC25. b Placebo control group; c CpG ODN 2395; d Aluminum hydroxide; e Buffer conditions (20 mM Na2PO4, 300 mM urea, 300 mM NaCl, pH 9.5); f Buffer conditions (20 mM Na2PO, 300 mM NaCl, pH 9.5).

[0439] CD1 mice CD1 IGS (or Cr1:CD1(ICR)) is an albino outcross mouse model commonly used in toxicology and pharmacology studies. A notable characteristic of CD1 IGS mice is their significant genetic diversity, similar to that within and between human populations. CD1 mice are immunized in a manner similar to that described for HLA-DR4 mice. This article provides a detailed description of experiments conducted using CD1 mice (Table 8).

[0440] Table 8. Immunity in CD1 mice (Experiment 8)

[0441] a Total volume 100µL, PBS diluent, 100µg antigen, administered subcutaneously twice, for mice aged 11 weeks at the time of the first vaccination (with an interval of approximately 2 weeks between vaccinations); b n=3 mice; c n=4 mice; d Placebo control group; e Aluminum hydroxide.

[0442] C57BL / 6 mice The C57BL / 6 mouse is the most widely used inbred strain in research and is commonly used as a model for human diseases. C57BL / 6 mice are immunized in a manner similar to that described for HLA-DR4 mice. This article provides a detailed description of experiments conducted using C57BL / 6 mice (Tables 9-11).

[0443] Table 9. C57BL / 6 a Immunity (Experiment 9)

[0444] a n = 15 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 6-7 weeks at the time of the first vaccination (with an interval of about 3 weeks between vaccinations), challenged with UPEC25 once, and immune samples were collected about 7 days after injection. b Placebo control group; c CpG ODN 2395.

[0445] Table 10. C57BL / 6 mice a Immunity (Experiment 10)

[0446] an = 20 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 6-7 weeks at the time of the first vaccination (with an interval of about 3 weeks between vaccinations), and immune samples were collected about 14 days after the second vaccination. b Placebo control group; c CpG ODN 2395; d Aluminum hydroxide.

[0447] Table 11. C57BL / 6 mice a immunity a (Experiment 11)

[0448] a n = 4 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 6 weeks at the time of the first vaccination (with an interval of about 3 weeks between vaccinations), and immune samples were collected about 12 days after the second vaccination. b Placebo control group; c CpG ODN 2395; d Aluminum hydroxide.

[0449] C3H / HeN mice C3H / HeN mice are Pde6brd1 Homozygous alleles, exhibiting early-onset severe retinal degeneration leading to blindness at weaning age. Despite an atherogenic diet, C3H / HeN mice do not develop aortic atherosclerosis compared to C57BL / 6J. C3H / HeN and C3H / HeJ mice show genetically mediated differences in their response to bacterial endotoxins (LPS). This response is associated with the TL4 protein (Toll-like receptor 4). C3H / HeN is Tlr4lps-n (Toll-like receptor 4; normal LPS response). This strain responds normally to LPS challenge and is sensitive to endotoxins. Conversely, the C3H / HeJ strain is Tlr4lps-d and is said to be endotoxin resistant. C3H / HeN mice were immunized in a manner similar to that described for HLA-DR4 mice. A detailed description of experiments conducted using C3H / HeN mice is provided in this paper (Table 12).

[0450] Table 12. C3H / HeN mice a Immunity (Experiment 12)

[0451] an = 10 mice per group, total volume 50µL, PBS diluent, 100µg antigen, administered intramuscularly twice, mice aged 6-7 weeks at the time of the first vaccination (with an interval of about 12 days between vaccinations), and immune samples were collected about 14 days after the second vaccination. b Placebo control group; c CpG ODN 2395; d Aluminum hydroxide.

[0452] Cellular Bead Array (CBA) The Cell Metrology Bead Array (CBA) assay (BD Biosciences) provides a method for capturing soluble analytes or a group of analytes using beads of known size and fluorescence, enabling detection of the analytes using flow cytometry. Each capture bead in the CBA kit has unique fluorescence and is coated with an antibody specific to soluble proteins. The detection reagent is a mixture of phycoerythrin (PE) conjugated antibodies that provides a fluorescence signal proportional to the amount of analyte bound.

[0453] When capture beads and detection reagents are incubated together with a standard or unknown sample containing the analyte to be identified, a sandwich complex (e.g., capture beads + analyte + detection reagent) is formed. These complexes are measured using flow cytometry to identify particles exhibiting the fluorescent properties of the beads and detection reagents, thereby enabling analyte identification.

[0454] An immune response to the compositions described herein is determined by measuring cytokine levels (e.g., IL-6, IL-17A, IL-2, TNF-α, IFN-γ, IL-4, and / or IL-10) using CBA.

[0455] Enzyme-linked immunosorbent assay (ELISA) An immune response to the compositions described herein is determined by measuring the sample optical density and / or antibody, antigen, protein, and / or glycoprotein titers (e.g., IgG1, IgG2b, IgA, and / or GTxMS-IgG) using standard ELISA and / or peptide-based ELISA. The protocols for standard ELISA and / or peptide ELISA are known to those skilled in the art. The following examples are provided as illustrations. It should be understood that the scope of this application is not limited to the examples provided herein.

[0456] In short, 100 µL of 2 pg / mL peptide (dissolved in 5 M urea) was added to each well of a 96-well EIA / RIA plate (Comin / Costar 3590) and incubated overnight at 4°C. All remaining steps were performed at room temperature. The plate was washed three times with PBS wash buffer (PBS containing 0.05% Tween 20), and then 200 µL / well sample buffer (PBS containing 0.05% Tween 20 and 1% bovine serum albumin) was added. After 90 minutes, the sample buffer was replaced with 100 µL / well PBS sample buffer. Primary antiserum was serially diluted 1:3 on the plate by adding 50 µL to the first row, mixing 10 times, and transferring 50 µL to the next row. The plate was incubated for 90 minutes, then washed three times, and 100 µL / well of the antibody, antigen, protein, and / or glycoprotein to be tested was added for reaction. After a 90-minute incubation period, the plate was washed four times and then 100 plc of TMB (BioFx; Surmodics, Eden Prairie, MN) was added per well.

[0457] The color was developed for 30 minutes, and the reaction was stopped by adding 100 pL of stop reagent (BioFx). The absorbance was measured at 450 nm, and the titer was calculated as the reciprocal of the dilution corresponding to 1.0 absorbance. Controls included standardized primary serum contained in each plate to monitor assay variability, and uncoated wells to subtract background. The limit of detection for this assay is the reciprocal of the initial serum dilution.

[0458] MILLIPEX® Multiplex Immunoassay for Assessing Secreted Cytokines The Millipore Sigma® multiplex immunoassay, based on the Luminex® xMAP® bead-based multiplex assay platform, provides a method for capturing soluble analytes or a group of analytes using magnetic microspheres of known size and fluorescence, enabling detection of the analytes using a Luminex® detection system. In short, each magnetic MagPlex® microsphere bead is fluorescently encoded with one of 500 specific ratios of two fluorophores and coated with an analyte-specific capture antibody. These microspheres are incubated with and washed with a sample, and then incubated with a detection reagent consisting of a mixture of a biotinylated analyte-specific detection antibody and an R-phycoerythrin-conjugated streptavidin reagent, which provides a fluorescence signal proportional to the amount of analyte bound. When the capture microspheres and detection reagent are incubated with a standard or unknown sample containing the identified analyte, a sandwich complex (e.g., capture microsphere + analyte + detection reagent) is formed. These complexes are measured using a Luminex MAGPIX® detection system to identify particles exhibiting the fluorescence properties of both the bead and the detection reagent, thus enabling analyte identification.

[0459] An immune response to the compositions described herein is determined by measuring cytokine levels (such as IL-6, IL-17A, IL-2, TNF-α, IFN-γ, IL-4, IL-22 and / or IL-10) using the MILLIPEX® multiplex immunoassay.

[0460] For the assay described in this article, 1 x 10^6 spleen lymphocytes were used in 0.2 mL. 6 (cells) and bladder lymphocytes (2 x 10 cells in 0.2 mL) 5 (Number of cells) were incubated with 12.5 pg of antigen at 37°C for 48 hours. At the end of the culture period, the culture plate was briefly centrifuged to precipitate the cells, and then the culture supernatant was removed. Samples were acquired on a Luminex MAGPIX® analyzer using Luminex® acquisition software, and the data were analyzed using Belysa® immunoassay curve fitting software.

[0461] spleen cell research Mice used for T-cell studies were euthanized, and their spleens were removed and processed to lyse red blood cells. Splenic lymphocytes (1 x 10^6 cells / mL in 0.2 mL RPMI-1640 containing 10% fetal bovine serum) were then added. 6 Cells were cultured with 12.5 pg of antigen for 48 hours, and the release of cytokines into the cell supernatant was measured according to the manufacturer’s protocol (BD Cell Measurement Bead Array (CBA), BD Biosciences, San Jose, CA).

[0462] Bladder and spleen tissue dissociation Dissect the bladders, combine them into groups (3-5 bladders per group), cut them into small pieces, and digest them at 37°C for 1 hour using Multi-Tissue Dissociation Kit 1 (Miltenyi) according to the manufacturer's instructions, with vigorous manual stirring every 15 minutes. Stop digestion by adding an equal volume of RPMI-1640 containing 10% fetal bovine serum. Disrupt the remaining tissue using the plunger of a 3cc syringe and pass it through a 70µm filter (Greiner Bio-One). Cells obtained from the combined bladder preparations were processed at 2 x 10⁻⁶ cells per group. 6 The final concentration of RPMI-1640 was resuspended at 10% fetal bovine serum.

[0463] Individual spleens were dissected, destroyed using the plunger of a 3cc syringe, and passed through a 70µm filter. They were then processed to remove red blood cells and resuspended in RPMI-1640 containing 10% fetal bovine serum. Cells obtained from the individual spleen preparations were processed at 1x10⁻¹⁰. 7The final concentration of RPMI-1640 was resuspended at 10% fetal bovine serum.

[0464] Flow cytometry assessment of activation-inducing markers (AIM) on restimulated lymphocytes Activation-inducible marker (AIM) proteins are a group of proteins expressed on immune cells upon contact with and / or binding to their homologous antigens and activation. AIM proteins are not expressed or are expressed only at low levels on quiescent, inactivated immune cells. In the experiments described herein, the combination of CD49d and CD11a was used to identify seed-induced effector / memory CD4+ and CD8+ T cells. The combination of CD49d and CD80 was used to identify seed-induced effector / memory B cells. Subsequently, the combination of AIM protein OX40 and PD-L1 was used to identify CD4+ T cells activated in vitro by the protein antigen. + T cells; subsequently, a combination of AIM protein CD69 and PD-L1 was used to identify CD8 cells activated by the protein antigen in vitro. + T cells; then, combinations of AIM proteins CD69 and PD-L1 or CD69 and CD86 were used to identify B cells activated by the protein antigens in vitro.

[0465] Splenic lymphocytes (1x10 in 0.2 mL) 6 (cells) and bladder lymphocytes (2 x 10 cells in 0.2 mL) 5 Cells were cultured at 37°C with 12.5 pg of antigen for 24–48 hours. At the end of the culture period, cells were washed in PBS and resuspended in an amine-reactive live / dead dye solution to remove dead cells, and Fc receptors were blocked with anti-mouse CD16 / CD32 antibody. Cells were incubated at 4°C in the dark for 25–30 minutes. Cells were then washed in FACS buffer (PBS supplemented with 2% fetal bovine serum and 0.095% sodium azide) and stained with a combination of fluorescent antibodies to identify T cells (CD3, CD4, CD8) and B cells (CD19, HLA-DR), and the expression of activation-inducing marker (AIM) proteins (CD11a, CD25, CD49d, OX40, PD-L1, CD69, CD80, and CD86) on these cells after restimulation with protein antigens. Antibodies were purchased from BD, BioLegend, and ThermoFisher, and the optimal staining concentration was empirically determined based on the manufacturer's recommended concentration. Cells were stained with fluorescent antibodies at 4°C for 25 to 30 minutes. Samples were acquired using NovoExpress software on an Agilent NovoCyte Quanteon flow cytometer, and data were analyzed using FlowJo (Treestar) software. AIM protein expression was assessed on live single cells.

[0466] Example 1: Immunized mice exhibited an innate immune response Experiment 1 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 1. Serum samples were obtained from the mice 24 hours after the second vaccination, and serum IL-6 concentrations were analyzed. Comparable levels of IL-6 were observed in all treatment groups (i.e., groups 2–5 in Table 1), while serum IL-6 levels in blank mice were below the lower limit of quantitation. Figure 1 Therefore, the data shows that immunity triggers an innate immune response.

[0467] Example 2: Immunized mice exhibited an adaptive immune response (B cell-mediated antibody production). Experiment 1 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 1.

[0468] Serum samples were obtained from mice in groups 1-2 and 4 (i.e., SEQ ID NO: 127, containing AlOH+dmLT or AlOH+CpG adjuvant) approximately 14 days after the second vaccination (i.e., approximately day 35), and serum IgG1 was analyzed by ELISA. Figure 2A ), serum IgG2b ( Figure 2B ), serum IgA ( Figure 2C ) and urine IgG(H)( Figure 2D The results showed that SEQ ID NO:127 immunization induced HLA-DR4 mice to produce IgG1 and IgG2b.

[0469] Approximately 14 days after the second vaccination (i.e., day 35), serum samples were obtained from mice in groups 1, 3, and 5 (i.e., SEQ ID NO: 128, containing AlOH+dmLT or AlOH+CpG adjuvant), and serum IgG1 was analyzed by ELISA. Figure 3A ) and serum IgG2b ( Figure 3B The results showed that SEQ ID NO:128 immunization induced HLA-DR4 mice to produce IgG1 and IgG2b.

[0470] Approximately 14 days after the second vaccination (i.e., day 35), serum and urine samples were obtained from mice in groups 1, 3, and 5 (i.e., SEQ ID NO: 128 with AlOH+dmLT or AlOH+CpG adjuvant), and serum IgA was analyzed by ELISA. Figure 4A ), urinary IgA ( Figure 4B ) and urine IgG(H)( Figure 4CThe results showed that, as demonstrated by urine and / or serum samples, SEQ ID NO:128 immunization induced the production of IgA and IgG (H) in HLA-DR4 mice.

[0471] Serum samples were obtained from mice in groups 1, 2, and 4 (i.e., SEQ ID NO: 127, containing AlOH+dmLT or AlOH+CpG adjuvant) approximately 14 days after the second vaccination (i.e., day 35) and analyzed by peptide ELISA in which wells were coated with certain polypeptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, and 218-219); antigens (e.g., SEQ ID NO: 127 and SEQ ID NO: 128); and controls (i.e., tetanus toxin, serum albumin only, and uncoated). Antibody production was largely unobserved in unvaccinated subjects (i.e., signal levels were lower or only slightly higher than those in the serum albumin-only control group 1), while significant peptide-specific antibody production was observed in many of the antigens or fragments thereof assessed in this study. Figure 5 ).

[0472] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in several antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0473] Experiment 2 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 2.

[0474] Serum samples were obtained from mice in groups A and DF approximately 14 days after the second vaccination (i.e., day 28), and serum IgG1 levels were analyzed by ELISA. Figure 9A , Figure 10A , Figure 11A ) and serum IgG2b ( Figure 9B , Figure 10B and Figure 11B The results showed that each of the SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135 immunizations induced the production of IgG1 and IgG2b in HLA-DR4 mice.

[0475] Serum samples were obtained from mice in groups A (i.e., placebo) and D (i.e., SEQ ID NO: 132) approximately 14 days after the second vaccination (i.e., day 28) and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, and 218-219), antigens (i.e., SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 135), and controls (i.e., tetanus toxin, serum albumin only, and uncoated). In subjects given placebo, substantially no antibody production was observed (i.e., signal was lower or only slightly higher than in the serum albumin only control in group A), while significant peptide-specific antibody production was observed in many of the antigens or antigen fragments thereof evaluated in this study. Figure 12 ).

[0476] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in several antibody levels, non-limiting examples including IgG1 and IgG2b.

[0477] Experiment 3 As described in this article, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 3.

[0478] Serum samples were collected from mice in the treatment group (Group A) on day 0 (the day of the first vaccination; samples collected before vaccination), day 21 (the day of the second vaccination), day 28, and day 41 (approximately 20 days after the second vaccination), and serum IgG1 was analyzed by ELISA. Figure 16A ) and IgG2b ( Figure 16B The results showed that immunization with SEQ ID NO:132 induced the production of IgG1 and IgG2b in HLA-DR4 mice.

[0479] Serum and urine samples were collected from mice in the treatment group (Group A) on day 0 (the day of the first vaccination; samples collected before vaccination) and approximately day 21 (the day of the second vaccination), day 28, and day 41 (20 days after the second vaccination), and serum IgA was analyzed by ELISA. Figure 17A ), urinary IgA ( Figure 17B ) and urine IgG(H)( Figure 17C The results showed that, as demonstrated by urine and / or serum samples, SEQ ID NO:132 immunization induced the production of IgA and IgG (H) in HLA-DR4 mice.

[0480] Serum samples were obtained from mice in the treatment group (i.e., group A) at approximately day 21 and 41 and analyzed by peptide ELISA, in which wells were coated with certain polypeptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 24, 40, 197-199, 163-168, 176-182, 188-192, 200-202, 206-207, 213-215, and 218-219), antigen (i.e., SEQ ID NO: 132), and controls (i.e., tetanus toxin, serum albumin only, and uncoated). Overall, antibody production was greater at approximately day 41 than at approximately day 21. Figure 18 ).

[0481] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in several antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0482] Experiment 4 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 4.

[0483] Serum and urine samples were obtained from mice in each treatment group (BF group) and control group (A group) at several time points after the initial vaccination, and the sample coated with SEQ ID NO:136 was analyzed by ELISA. Figures 28A-28E ), SEQ ID NO:137 Figures 29A-29E ), SEQ ID NO:138 ( Figures 30A-30E )、SE ID NO:139( Figures 31A-31E ) and SEQ ID NO:140 ( Figures 32A-32E Serum IgG1, serum IgG2b, serum IgA, urine IgA and urine IgG(H) in the wells.

[0484] In some embodiments, results showed that, compared to the control group, all evaluated immunizations induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice approximately 5 weeks after the initial vaccination. In some embodiments, results showed that, compared to the control group, most evaluated immunizations induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice approximately 2 weeks after the second vaccination. In some embodiments, results showed that, compared to the control group, most evaluated immunizations induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice approximately 2 weeks after the initial vaccination.

[0485] In some implementations, results showed that, compared with the control group, all assessed immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice approximately 5 weeks after initial vaccination.

[0486] On day 36 after the first vaccination (approximately 2 weeks after the second vaccination), samples were collected from the control group (i.e., group A) and the treatment group B. Figure 33A ), D( Figure 33B ) and F( Figure 33C Serum samples were obtained from mice in each of the groups and analyzed by peptide ELISA, wherein wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 136-140) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen used in the BF group and certain exemplary antigen fragments.

[0487] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0488] Experiment 5 As described in this article, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 5.

[0489] Serum and urine samples were obtained from mice in each treatment group (i.e., BC and EF groups) and the control group (i.e., A and D groups) 33 days after the initial vaccination (approximately 2 weeks after the second vaccination) and were analyzed by ELISA for mice coated with SEQ ID NO:136 ( Figures 36A-36E ) or SEQ ID NO:138( Figures 37A-37E Serum IgG1, serum IgG2b, serum IgA, urine IgA and urine IgG(H) in the wells.

[0490] In some embodiments, results showed that, compared with the control group, all evaluated immunizations induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice 33 days after the initial vaccination (approximately 2 weeks after the second vaccination). In some embodiments, results showed that, compared with the control group, most evaluated immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 33 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0491] On day 33 after the first vaccination (approximately 2 weeks after the second vaccination), samples were collected from treatment groups B and D. Figure 38A ) and treatment groups E and F ( Figure 38B Serum samples were obtained from mice and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, 136, and 138) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in exemplary vaccine antigens.

[0492] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0493] Experiment 6 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 6.

[0494] Serum and urine samples were obtained from mice in each treatment group (BF group) and control group (A group) 34 days after the initial vaccination (approximately 2 weeks after the second vaccination), and serum IgG1, serum IgG2b, serum IgA, urine IgA, and urine IgG(H) were analyzed by ELISA. Figures 41A-41E ).

[0495] In some embodiments, results showed that, compared with the control group, the assessed immunization induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice 34 days after the initial vaccination (approximately 2 weeks after the second vaccination). In some embodiments, results showed that, compared with the control group, the assessed immunization induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 34 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0496] On day 34 after the first vaccination (approximately 2 weeks after the second vaccination), samples were collected from the control group (Group A) and the treatment group B. Figure 42A ), C( Figure 42B ), D( Figure 42C ) and E( Figure 42DSerum samples were obtained from mice for each of the following assays and analyzed by peptide ELISA, wherein wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 141-145) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in the exemplary vaccine antigens.

[0497] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0498] Experiment 7 As described herein, HLA-DR4 mice were immunized using the vaccine compositions shown in Table 7.

[0499] Serum and urine samples were obtained from mice in each treatment group (i.e., groups BC, E, and G) and each control group (i.e., groups A, D, and F) 35 days after the initial vaccination (approximately 2 weeks after the second vaccination), and serum IgG1, serum IgG2b, serum IgA, urine IgA, and urine IgG(H) in wells coated with SEQ ID NO: 138 (1) or (2) were analyzed by ELISA, where (1) and (2) varied depending on buffer conditions, as described in Table 7. Figures 45A-45E ).

[0500] In some embodiments, results showed that, compared with the control group, certain assessed immunizations induced the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice 35 days after the initial vaccination (approximately 2 weeks after the second vaccination). In some embodiments, results showed that certain assessed immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 35 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0501] On day 35 after the first vaccination (approximately 2 weeks after the second vaccination), samples were collected from the control group (Group A) and each treatment group (Group B). Figure 46Serum samples were obtained from mice and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 16-17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, 138(1), and 138(2), where (1) and (2) vary depending on buffer conditions, as described in Table 7) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in exemplary vaccine antigens.

[0502] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0503] Experiment 8 As described herein, CD1 mice were immunized using the vaccine compositions shown in Table 8.

[0504] Serum samples were obtained from mice in each AC group approximately 14 days after the second vaccination (i.e., around day 28), and serum IgG1 was analyzed by ELISA. Figure 19A and Figure 21A ) and serum IgG2b ( Figure 19B and Figure 21B The results showed that immunization with each of SEQ ID NO:132 and SEQ ID NO:135 induced CB1 mice to produce IgG1 and IgG2b.

[0505] Serum samples were obtained from mice in each AC group approximately 14 days after the second vaccination (i.e., around day 28), and serum IgA was analyzed by ELISA. Figure 20A and Figure 22A ), urinary IgA ( Figure 20B and Figure 22B ) and urine IgG(H)( Figure 20C and Figure 22C The results showed that SEQ ID NO:132 immunization induced the production of IgA and IgG(H) in CB1 mice, as demonstrated by urine and / or serum samples.

[0506] Serum samples were obtained from mice in group A (i.e., placebo) and group B (i.e., SEQ ID NO: 132) on day 28 (approximately 14 days after the second vaccination) and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17-20, 23-25, 27, 32, 35, 37, 39-40), antigens (i.e., SEQ ID NO: 132 and SEQ ID NO: 135), and controls (i.e., tetanus toxin, serum albumin only, and uncoated). Substantial antibody production was observed in the placebo group (i.e., signal was lower or only slightly higher than in the serum albumin only control in group A), while significant peptide-specific antibody production was observed in many of the antigens or antigen fragments thereof evaluated in this study. Figure 23 ).

[0507] Serum samples were obtained from mice in groups A (i.e., placebo) and C (i.e., SEQ ID NO: 135) on day 28 (approximately 14 days after the second vaccination) and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17-20, 23-25, 27, 32, 35, 37, 39-40), antigens (i.e., SEQ ID NO: 132 and SEQ ID NO: 135), and controls (i.e., tetanus toxin, serum albumin only, and uncoated). Substantial antibody production was observed in the placebo group (i.e., signal was lower or only slightly higher than in the serum albumin only control in group A), while significant peptide-specific antibody production was observed in many of the antigens or antigen fragments thereof evaluated in this study. Figure 24 ).

[0508] Experiment 9 As described herein, C57BL / 6 mice were immunized using the vaccine compositions shown in Table 9.

[0509] Serum and urine samples were obtained from mice in each treatment group (BE group) and control group (A group) 34 days after the initial vaccination (approximately 2 weeks after the second vaccination), and serum and urine samples were analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urine IgA, and urine IgG(H). Figures 51A-51E ).

[0510] In some embodiments, results showed that, compared with the control group, certain assessed immunizations induced the production of IgG1, IgG2b, and IgA in the serum of C57BL / 6 mice 34 days after the initial vaccination (approximately 2 weeks after the second vaccination). In some embodiments, results showed that certain assessed immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 34 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0511] On day 34 after the first vaccination (approximately 2 weeks after the second vaccination), samples were collected from the control group (Group A) and treatment groups B and D. Figure 52A ) and treatment groups C and E ( Figure 52B Serum samples were obtained from mice for each of the following assays and analyzed by peptide ELISA, wherein wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 25, 27, 195, 197-199, 163-168, 176-182, 188-192, 207, 213-215, 218-219, 200-203, and 136-139) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in the exemplary vaccine antigens.

[0512] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0513] Experiment 10 As described herein, C57BL / 6 mice were immunized using the vaccine compositions shown in Table 10.

[0514] Serum and urine samples were obtained from mice in each treatment group (i.e., groups B and D) and the control group (i.e., groups A and C) 33 days after the initial vaccination (approximately 12 days after the second vaccination), and serum IgG1, serum IgG2b, serum IgA, urine IgA, and urine IgG(H) were analyzed by ELISA. Figures 59A-59E ).

[0515] In some embodiments, results showed that, compared with the control group, certain assessed immunizations induced the production of IgG1, IgG2b, and IgA in the serum of C57BL / 6 mice 33 days after the initial vaccination (approximately 12 days after the second vaccination). In some embodiments, results showed that certain assessed immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 33 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0516] On day 33 after the first vaccination (approximately 12 days after the second vaccination), samples were collected from control group A and treatment group B. Figure 60A ) and control group C and treatment group D ( Figure 60B Serum samples were obtained from mice and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219, and 225) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in exemplary vaccine antigens.

[0517] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0518] Experiment 11 As described herein, C57BL / 6 mice were immunized using the vaccine compositions shown in Table 11.

[0519] Serum and urine samples were obtained from mice in each treatment group (i.e., groups A, C, E, G, and I) and the control / placebo group (i.e., groups B, D, F, and H) 12 days after the second vaccination (i.e., 33 days after the first vaccination), and serum IgG1, serum IgG2b, serum IgA, urine IgA, and urine IgG(H) were analyzed by ELISA. Figures 62A-62E ).

[0520] In some embodiments, results showed that the assessed immunization induced the production of IgG1, IgG2b, and IgA in the serum of C57BL / 6 mice 12 days after the second vaccination, compared to the control group. In some embodiments, results showed that the assessed immunization induced the production of IgA and IgG(H) in the urine of C57BL / 6 mice 12 days after the initial vaccination, compared to the control group. In some embodiments, results showed that the use of adjuvants is essential for stimulating the production of IgG1, IgG2b, IgA, and / or IgG(H) when the antigen is administered parenterally (e.g., intramuscularly) (see Group A and any of Groups C, E, G, and I).

[0521] Serum samples were obtained from C57BL / 6 mice in each treatment group (i.e., groups A, C, E, G, and I) and control / placebo groups (i.e., groups B, D, F, and H) 12 days after the second vaccination (i.e., 33 days after the first vaccination) and analyzed by peptide ELISA, wherein wells were coated with certain peptides and / or antigen fragments (i.e., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219, and 225) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment used in the exemplary vaccine compositions of this disclosure.

[0522] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2b, IgA, and IgG(H).

[0523] Experiment 12 As described herein, C3H / HeN mice were immunized using the vaccine compositions shown in Table 12.

[0524] Serum and urine samples were obtained from mice in each treatment group (i.e., groups B and D) and control group (i.e., groups A and C) 33 days after the initial vaccination (approximately 12 days after the second vaccination), and serum IgG1, serum IgG2a, serum IgA, urine IgA, and urine IgG(H) were analyzed by ELISA. Figures 56A-56E ).

[0525] In some embodiments, results showed that, compared with the control group, certain assessed immunizations induced the production of IgG1, IgG2a, and IgA in the serum of C3H / HeN mice 33 days after the initial vaccination (approximately 12 days after the second vaccination). In some embodiments, results showed that certain assessed immunizations induced the production of IgA and IgG(H) in the urine of HLA-DR4 mice 33 days after the initial vaccination (approximately 2 weeks after the second vaccination).

[0526] On day 33 after the first vaccination (approximately 12 days after the second vaccination), samples were collected from control group A and treatment group B. Figure 57A ) and control group C and treatment group D ( Figure 57BSerum samples were obtained from mice and analyzed by peptide ELISA, in which wells were coated with certain peptides and / or antigen fragments (e.g., SEQ ID NO: 17, 20, 25, 27, 40, 163-168, 176-182, 188-192, 195, 197-202, 207, 213-215, 218-219, and 225) and controls (i.e., tetanus toxin, serum albumin only, and uncoated). These data describe the generation of antibodies in wells containing each antigen and / or fragment thereof used in exemplary vaccine antigens.

[0527] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by B cell activation, as indicated by an increase in certain antibody levels, non-limiting examples including IgG1, IgG2a, IgA, and IgG(H).

[0528] Example 3: Immunized mice exhibited an adaptive immune response (T cell-mediated antibody production). Experiment 1 T cell-mediated adaptive immune responses were assessed by analyzing spleen cells obtained from HLA-DR4 mice immunized with the vaccine compositions shown in Table 1, as described elsewhere in this document.

[0529] Approximately 22 days after the second vaccination, spleen cells were obtained from mice in groups 1–5, stimulated with SEQ ID NO:127 and SEQ ID NO:128, and analyzed using a Th1 / 17 / pro-inflammatory cytokine IL-17A assay via a cell bead array (CBA) using a Th1 / 2 / 17 kit (BD Biosciences). Figure 6A ), IL-2 Figure 6B ) and IL-6 ( Figure 6C The results showed that a strong response (i.e., high concentrations of antibodies) to the stimulation of antigens SEQ ID NO:127 and SEQ ID NO:128 was observed in spleen cells from the treatment group mice (i.e., groups 2-5).

[0530] Approximately 22 days after the second vaccination, spleen cells were obtained from mice in groups 1–5, stimulated with SEQ ID NO:127 and SEQ ID NO:128, and analyzed using a Th1 / 2 / 17 kit (BD Biosciences) via a cell dosing bead array (CBA) to measure the Th1 / pro-inflammatory cytokine TNF-α (TNF-α). Figure 7A ) and IFN-γ Figure 7BThe results showed that the production of TNF-α in spleen cells from the treatment group mice (i.e., groups 2-5) was a response to stimulation by antigens SEQ ID NO:127 and SEQ ID NO:128.

[0531] Approximately 22 days after the second vaccination, spleen cells were obtained from mice in groups 1–5, stimulated with SEQ ID NO:127 and SEQ ID NO:128, and analyzed using a Th1 / 2 / 17 kit (BD Biosciences) via a cell dosing bead array (CBA) to measure the Th2 / pro-inflammatory cytokine IL-4 (IL-4). Figure 8A ) and IL-10 ( Figure 8B The production of IL-4 was observed in spleen cells from the treatment group mice (i.e., groups 2–5) as a response to stimulation by antigens SEQ ID NO:127 and SEQ ID NO:128.

[0532] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by T cell activation, as indicated by elevated levels of several antibodies and / or cytokines, non-limiting examples including IL-17A, IL-2, IL-6, TNF-α, IFN-γ, IL-4, and IL-10.

[0533] Experiment 2 T cell-mediated adaptive immune responses were assessed by analyzing spleen cells obtained from HLA-DR4 mice immunized with the vaccine compositions shown in Table 2, as described elsewhere in this document.

[0534] Approximately 14 days after the second vaccination, spleen cells were obtained from mice in the AF group, stimulated with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135, and analyzed using a Th1 / Th17 / pro-inflammatory cytokine IL-17A assay via a cell bead array (CBA) using a Th1 / 2 / 17 kit (BD Biosciences). Figure 13A ), IL-2 Figure 13B ) and IL-6 ( Figure 13C The results showed that responses to antigens SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135 were observed in spleen cells from mice in the treatment group.

[0535] Approximately 14 days after the second vaccination, spleen cells were obtained from mice in groups A and DF, stimulated with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135, and analyzed using a Th1 / 2 / 17 kit (BD Biosciences) via a cell dosing bead array (CBA) to measure the Th1 / pro-inflammatory cytokine TNF-α (TNF-α). Figure 14A ) and IFN-γ Figure 14B The results showed that the production of TNF-α and IFN-γ in spleen cells from the treatment group mice (i.e., the DF group) was a response to stimulation by antigens SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135.

[0536] Approximately 14 days after the second vaccination, spleen cells were obtained from mice in groups A and DF, stimulated with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135, and analyzed using a Th1 / 2 / 17 kit (BD Biosciences) via a cell dosing bead array (CBA) to measure the Th2 / anti-inflammatory cytokine IL-4 (IL-4). Figure 15A ) and IL-10 ( Figure 15B The production of IL-10 was observed in spleen cells from the treatment group mice (i.e., the DF group). Overall, the results indicated that IL-10 production was a response to stimulation by antigens SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135.

[0537] Therefore, in some embodiments, the antigens and / or antigen fragments of this disclosure trigger an adaptive immune response mediated by T cell activation, as indicated by elevated levels of several antibodies and / or cytokines, non-limiting examples including IL-17A, IL-2, IL-6, TNF-α, IFN-γ, IL-4, and IL-10.

[0538] Experiment 4 T cell-mediated adaptive immune responses were assessed by analyzing spleen cells obtained from HLA-DR4 mice immunized with the vaccine compositions shown in Table 4, as described elsewhere in this document.

[0539] Spleen cells were obtained from AF group mice 49 days after the first vaccination (approximately 30 days after the second vaccination) and approximately 3 days after UPEC25 challenge. These cells were stimulated with or without SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140, and cytokine levels were measured using the MagPix® system. The results showed that cytokine responses to certain antigenic stimuli were observed in the treated mice, with the strongest responses observed in IFN-γ, IL-4, IL-6, IL-22, IL-17a, and IL-10.

[0540] Using combinations of AIM protein markers OX40 and PD-L1, CD69 and PD-L1, and CD25 and PD-L1, splenocytes obtained from HLA-DR4 mice in all treatment groups (BF group) and the control group (A group) were subjected to antigen-specific CD44 antigen-specific assays by flow cytometry. + T cells, CD8 + Identification of T cells and B cells, wherein the spleen cells are activated in vitro by exemplary antigens: SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140; or without stimulation ( Figures 35A-35C ).

[0541] Therefore, in some embodiments, the antigens of this disclosure trigger an adaptive immune response mediated by T cell activation, as indicated by an increase in certain exemplary cytokines and / or certain exemplary T cell levels.

[0542] Experiment 5 T cell-mediated adaptive immune responses were assessed by analyzing spleen cells obtained from HLA-DR4 mice immunized with the vaccine compositions shown in Table 5, as described elsewhe...

Claims

1. A polypeptide comprising formula (I) or a salt or solvate thereof: (I)， in: B 1 B 2 B 3 L 1 L 2 L 3 L 4 L 5 L 6 T 1 T 2 and T 3 Each occurrence of can either exist or not exist; B 1 B 2 and B 3 Each occurrence of (if present) independently contains an immunogenic fragment of a bacterial surface protein; T 1 T 2 and T 3 Each occurrence (if present) independently contains an immunogenic fragment of the iron receptor protein. Among them, T 1 T 2 and T 3 At least two of them exist or T exists. 1 T 2 and T 3 At least one of them exists and n is at least 2; L 1 L 2 L 3 L 4 L 5 and L 6 Each occurrence of (if present) is an independent polypeptide consisting of 1-10 amino acids. Where L 1 L 2 L 3 L 4 L 5 and L 6 At least one of them exists, and Among them B 1 B 2 B 3 T 1 T 2 and T 3 Every time L appeared, it was 1 L 2 L 3 L 4 L 5 and L 6 At least one of them is separated from the other; and n is an integer selected from 1, 2, 3, 4, and 5.

2. The polypeptide according to claim 1, wherein B 1 B 2 Or B 3 At least one of them exists.

3. The polypeptide according to claim 1 or 2, wherein each bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

4. The polypeptide of claim 3, wherein each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:195, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:

41.

5. The polypeptide according to claim 3 or 4, wherein each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:

40.

6. The polypeptide according to any one of claims 1-5, wherein each iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:

15.

7. The polypeptide according to claim 6, wherein each immunogenic fragment of the iron receptor protein is independently selected from SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ IDThe polypeptides of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

8. The polypeptide according to claim 6 or 7, wherein each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:85, and SEQ ID NO:

88.

9. The polypeptide according to any one of claims 1-8, wherein L 1 L 2 L 3 L 4 L 5 and L 6 Each (if present) independently comprises a polypeptide, wherein each amino acid residue side chain contains a neutral (i.e. uncharged) substituent.

10. The polypeptide according to any one of claims 1-9, wherein L 1 L 2 L 3 L 4 L 5 and L 6 Each of these (if present) is an independent polypeptide containing 4 to 6 amino acids.

11. The polypeptide according to any one of claims 1-10, wherein L 1 L 2 L 3 L 4 L 5 and L 6 Each of (if present) is independently selected from GSGS (SEQ ID NO:124), GPGP (SEQ ID NO:125), LLSVGG (SEQ ID NO:126), and (SGSG). 1-2 (SEQ ID NO:146-147), SSSS (SEQ ID NO:156), GGGS (SEQ ID NO:157), GGC (SEQ ID NO:158), GGS (SEQ ID NO:159), (GGC)8 (SEQ ID NO:160), (GGGGS)3 (SEQ ID NO:161), and GGAAY (SEQ ID NO:162).

12. The polypeptide according to any one of claims 1-11, wherein n is 3.

13. The polypeptide according to any one of claims 1-12, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the polypeptide selected from SEQ ID NO:130 and SEQ ID NO:

129.

14. The polypeptide according to any one of claims 1-11, wherein n is 4.

15. The polypeptide according to any one of claims 1-11 and 14, wherein the polypeptide is combined with a peptide selected from SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:129, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:23 ... The polypeptides of SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259 and SEQ ID NO:260 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

16. A polypeptide or a salt or solvate thereof, said polypeptide being selected from: (a)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 ; (b)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 ; (c)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 ; (d)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 ; (e)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 1 -L 10 -B 6 -L 11 -T 2 -L 12 -B 7 -L 13 -T 3 -L 14 -B 8 -L 15 -T 4 -L 16 -B 9 -L 17 -T 1 -L 18 -B 10 -L 19 -T 2 -L 20 -B 11 -L 21 -T 3 -L 22 -B 12 -L 23 -T 4 ; (f)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 -L 25 -T 13 ; (g)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L 6 -T 7 -L 7 -T 8 -L 8 -T 9 -L 9 -T 10 -L 10 -T 11 -L 11 -T 12 -L 12 -T 13 ; (h)B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 -B 4 -L 7 -T 4 -L 8 -B 5 -L 9 -T 5 -L 10 -B 6 -L 11 -T 6 -L 12 -B 7 -L 13 -T 7 -L 14 -B 8 -L 15 -T 8 -L 16 -B 9 -L 17 -T 9 -L 18 -B 10 -L 19 -T 10 -L 20 -B 11 -L 21 -T 11 -L 22 -B 12 -L 23 -T 12 -L 24 -B 13 ; (i)B 1 -L 1 -B 2 -L 2 -B 3 -L 3 -B 4 -L 4 -T 1 -L 5 -T 2 -L 6 -T 3 -L 7 -T 4 -L 8 -B 5 -L 9 -B 6 -L 10 -B 7 -L 11 -B 8 -L 12 -T 5 -L 13 -T 6 -L 14 -T 7 -L 15 -T 8 -L 16 -B 9 -L 17 -B 10 -L 18 -B 11 -L 19 -B 12 -L 20 -T 9 -L 21 -T 10 -L 22 -T 11 -L 23 -T 12 ; and (j)T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -B 1 -L 5 -B 2 -L 6 -B 3 -L 7 -B 4 -L 8 -T 5 -L 9 -T 6 -L 10 -T 7 -L 11 -T 8 -L 12 -B 5 -L 13 -B 6 -L 14 -B 7 -L 15 -B 8 -L 16 -T 9 -L 17 -T 10 -L 18 -T 11 -L 19 -T 12 -L 20 -B 9 -L 21 -B 10 -L 22 -B 11 -L 23 -B 12 ; in: Each occurrence of B (if present) independently contains an immunogenic fragment of a bacterial surface protein. Each B instance has a C-terminus and an N-terminus; Each occurrence of T independently contains an immunogenic fragment of the iron receptor protein. Each T instance has a C-terminus and an N-terminus; Each occurrence of L independently comprises a polypeptide of 1-10 amino acids. Each L instance has a C-terminus and an N-terminus; Each B instance is covalently linked to one or two independent L instances via a covalent peptide bond between the C-terminus of B and the N-terminus of L and / or between the N-terminus of B and the C-terminus of L. Each T instance is covalently linked to one or two independent L instances via a covalent peptide bond between the C-terminus of T and the N-terminus of L and / or between the N-terminus of T and the C-terminus of L.

17. The polypeptide of claim 16, wherein each bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

18. The polypeptide of claim 17, wherein each immunogenic fragment of the bacterial surface protein is independently selected from SEQ ID NO:16, SEQ ID NO:195, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:

195. The polypeptide of NO:41 has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

19. The polypeptide according to claim 17 or 18, wherein each immunogenic fragment of the bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:

40.

20. The polypeptide according to any one of claims 16-19, wherein each iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

15.

21. The polypeptide according to claim 20, wherein each immunogenic fragment of the iron receptor protein is independently selected from SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ IDThe polypeptides of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology.

22. The polypeptide of claim 20 or 21, wherein each immunogenic fragment of the iron receptor protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:85, and SEQ ID NO:

88.

23. The polypeptide according to any one of claims 16-22, wherein L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each (if present) independently comprises a polypeptide, wherein each amino acid residue side chain contains a neutral (i.e., uncharged) substituent.

24. The polypeptide according to any one of claims 16-23, wherein L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each of these (if present) is an independent polypeptide consisting of 4 to 6 amino acids.

25. The polypeptide according to any one of claims 16-24, wherein L 1 L 2 L 3 L 4 L 5 L 6 L 7 L 8 L 9 L 10 L 11 L 12 L 13 L 14 L 15 L 16 L 17 L 18 L 19 、 L 20 L 21 L 22 L 23 L 24 and L 25 Each of them (if it exists) is independently selected from GSGS (SEQ ID NO:124), GPGP (SEQ ID NO:125), LLSVGG (SEQ ID NO:126), and (SGSG). 1-2 (SEQ ID NO:146-147), SSSS (SEQ ID NO:156), GGGS (SEQ ID NO:157), GGC (SEQ ID NO:158), GGS (SEQ ID NO:159), (GGC)8 (SEQ ID NO:160), (GGGGS)3 (SEQ ID NO:161), and GGAAY (SEQ ID NO:162).

26. The polypeptide according to any one of claims 16-25, wherein the polypeptide is combined with a sample selected from SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231 ... ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259 and SEQ ID The polypeptide of NO:260 has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

27. The polypeptide according to any one of claims 1-26, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:

225.

28. The polypeptide according to any one of claims 1-26, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:

226.

29. An isolated messenger ribonucleic acid (mRNA) encoding a polypeptide according to any one of claims 1-28.

30. The isolated mRNA of claim 29, wherein the mRNA is codon-optimized for expression in organisms that: (a) prokaryotes; or (b) Mammals.

31. The isolated mRNA according to claim 30, wherein one of the following applies: (a) The prokaryote is *Escherichia coli*; or (b) The mammal in question is a human.

32. An isolated deoxyribonucleic acid (DNA) encoding the polypeptide of any one of claims 1-28.

33. The isolated DNA of claim 32, wherein the DNA is codon-optimized for expression in organisms that: (a) prokaryotes; or (b) Mammals.

34. The isolated DNA according to claim 33, wherein one of the following applies: (a) The prokaryote is *Escherichia coli*; or (b) The mammal in question is a human.

35. An isolated polynucleotide encoding the mRNA of any one of claims 29-31, wherein the polynucleotide comprises one or more promoters and / or polyadenylation signals operatively linked to a sequence encoding the mRNA.

36. A vector comprising the isolated mRNA of any one of claims 29-31, the isolated DNA of any one of claims 32-34, and / or the isolated polynucleotide of claim 35.

37. The vector according to claim 36, wherein the vector is a viral vector.

38. The vector of claim 37, wherein the viral vector is adeno-associated virus (AAV), optionally wherein the AAV is AAV9.

39. The vector according to claim 36, wherein the vector is a bacterial expression vector.

40. The vector according to claim 39, wherein the bacterial expression vector is an Escherichia coli vector.

41. A lipid nanoparticle (LNP) composition comprising isolated mRNA as described in any one of claims 29-31, isolated DNA as described in any one of claims 32-34, and / or isolated polynucleotide as described in claim 35.

42. The LNP of claim 41, wherein at least one of the following applies: (a) The ratio of lipids to isolated mRNA or DNA in the LNP is approximately 5:1 to approximately 25:1; and (b) The ratio of lipids to isolated polynucleotides in the LNP is about 5:1 to about 25:

1.

43. The LNP according to claim 41 or 42, wherein the LNP comprises: (a) at least one ionizable lipid; (b) at least one accessory lipid; (c) cholesterol or its modified derivatives, and any combination thereof; and (d) At least one conjugated lipid.

44. The LNP of claim 43, wherein the ionizable lipid is selected from at least one of the following: DLinDMA, DLenDMA, DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DLin-KDMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, DLin-EG-DMA, DODAC, DODMA, DSDMA, DOTMA, DDAB, DOTAP, DC-Chol, DMRIE, DOSPA, DOGS, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, and DLincarbDAP.

45. The LNP according to claim 43 or 44, wherein the at least one ionizable lipid comprises about 50 mol% to about 90 mol% of the LNP.

46. ​​The LNP according to any one of claims 43-45, wherein the auxiliary lipid is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC).

47. The LNP according to any one of claims 43-46, wherein the at least one auxiliary lipid accounts for about 1 to about 25 mol of the LNP.

48. The LNP according to any one of claims 43-47, wherein the cholesterol accounts for about 20 to about 60 mol of the LNP.

49. The LNP according to any one of claims 43-48, wherein the conjugated lipid is selected from at least one of the following: 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (PEG-DMG), 1,2-distearate-sn-glycerol-methoxy polyethylene glycol (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol-methoxy polyethylene glycol (PEG-DPG), mPEG-OH, mPEG-AA (mPEG-CM), mPEG-CH2CH2CH2-NH 2、 mPEG-DMG, mPEG-N,N-bis(tetradecyl)acetamide (ALC-0159), mPEG-DSPE and mPEG-DPPE.

50. The LNP according to any one of claims 43-49, wherein the at least one conjugated lipid comprises about 0.1 to about 5 mol of the LNP.

51. The LNP according to any one of claims 41-50, wherein the isolated mRNA, isolated DNA and / or isolated polynucleotides are at least partially encapsulated in the LNP.

52. A pharmaceutical composition comprising the LNP of any one of claims 41-51 and a pharmaceutically acceptable carrier.

53. A vaccine composition comprising any one of claims 41-51 LNP and / or the pharmaceutical composition of claim 52.

54. A vaccine composition comprising a polypeptide according to any one of claims 1-28 and at least one pharmaceutically acceptable excipient.

55. The vaccine composition according to claim 54, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:

225.

56. The vaccine composition according to claim 54, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:

226.

57. The vaccine composition according to any one of claims 54 to 56, further comprising an adjuvant.

58. The vaccine composition of claim 57, wherein the adjuvant comprises at least one selected from the group consisting of aluminum hydroxide (AlOH), double-mutant heat-labile toxin (dmLT), CpG, and oil-in-water emulsion adjuvants.

59. The vaccine composition according to claim 58, wherein at least one of the following applies: (a) The vaccine contains about 10 µg to about 100 µg of the polypeptide; (b) The vaccine contains approximately 100 µg to approximately 6000 µg of the CpG; (c) The vaccine contains about 250 µg to about 750 µg of the AlOH; (d) The vaccine comprises approximately 1 µg to approximately 25 µg of the dmLT; and (e) The vaccine comprises about 25% (v / v) to about 75% (v / v) of the oil-in-water emulsion adjuvant.

60. A vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with SEQ ID NO:225 or SEQ ID NO:226, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide. (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, wherein the polysorbate 80 comprises about 0.9% (v / v) of the vaccine composition; The polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in phosphate-buffered saline (PBS) solution.

61. A vaccine composition comprising: (a) A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to SEQ ID NO:225 or SEQ ID NO:226, wherein the vaccine composition comprises about 10 µg to about 100 µg of the polypeptide; (b) α-Tocopherol, wherein the α-tocopherol constitutes about 2.5% (v / v) of the vaccine composition; (c) Squalene, wherein the squalene comprises about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80, wherein the polysorbate 80 comprises about 0.9% (v / v) of the vaccine composition; and (e) CpG, wherein the vaccine comprises about 100 µg to about 6000 µg of the CpG; The polypeptide, α-tocopherol, squalene, polysorbate and CpG are dissolved or suspended in phosphate-buffered saline (PBS) solution.

62. A method for treating, preventing, and / or improving bacterial infections in a subject with such need, the method comprising administering to the subject at least one selected from: (a) the polypeptide according to any one of claims 1-28; (b) the isolated mRNA according to any one of claims 29-31; (c) The isolated DNA according to any one of claims 32-34; (d) The isolated polynucleotide as described in claim 35; (e) The carrier according to any one of claims 36-40; (f) The LNP according to any one of claims 41-51; (g) the pharmaceutical composition according to claim 52; and (h) The vaccine composition according to any one of claims 53-61.

63. The method of claim 62, wherein the bacterial infection is a urinary tract infection (UTI).

64. The method of claim 62, wherein the bacterial infection includes urinary tract infection (UTI) and sepsis.

65. The method of claim 62, wherein the bacterial infection is sepsis.

66. The method according to claim 64 or 65, wherein the sepsis is neonatal sepsis.

67. The method of claim 62, wherein the bacterial infection is pneumonia.

68. The method according to any one of claims 62-67, wherein the subject is a mammal.

69. The method of claim 68, wherein the mammal is a human.

70. The method according to any one of claims 62-69, wherein the subject is pregnant with a fetus.

71. The method of claim 70, wherein the bacterial infection is treated, prevented, and / or improved in at least one of the subject and their fetus or newborn.

72. The method of claim 70, wherein the bacterial infection is treated, prevented, and / or improved in both the subject and their fetus or newborn.

73. A method for generating immunity against infection with one or more pathogenic bacteria in a subject, the method comprising administering to the subject at least one selected from: (a) the polypeptide according to any one of claims 1-28; (b) the isolated mRNA according to any one of claims 29-31; (c) The isolated DNA according to any one of claims 32-34; (d) The isolated polynucleotide as described in claim 35; (e) The carrier according to any one of claims 36-40; (f) The LNP according to any one of claims 41-51; (g) the pharmaceutical composition of claim 52; and (h) The vaccine composition according to any one of claims 53-61.

74. The method of claim 73, wherein the one or more pathogenic bacteria comprises at least one selected from the group consisting of Escherichia coli (Escherichia coli). Escherichia coli Klebsiella pneumoniae ( Klebsiella pneumoniae ), Proteus mirabilis ( Proteus mirabilis ), Shigella dysenteriae ( Shigella dysenteriae Salmonella enterica () Salmonella enterica Streptococcus pneumoniae () Salmonella enterica Haemophilus influenzae ( ) Haemophilus influenzae Chlamydia pneumoniae ( Chlamydophila pneumoniae Mycoplasma pneumoniae ( ) Mycoplasma pneumoniae Staphylococcus aureus ( Staphylococcus aureus ), Moraxella catarrhalis ( Moraxella catarrhalis ) and Legionella pneumophila ( Legionella pneumophila Streptococcus pyogenes ( Legionella pneumophila ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Salmonella Bongo ( Salmonella bongori ).

75. The method according to claim 73 or 74, wherein the immunity prevents bacterial infection.

76. The method of claim 75, wherein the bacterial infection is a urinary tract infection (UTI).

77. The method of claim 75, wherein the bacterial infection includes urinary tract infection (UTI) and sepsis.

78. The method of claim 75, wherein the bacterial infection is sepsis.

79. The method according to claim 77 or 78, wherein the sepsis is neonatal sepsis.

80. The method of claim 75, wherein the bacterial infection is pneumonia.

81. The method according to any one of claims 73-80, wherein the subject is a mammal.

82. The method of claim 81, wherein the mammal is a human.

83. The method according to any one of claims 73-82, wherein the subject is pregnant with a fetus.

84. The method of claim 83, wherein immunity against infection with one or more pathogenic bacteria is generated in at least one of the subject and their fetus or newborn.

85. The method according to claim 83 or 84, wherein immunity against infection with one or more pathogenic bacteria is generated in both the subject and their fetus or newborn.

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