Compositions and methods for generating immunity against bacterial infections

Abstract

This disclosure relates, in part, to a vaccine composition comprising a polypeptide of formula (I) or a salt or solvate thereof, and at least one pharmaceutically acceptable excipient. This disclosure further relates to isolated mRNA and / or isolated polynucleotides encoding a polypeptide of formula (I), vectors and / or LNPs comprising the same, and pharmaceutically acceptable compositions thereof. This disclosure further relates to a method for treating, preventing and / or improving a bacterial infection in a subject where such treatment is needed, and / or a method for generating immunity against infection by one or more pathogenic bacteria, the method comprising administering to the subject at least one composition of this disclosure. In certain embodiments, the bacterial infection is a urinary tract infection, sepsis (e.g., neonatal sepsis), or pneumonia. TIFF2026521465000060.tif146130

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 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, both filed under 35 U.S.C. § 119(e), all of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings The XML file named "375836_7000WO1_SequenceListing.xml", created on June 5, 2024, and containing 245KB, is thus incorporated into this specification by reference in its entirety. [Background technology]

[0003] background Bacterial infections (e.g., urinary tract infections (UTIs), sepsis, and pneumonia) pose a significant threat to human health and result in substantial morbidity and mortality rates globally, including infant mortality (e.g., neonatal sepsis).

[0004] Infections such as UTIs, sepsis (e.g., neonatal sepsis), and pneumonia can be caused by any of several bacteria, viruses, and / or fungi, but these infections most often have a bacterial origin (e.g., Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are generally considered to be the most common causes of UTIs). Therefore, antibiotics (e.g., ciprofloxacin) are commonly prescribed as first-line treatment. However, in addition to the risks associated with excessive reliance on antibiotics (e.g., the development of antibiotic resistance), such antibiotics may cause side effects, non-limited, including nausea, vomiting, stomach pain, heartburn, diarrhea, and fatigue.

[0005] Thus, there is a need in the art for compositions and methods for preventing bacterial infections (e.g., UTI, sepsis, and / or pneumonia) and / or generating immunity against infection by one or more bacterial pathogens (including the non-limiting example of Escherichia coli). This disclosure addresses this need. SUMMARY OF THE INVENTION

[0006] Summary In one aspect, the disclosure provides a polypeptide comprising formula (I): (B 3 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 ) n (I) or a salt or solvate thereof, wherein n and each occurrence of B 1 , B <U+ 2 , B <U+ 3 [[ID=U+0000017]] 1 [[ID=4%]] <U+ 2 <U+ 3 <U+ 4 <U+ 5 <U+ 6 <U+ 1 <U+OO00024> <U+ 3 <U+ 1 <U+ 1 <U+ 1 <U+ 2 <U+ 2 <U+ 3 <U+ 2 ; <U+ 4 are defined elsewhere in this specification.

[0007] In another aspect, the disclosure provides 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 -L7 -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 Provided by むむポリペプチドを, formula Chinese, 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 , 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 This is defined elsewhere in this specification.

[0008] In another context, this disclosure is 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 Provides a polypeptide containing, in the formula, L 1 , L 2 , L 3 , L 4 , 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 T12 This is defined elsewhere in this specification.

[0009] In another context, this disclosure is 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 Provides a polypeptide containing, in the formula, B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 , 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 , L17 , T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , and T 9 This is defined elsewhere in this specification.

[0010] In another context, this disclosure is 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 Provides a polypeptide containing, in the formula, 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 This is defined elsewhere in this specification.

[0011] In another context, this disclosure is 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 Provided by むむポリペプチドを, formula Chinese, 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 L19 , L 20 , L 21 , L 22 , L 23 , T 1 , T 2 , T 3 , and T 4 This is defined elsewhere in this specification.

[0012] In another context, this disclosure is 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 Provides a polypeptide containing, in the formula, B 1B 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 This is defined elsewhere in this specification.

[0013] In another context, this disclosure is T 1 -L 1 -T 2 -L 2 -T 3 -L 3 -T 4 -L 4 -T 5 -L 5 -T 6 -L6 -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 Provides a polypeptide containing, in the formula, 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 This is defined elsewhere in this specification.

[0014] In another context, this disclosure is 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 Provided by むむポリペプチドを, formula Chinese, 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 T 1 T2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 This is defined elsewhere in this specification.

[0015] In another context, this disclosure is 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 Provides a polypeptide containing, in the formula, B 1 B 2 B 3B 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 This is defined elsewhere in this specification.

[0016] In another context, this disclosure is 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 -L9 -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 Provided by むむポリペプチドを, formula Chinese, 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 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 This is defined elsewhere in this specification.

[0017] In another context, the Disclosure provides isolated messenger ribonucleic acid (mRNA) encoding the polypeptide of the Disclosure.

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

[0019] In another aspect, the present disclosure provides isolated polynucleotides encoding mRNA of the present disclosure, wherein the polynucleotide comprises one or more promoter and / or polyadenylation signals functionally linked to the mRNA-coding sequence.

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

[0021] In another aspect, the Disclosure provides lipid nanoparticle (LNP) compositions comprising isolated mRNA, isolated DNA, and / or isolated polynucleotides.

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

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

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

[0025] In another context, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising the step of administering the polypeptide of the Disclosure to the subject.

[0026] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising administering the subject isolated mRNA of the Disclosure.

[0027] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising administering the isolated DNA of the Disclosure to the subject.

[0028] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising administering the subject an isolated polynucleotide of the Disclosure.

[0029] In another context, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising the step of administering the vector of the Disclosure to the subject.

[0030] In another context, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising the step of administering the LNP of the Disclosure to the subject.

[0031] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising the step of administering a pharmaceutical composition of the Disclosure to the subject.

[0032] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed, the method comprising the step of administering the subject a vaccine composition of the Disclosure.

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

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

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

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

[0037] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria, the method comprising administering the vector of the Disclosure to the subject.

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

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

[0040] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria, the method comprising administering the subject a vaccine composition of the Disclosure. [Brief explanation of the drawing]

[0041] The drawings illustrate, but are not limited to, various embodiments of this application.

[0042] [Figure 1] A graph is provided showing serum IL-6 concentrations 24 hours after the second vaccination in HLA-DR4 mice, where the mice were vaccinated as follows according to the vaccination protocol described elsewhere in this specification (experiment number 1): 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). [Figure 2A] Figures 2A–2D provide graphs showing the serum titers of IgG1 (Figure 2A), IgG2b (Figure 2B), as well as the levels of serum IgA (Figure 2C) and urinary IgG(H) (Figure 2D) in HLA-DR4 mice approximately 14 days (i.e., day 35) after a second vaccination using no vaccination (i.e., naive; 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, in HLA-DR4 mice following the vaccination protocol (experiment number 1) described elsewhere in this specification (experiment number 1). [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 2C] Please refer to the explanation in Figure 2A. [Figure 2D] Please refer to the explanation in Figure 2A. [Figure 3]Figures 3A-3B provide graphs showing serum titers of IgG1 (Figure 3A) and IgG2b (Figure 3B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days (i.e., day 35) after a second vaccination using no vaccination (i.e., naive; group 1); SEQ ID NO: 128 (AlOH + dmLT adjuvant; group 3); and SEQ ID NO: 128 (AlOH + CpG adjuvant; group 5), following the vaccination protocol described elsewhere in this specification (experiment number 1). [Figure 4A] Figures 4A–4C provide graphs showing serum (Figure 4A) and urine (Figure 4B–4C) levels of IgA (Figure 4A–4B) and IgG(H) (Figure 4C) as determined by standard ELISA in HLA-DR4 mice approximately 14 days (i.e., day 35) after a second vaccination using no vaccination (i.e., naive; group 1); SEQ ID NO:128 (AlOH+dmLT adjuvant; group 3); and SEQ ID NO:128 (AlOH+CpG adjuvant; group 5), following the vaccination protocol described elsewhere in this specification (experiment number 1). [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 4C] Please refer to the explanation in Figure 4A. [Figure 5]We provide a bar graph showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 14 days after a second vaccination using no vaccination (Group 1); SEQ ID NO: 127 (AlOH + dmLT adjuvant; Group 2); and SEQ ID NO: 128 (AlOH + CpG adjuvant; Group 4), following the vaccination protocol (Experiment No. 1) described elsewhere in this specification, with wells coated with certain polypeptides (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 no coating). Three bars are shown for each coating along the x-axis (e.g., SEQ ID NO: 17), where the left, center, and right bars represent data corresponding to groups 1, 2, and 4 of experiment number 1, respectively. [Figure 6A] Figures 6A–6C provide bar graphs showing the production of Th1 / Th17 / inflammatory cytokines IL-17A (Figure 6A), IL-2 (Figure 6B), and IL-6 (Figure 6C) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) (BD Biosciences), approximately 22 days (i.e., day 43) after a second vaccination immunization using SEQ ID NO:127 (AlOH+dmLT adjuvant; Group 1); 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), and after restimulation using SEQ ID NO:127 and SEQ ID NO:128. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C] Please refer to the explanation in Figure 6A. [Figure 7A]Figures 7A-7B provide bar graphs showing the production of Th1 / inflammatory cytokines TNF-α (Figure 7A) and IFN-γ (Figure 7B) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) (BD Biosciences), approximately 22 days (i.e., day 43) after a second vaccination immunization using SEQ ID NO:127 (AlOH+dmLT adjuvant; Group 1); 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), and after restimulation using SEQ ID NO:127 and SEQ ID NO:128. [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 8A] Figures 8A-8B provide bar graphs showing the production of Th2 / anti-inflammatory cytokines IL-4 (Figure 8A) and IL-10 (Figure 8B) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization with SEQ ID NO:127 (AlOH+dmLT adjuvant; 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), and restimulation with SEQ ID NO:127 and SEQ ID NO:128 approximately 22 days after the second vaccination (i.e., day 43). [Figure 8B] Please refer to the explanation in Figure 8A. [Figure 9A]Figures 9A–9B provide graphs showing serum titers of IgG1 (Figure 9A) and IgG2b (Figure 9B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days (i.e., day 28) after a second vaccination with placebo (i.e., AlOH + CpG adjuvant; group A) or SEQ ID NO: 132 (AlOH + CpG adjuvant; group D), following the vaccination protocol (experiment number 2) described elsewhere in this specification. [Figure 9B] Please refer to the explanation in Figure 9A. [Figure 10A] Figures 10A–10B provide graphs showing serum titers of IgG1 (Figure 10A) and IgG2b (Figure 10B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days (i.e., day 28) after a second vaccination with placebo (i.e., AlOH + CpG adjuvant; group A) or SEQ ID NO: 133 (AlOH + CpG adjuvant; group E), following the vaccination protocol (experiment number 2) described elsewhere in this specification. [Figure 10B] Please refer to the explanation in Figure 10A. [Figure 11A] Figures 11A–11B provide graphs showing serum titers of IgG1 (Figure 11A) and IgG2b (Figure 11B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days (i.e., day 28) after a second vaccination with placebo (i.e., AlOH + CpG adjuvant; group A) or SEQ ID NO: 135 (AlOH + CpG adjuvant; group F), following the vaccination protocol (experiment number 2) described elsewhere in this specification. [Figure 11B] Please refer to the explanation in Figure 11A. [Figure 12]We provide a bar graph showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 28 days after initial vaccination with placebo (i.e., AlOH + CpG adjuvant; group A); or SEQ ID NO: 132 (AlOH + CpG adjuvant; group D), following the vaccination protocol (experiment number 2) described elsewhere in this specification, with wells coated with certain polypeptides (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 control (i.e., tetanus toxin (TTX), serum albumin (SA), and no coating). Two bars are shown for each coating along the x-axis (e.g., SEQ ID NO: 17), where the left and right bars represent data corresponding to groups A and D of experiment number 2, respectively. [Figure 13A] Figures 13A-13C show the results of cytometric bead array (CBA) (BD) immunization with 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); followed by restimulation with SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 135. This document provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines IL-17A (Figure 13A), IL-2 (Figure 13B), and IL-6 (Figure 13C) by splenocytes from HLA-DR4 mice, as determined by Biosciences. [Figure 13B] Please refer to the explanation in Figure 13A. [Figure 13C] Please refer to the explanation in Figure 13A. [Figure 14A]Figures 14A-14B provide bar graphs showing the production of Th1 / inflammatory cytokines TNF-α (Figure 14A) and IFN-γ (Figure 14B) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization with 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 restimulation with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135. [Figure 14B] Please refer to the explanation in Figure 14A. [Figure 15A] Figures 15A-15B provide bar graphs showing the production of Th2 / anti-inflammatory cytokines IL-4 (Figure 15A) and IL-10 (Figure 15B) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization with 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 restimulation with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135. [Figure 15B] Please refer to the explanation in Figure 15A. [Figure 16A] Figures 16A and 16B provide graphs showing serum titers of IgG1 (Figure 16A) and IgG2b (Figure 16B) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination using SEQ ID NO: 132 (AddaS03 (trademark) + CpG adjuvant) according to the vaccination protocol (experiment number 3) described elsewhere in this specification. [Figure 16B]Please refer to the explanation in Figure 16A. [Figure 17A] Figures 17A–17C provide graphs showing serum (Figure 17A–17B) and urine (Figure 17B–17C) levels of IgA (Figure 17A–17B) and IgG(H) (Figure 17C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination using SEQ ID NO: 132 (AddaS03(trademark) + CpG adjuvant) according to the vaccination protocol (experiment number 3) described herein. [Figure 17B] Please refer to the explanation in Figure 17A. [Figure 17C] Please refer to the explanation in Figure 17A. [Figure 18] We provide bar graphs showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 21 and 41 days after vaccination with SEQ ID NO: 132 (AddaS03® + CpG adjuvant), following the vaccination protocol (experiment number 3) described elsewhere in this specification, with wells coated with certain polypeptides (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 no coating). Two bars are shown for each coating (e.g., SEQ ID NO: 17) along the x-axis, where the left and right bars represent data corresponding to samples collected from mice 21 days and 41 days after vaccination, respectively. [Figure 19] Figures 19A and 19B provide graphs showing serum titers of IgG1 (Figure 19A) and IgG2b (Figure 19B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days after a second vaccination with placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO: 132 (AlOH adjuvant; group B), following the vaccination protocol (experiment number 8) described herein. [Figure 20A] Figures 20A–20C provide graphs showing serum (Figure 20A) and urine (Figures 20B–20C) levels of IgA (Figures 20A–20B) and IgG(H) (Figure 20C) in HLA-DR4 mice approximately 14 days after a second vaccination with 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 (experiment number 8) described herein. [Figure 20B] Please refer to the explanation in Figure 20A. [Figure 20C] Please refer to the explanation in Figure 20A. [Figure 21A] Figures 21A and 21B provide graphs showing serum titers of IgG1 (Figure 21A) and IgG2b (Figure 21B) as determined by standard ELISA in HLA-DR4 mice approximately 14 days after a second vaccination with placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO: 135 (AlOH adjuvant; group C), following the vaccination protocol (experiment number 8) described herein. [Figure 21B] Please refer to the explanation in Figure 21A. [Figure 22A] Figures 22A–22C provide graphs showing serum (Figure 22A) and urine (Figures 22B–22C) levels of IgA (Figures 22A–22B) and IgG(H) (Figure 22C) as determined by standard ELISA in HLA-DR4 mice approximately 14 days after a second vaccination with placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO: 135 (AlOH adjuvant; group C), following the vaccination protocol (experiment number 8) described herein. [Figure 22B] Please refer to the explanation in Figure 22A. [Figure 22C] Please refer to the explanation in Figure 22A. [Figure 23]We provide a bar graph showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 14 days after a second vaccination with placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO: 132 (AlOH adjuvant; group B), following the vaccination protocol (experiment number 8) described elsewhere herein, with wells coated with certain polypeptides (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 no coating). Two bars are shown for each coating along the x-axis (e.g., SEQ ID NO: 17), where the left and right bars represent the data corresponding to groups A and B of experiment number 8, respectively. [Figure 24] We provide a bar graph showing the results of peptide ELISA using serum from CD1 mice approximately 14 days after a second vaccination with placebo (i.e., AlOH adjuvant; group A) or SEQ ID NO: 135 (AlOH adjuvant; group C), following the vaccination protocol (experiment number 8) described elsewhere in this specification, with wells coated with certain polypeptides (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 no coating). Two bars are shown for each coating along the x-axis (e.g., SEQ ID NO: 17), where the left and right bars represent the data corresponding to groups A and C of experiment number 8, respectively. [Figure 25A]Figures 25A–25C provide bar graphs showing the production of Th1 / Th17 / inflammatory cytokines IL-17A (Figure 25A), IL-2 (Figure 25B), and IL-6 (Figure 25C) by CD1 splenocytes, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization 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) and subsequent restimulation with SEQ ID NO:132 or SEQ ID NO:135. [Figure 25B] Please refer to the explanation in Figure 25A. [Figure 25C] Please refer to the explanation in Figure 25A. [Figure 26A] Figures 26A-26B provide bar graphs showing the production of Th1 / inflammatory cytokines TNF-α (Figure 26A) and IFN-γ (Figure 26B) by CD1 splenocytes, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization 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); and restimulation with SEQ ID NO:132 or SEQ ID NO:135. [Figure 26B] Please refer to the explanation in Figure 26A. [Figure 27A] Figures 27A-27B provide bar graphs showing the production of Th2 / anti-inflammatory cytokines IL-4 (Figure 27A) and IL-10 (Figure 27B) by CD1 splenocytes, as determined by cytometric bead array (CBA) (BD Biosciences), after immunization 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); and restimulation with SEQ ID NO:132 or SEQ ID NO:135. [Figure 27B] Please refer to the explanation in Figure 27A. [Figure 28A] Figures 28A–28E provide graphs showing serum titers or levels of IgG1 (Figure 28A), IgG2b (Figure 28B), and IgA (Figure 28C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 136 (AddaS03(trademark) + CpG adjuvant) or placebo, according to the vaccination protocol (experiment number 4) described herein, as well as urinary levels of IgA (Figure 28D) and IgG(H) (Figure 28E). [Figure 28B] Please refer to the explanation in Figure 28A. [Figure 28C] Please refer to the explanation in Figure 28A. [Figure 28D] Please refer to the explanation in Figure 28A. [Figure 28E] Please refer to the explanation in Figure 28A. [Figure 29A] Figures 29A–29E provide graphs showing serum titers or levels of IgG1 (Figure 29A), IgG2b (Figure 29B), and IgA (Figure 29C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 137 (AddaS03(trademark) + CpG adjuvant) or placebo, according to the vaccination protocol (experiment number 4) described herein, as well as urinary levels of IgA (Figure 29D) and IgG(H) (Figure 29E). [Figure 29B] Please refer to the explanation in Figure 29A. [Figure 29C] Please refer to the explanation in Figure 29A. [Figure 29D] Please refer to the explanation in Figure 29A. [Figure 29E] Please refer to the explanation in Figure 29A. [Figure 30A]Figures 30A–30E provide graphs showing serum titers or levels of IgG1 (Figure 30A), IgG2b (Figure 30B), and IgA (Figure 30C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 138 (AddaS03(trademark) + CpG adjuvant) or placebo, according to the vaccination protocol (experiment number 4) described herein, as well as urinary levels of IgA (Figure 30D) and IgG(H) (Figure 30E). [Figure 30B] Please refer to the explanation in Figure 30A. [Figure 30C] Please refer to the explanation in Figure 30A. [Figure 30D] Please refer to the explanation in Figure 30A. [Figure 30E] Please refer to the explanation in Figure 30A. [Figure 31A] Figures 31A–31E provide graphs showing serum titers or levels of IgG1 (Figure 31A), IgG2b (Figure 31B), and IgA (Figure 31C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 139 (AddaS03(trademark) + CpG adjuvant) or placebo, according to the vaccination protocol (experiment number 4) described herein, as well as urinary levels of IgA (Figure 31D) and IgG(H) (Figure 31E). [Figure 31B] Please refer to the explanation in Figure 31A. [Figure 31C] Please refer to the explanation in Figure 31A. [Figure 31D] Please refer to the explanation in Figure 31A. [Figure 31E] Please refer to the explanation in Figure 31A. [Figure 32A]Figures 32A–32E provide graphs showing serum titers or levels of IgG1 (Figure 32A), IgG2b (Figure 32B), and IgA (Figure 32C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 140 (AddaS03(trademark) + CpG adjuvant) or placebo, according to the vaccination protocol (experiment number 4) described herein, as well as urinary levels of IgA (Figure 32D) and IgG(H) (Figure 32E). [Figure 32B] Please refer to the explanation in Figure 32A. [Figure 32C] Please refer to the explanation in Figure 32A. [Figure 32D] Please refer to the explanation in Figure 32A. [Figure 32E] Please refer to the explanation in Figure 32A. [Figure 33A] Figures 33A–33C provide bar graphs showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 14 days after a second vaccination using 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), following the vaccination protocol (experiment number 4) described elsewhere herein, with wells coated with a specific polypeptide (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 a control (i.e., tetanus toxin (TTX), serum albumin (SA), and no coating). Two bars are shown for each coating (e.g., SEQ ID NO: 17) along the x-axis, where the left and right bars represent the data corresponding to groups A and B (Figure 33A); groups A and D (Figure 33B); and groups A and F (Figure 33C) of experiment number 4, respectively. [Figure 33B] Please refer to the explanation in Figure 33A. [Figure 33C]Please refer to the explanation in Figure 33A. [Figure 34A] Figures 34A-34F show the results of immunization using placebo (AddaS03(trademark)+CpG; group A); SEQ ID NO:136 (AddaS03(trademark)+CpG; group B); SEQ ID NO:137 (AddaS03(trademark)+CpG; group C); SEQ ID NO:138 (AddaS03(trademark)+CpG; group D); SEQ ID NO:139 (AddaS03(trademark)+CpG; group E); and SEQ ID NO:140 (AddaS03(trademark)+CpG; group F) (experiment number 4). Approximately 49 days after the initial vaccination and approximately 3 days after UPEC25 induction, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID This document provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: 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) by splenocytes from HLA-DR4 mice, as determined by a cytometric bead array (CBA) (MAGPIX® system), with or without restimulation using NO:140. [Figure 34B] Please refer to the explanation in Figure 34A. [Figure 34C] Please refer to the explanation in Figure 34A. [Figure 34D] Please refer to the explanation in Figure 34A. [Figure 34E] Please refer to the explanation in Figure 34A. [Figure 34F] Please refer to the explanation in Figure 34A. [Figure 35A]Figures 35A–35C provide bar graphs showing the rates of the activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 35A), CD69 and PD-L1 (Figure 35B), and CD25 and PD-L1 (Figure 35C) in splenocytes approximately 3 days after induction with UPEC25, and approximately 49 days after initial vaccination with 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); after restimulation with SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:139; and SEQ ID NO:140; or without stimulation. [Figure 35B] Please refer to the explanation in Figure 35A. [Figure 35C] Please refer to the explanation in Figure 35A. [Figure 36A] Figures 36A–36E provide graphs showing serum titers or levels of IgG1 (Figure 36A), IgG2b (Figure 36B), and IgA (Figure 36C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 136 (Group B); SEQ ID NO: 136 (Group E); or placebo (Group A), according to the vaccination protocol (Experiment No. 5) described herein, as well as urinary levels of IgA (Figure 36D) and IgG(H) (Figure 36E). [Figure 36B] Please refer to the explanation in Figure 36A. [Figure 36C] Please refer to the explanation in Figure 36A. [Figure 36D] Please refer to the explanation in Figure 36A. [Figure 36E] Please refer to the explanation in Figure 36A. [Figure 37A]Figures 37A–37E provide graphs showing serum titers or levels of IgG1 (Figure 37A), IgG2b (Figure 37B), and IgA (Figure 37C) in HLA-DR4 mice as determined by standard ELISA, as a function of time (days) after vaccination with SEQ ID NO: 138 (Group C); SEQ ID NO: 138 (Group F); or placebo (Group D), according to the vaccination protocol (Experiment No. 5) described herein, as well as urinary levels of IgA (Figure 37D) and IgG(H) (Figure 37E). [Figure 37B] Please refer to the explanation in Figure 37A. [Figure 37C] Please refer to the explanation in Figure 37A. [Figure 37D] Please refer to the explanation in Figure 37A. [Figure 37E] Please refer to the explanation in Figure 37A. [Figure 38A] Figures 38A–38B provide bar graphs showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 14 days after a second vaccination 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), following the vaccination protocol (experiment number 5) described elsewhere in this specification, where wells were coated with certain polypeptides (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 no coating). Two bars are shown for each coating (e.g., SEQ ID NO: 17) along the x-axis, where the left and right bars represent data corresponding to groups B and C (Figure 38A) and groups E and F (Figure 38B) of experiment number 5, respectively. [Figure 38B] Please refer to the explanation in Figure 38A. [Figure 39A]Figures 39A-39F show the results of immunization using 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 number 5), with SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, and SEQ ID The bar graphs provide the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ (Figure 39A), IL-4 (Figure 39B), IL-6 (Figure 39C), IL-22 (Figure 39D), IL-10 (Figure 39E), and IL-17A (Figure 39F) by splenocytes from HLA-DR4 mice, as determined by cytometric bead array (CBA) approximately 33 days after initial vaccination, either after restimulation with NO:140 or without stimulation. [Figure 39B] Please refer to the explanation in Figure 39A. [Figure 39C] Please refer to the explanation in Figure 39A. [Figure 39D] Please refer to the explanation in Figure 39A. [Figure 39E] Please refer to the explanation in Figure 39A. [Figure 39F] Please refer to the explanation in Figure 39A. [Figure 40A] Figures 40A–40C provide bar graphs showing the rates of the activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 40A), CD69 and PD-L1 (Figure 40B), and CD69 and CD86 (Figure 40C) in splenocytes approximately 7 days after induction with UPEC25, and approximately 53 days after initial vaccination 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); after restimulation with SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; and SEQ ID NO:139; or without stimulation. [Figure 40B] Please refer to the explanation in Figure 40A. [Figure 40C] Please refer to the explanation in Figure 40A. [Figure 41A] Figures 41A–41E provide graphs showing serum titers or levels of IgG1 (Figure 41A), IgG2b (Figure 41B), and IgA (Figure 41C), as determined by standard ELISA, in HLA-DR4 mice approximately 34 days after vaccination with 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 to placebo (Group A), according to the vaccination protocol (Experiment No. 6) described herein. [Figure 41B] Please refer to the explanation in Figure 41A. [Figure 41C] Please refer to the explanation in Figure 41A. [Figure 41D] Please refer to the explanation in Figure 41A. [Figure 41E] Please refer to the explanation in Figure 41A. [Figure 42A]Figures 42A–42D show wells coated with certain polypeptides (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 no coating), following the vaccination protocol (experiment number 6) described elsewhere in this specification: placebo (group A) and SEQ ID NO: 141 (group B) (Figure 42A); SEQ ID NO: 142 (group C) (Figure 42B); SEQ ID NO: 143 (group D) (Figure 42C); or SEQ ID A bar graph is provided showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 34 days after initial vaccination with NO:144 (Group E) (Figure 42D). Two bars are shown for each coating (e.g., SEQ ID NO:17) along the x-axis, where the left and right bars represent data corresponding to groups A and B (Figure 42A), groups A and C (Figure 42B), groups A and D (Figure 42C), and groups A and E (Figure 42D) of experiment number 6, respectively. [Figure 42B] Please refer to the explanation in Figure 42A. [Figure 42C] Please refer to the explanation in Figure 42A. [Figure 42D] Please refer to the explanation in Figure 42A. [Figure 43A]Figures 43A-43F show the results of immunization using 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 specification (experiment number 6). Approximately 47 days after the initial vaccination and approximately 7 days after UPEC25 induction, SEQ ID NO:141; SEQ ID NO:142; SEQ ID NO:143; SEQ ID NO:144; SEQ ID NO:145; and SEQ ID This document provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ (Figure 43A), IL-4 (Figure 43B), IL-6 (Figure 43C), IL-22 (Figure 43D), IL-10 (Figure 43E), and IL-17A (Figure 43F) by splenocytes from HLA-DR4 mice restimulated with NO:137, as determined by cytometric bead array (CBA) Magpix®. [Figure 43B] Please refer to the explanation in Figure 43A. [Figure 43C] Please refer to the explanation in Figure 43A. [Figure 43D] Please refer to the explanation in Figure 43A. [Figure 43E] Please refer to the explanation in Figure 43A. [Figure 43F] Please refer to the explanation in Figure 43A. [Figure 44A]Figures 44A–44C provide bar graphs showing the rates of the activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 44A), CD69 and PD-L1 (Figure 44B), and CD69 and CD86 (Figure 44C) in splenocytes approximately 7 days after induction with UPEC25, and approximately 47 days after initial vaccination 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); after restimulation with SEQ ID NO:141; SEQ ID NO:142; SEQ ID NO:143; SEQ ID NO:144; SEQ ID NO:145; or without stimulation. [Figure 44B] Please refer to the explanation in Figure 44A. [Figure 44C] Please refer to the explanation in Figure 44A. [Figure 45A] Figures 45A–45E provide graphs showing serum titers or levels of IgG1 (Figure 45A), IgG2b (Figure 45B), and IgA (Figure 45C), as well as urinary levels of IgA (Figure 45D) and IgG(H) (Figure 45E), in HLA-DR4 mice approximately 35 days after vaccination with 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 determined by standard ELISA according to the vaccination protocol (Experiment No. 7) described herein. [Figure 45B] Please refer to the explanation in Figure 45A. [Figure 45C] Please refer to the explanation in Figure 45A. [Figure 45D] Please refer to the explanation in Figure 45A. [Figure 45E] Please refer to the explanation in Figure 45A. [Figure 46]We provide a bar graph showing the results of peptide ELISA using serum from HLA-DR4 mice approximately 35 days after initial vaccination with placebo (group A); SEQ ID NO: 138 (group B); or SEQ ID NO: 138 (group C), following the vaccination protocol (experiment number 7) described elsewhere in this specification, with wells coated with a specific polypeptide (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 a control (i.e., tetanus toxin (TTX), serum albumin (SA), and no coating). Three bars are shown for each coating along the x-axis (for example, SEQ ID NO: 17), where the left, center, and right bars represent the data corresponding to groups A, B, and C of experiment number 7, respectively. [Figure 47A] Figures 47A-47F show that immunization was performed using 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 specification (experiment number 7), and approximately 55 days after the initial vaccination and approximately 7 days after UPEC25 induction, SEQ ID NO:138(1); SEQ ID NO:138(2); or SEQ ID This document provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ (Figure 47A), IL-4 (Figure 47B), IL-6 (Figure 47C), IL-22 (Figure 47D), IL-10 (Figure 47E), and IL-17A (Figure 47F) by splenocytes of HLA-DR4 mice restimulated with NO:139, as determined by cytometric bead array (CBA) Magpix®. [Figure 47B] Please refer to the explanation in Figure 47A. [Figure 47C] Please refer to the explanation in Figure 47A. [Figure 47D]Please refer to the explanation in Figure 47A. [Figure 47E] Please refer to the explanation in Figure 47A. [Figure 47F] Please refer to the explanation in Figure 47A. [Figure 48A] Figures 48A-48F show that immunization was performed using 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 specification (experiment number 7), with SEQ ID NO:138(1); or SEQ ID NO:138(2) (group G) approximately 55 days after the initial vaccination and approximately 7 days after UPEC25 induction. This document provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: IFN-γ (Figure 48A), IL-4 (Figure 48B), IL-6 (Figure 48C), IL-22 (Figure 48D), IL-10 (Figure 48E), and IL-17A (Figure 48F) by bladder cells of HLA-DR4 mice restimulated with NO:138(2), as determined by cytometric bead array (CBA) Magpix®. [Figure 48B] Please refer to the explanation in Figure 48A. [Figure 48C] Please refer to the explanation in Figure 48A. [Figure 48D] Please refer to the explanation in Figure 48A. [Figure 48E] Please refer to the explanation in Figure 48A. [Figure 48F] Please refer to the explanation in Figure 48A. [Figure 49A]Figures 49A–49C provide bar graphs showing the rates of activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 49A), CD69 and PD-L1 (Figure 49B), and CD69 and CD86 (Figure 49C) in splenocytes of HLA-DR4 mice approximately 7 days after induction with UPEC25, and approximately 55 days after initial vaccination with 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), after restimulation with SEQ ID NO:138(1); SEQ ID NO:138(2); SEQ ID NO:139; or without stimulation. [Figure 49B] Please refer to the explanation in Figure 49A. [Figure 49C] Please refer to the explanation in Figure 49A. [Figure 50A] Figures 50A–50B provide bar graphs showing the rates of the activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 50A) and CD69 and PD-L1 (Figure 50B) in bladder cells of HLA-DR4 mice approximately 7 days after induction with UPEC25, and approximately 55 days after initial vaccination with 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); after restimulation with SEQ ID NO:138(1); SEQ ID NO:138(2); or without stimulation. [Figure 50B] Please refer to the explanation in Figure 50A. [Figure 51A]Figures 51A–51E provide graphs showing serum titers or levels of IgG1 (Figure 51A), IgG2b (Figure 51B), and IgA (Figure 51C), as well as urinary levels of IgA (Figure 51D) and IgG(H) (Figure 51E), in C57BL / 6 mice approximately 34 days after vaccination 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), as determined by standard ELISA according to the vaccination protocol (Experiment No. 9) described herein. [Figure 51B] Please refer to the explanation in Figure 51A. [Figure 51C] Please refer to the explanation in Figure 51A. [Figure 51D] Please refer to the explanation in Figure 51A. [Figure 51E] Please refer to the explanation in Figure 51A. [Figure 52A] Figures 52A–52B show wells coated with certain polypeptides (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 no coating), following the vaccination protocol (experiment number 9) described elsewhere in this specification: 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 A bar graph is provided showing the results of peptide ELISA using serum from C57BL / 6 mice approximately 34 days after initial vaccination with NO:139 (group E). Three bars are shown along the x-axis for each coating (e.g., SEQ ID NO:17), where the left, center, and right bars represent data corresponding to groups A, B, and D (Figure 52A) and groups A, C, and E (Figure 52B) of experiment number 9, respectively. [Figure 52B]Please refer to the explanation in Figure 52A. [Figure 53A] Figures 53A-53F show immunization with 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 specification (experiment number 9); approximately 52 days after the initial vaccination and approximately 7 days after UPEC25 induction, SEQ ID NO: 136; SEQ ID NO: 137; SEQ ID NO: 138; (Figure 52B); or SEQ ID The present invention provides bar graphs showing the production of Th1 / Th17 / inflammatory cytokines or Th2 / anti-inflammatory cytokines: 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) by splenocytes from C57BL / 6 mice, as determined by a cytometric bead array (CBA) Magpix®, with or without stimulation using NO:139. [Figure 53B] Please refer to the explanation in Figure 53A. [Figure 53C] Please refer to the explanation in Figure 53A. [Figure 53D] Please refer to the explanation in Figure 53A. [Figure 53E] Please refer to the explanation in Figure 53A. [Figure 53F] Please refer to the explanation in Figure 53A. [Figure 54A]Figures 54A–54B provide bar graphs showing the rates of the activation-inducing markers (AIMs) OX40 and PD-L1 (Figure 54A), as well as CD86 and CD69 (Figure 54B), in splenocytes of C57BL / 6 mice approximately 7 days after induction with UPEC25, and approximately 52 days after initial vaccination 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); after restimulation with SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:139; or without stimulation. [Figure 54B] Please refer to the explanation in Figure 54A. [Figure 55] This document provides bar graphs showing the rates of the activation-inducing markers (AIM) OX40 and PD-L1 in bladder cells of C57BL / 6 mice approximately 7 days after induction with UPEC25, and approximately 52 days after initial vaccination 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), after restimulation with SEQ ID NO:136; SEQ ID NO:138; or without stimulation. [Figure 56A] Figures 56A–56E provide graphs showing serum titers or levels of IgG1 (Figure 56A), IgG2a (Figure 56B), and IgA (Figure 56C), as well as urinary levels of IgA (Figure 56D) and IgG(H) (Figure 56E), in C3H / HeN mice approximately 33 days after initial vaccination with placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D), as determined by standard ELISA according to the vaccination protocol (Experiment No. 12) described herein. [Figure 56B] Please refer to the explanation in Figure 56A. [Figure 56C]Please refer to the explanation in Figure 56A. [Figure 56D] Please refer to the explanation in Figure 56A. [Figure 56E] Please refer to the explanation in Figure 56A. [Figure 57A] Figures 57A–57B provide bar graphs showing the results of peptide ELISA using serum from C3H / HeN mice approximately 33 days after initial vaccination using 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), following the vaccination protocol (experiment number 12) described elsewhere herein. Wells were coated with certain polypeptides (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 no coating). Two bars are shown for each coating (e.g., SEQ ID NO: 17) along the x-axis, where the left and right bars represent data corresponding to groups A and B (Figure 57A) and groups C and D (Figure 57B) of experiment number 12, respectively. [Figure 57B] Please refer to the explanation in Figure 57A. [Figure 58] As described elsewhere in this specification (experiment number 12), we provide bar graphs showing IFN-γ production in the spleen, lymph nodes, and bladder of C3H / HeN mice, as determined by cytometric bead array (CBA) Magpix®, during restimulation with SEQ ID NO:225 or without stimulation (i.e., unstimulated), approximately 33 days after initial vaccination. [Figure 59A]Figures 59A–59E provide graphs showing serum titers or levels of IgG1 (Figure 59A), IgG2b (Figure 59B), and IgA (Figure 59C), as well as urinary levels of IgA (Figure 59D) and IgG(H) (Figure 59E), in C57BL / 6 mice approximately 33 days after initial vaccination with placebo (Group A); SEQ ID NO:225 (Group B); placebo (Group C); and SEQ ID NO:225 (Group D), as determined by standard ELISA according to the vaccination protocol (Experiment No. 10) described herein. [Figure 59B] Please refer to the explanation in Figure 59A. [Figure 59C] Please refer to the explanation in Figure 59A. [Figure 59D] Please refer to the explanation in Figure 59A. [Figure 59E] Please refer to the explanation in Figure 59A. [Figure 60A] Figures 60A–60B provide bar graphs showing the results of peptide ELISA using serum from C57BL / 6 mice approximately 33 days after initial vaccination with placebo (group A; Figure 60A); SEQ ID NO: 225 (group B; Figure 60A); placebo (group C; Figure 60B); and SEQ ID NO: 225 (group D; Figure 60B), following the vaccination protocol described elsewhere in this specification (experiment number 10). Wells were coated with certain polypeptides (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 no coating). Two bars are shown for each coating (e.g., SEQ ID NO: 17) along the x-axis, where the left and right bars represent data corresponding to groups A and B (Figure 60A) and groups C and D (Figure 60B) of experiment number 10, respectively. [Figure 60B] Please refer to the explanation in Figure 60A. [Figure 61]As described elsewhere in this specification (experiment number 10), we provide bar graphs showing IFN-γ production in the spleen, lymph nodes, and bladder of C57BL / 6 mice, as determined by a cytometric bead array (CBA) Magpix®, approximately 33 days after initial vaccination, immunized with placebo (group A); SEQ ID NO:225 (group B); placebo (group C); and SEQ ID NO:225 (group D); and with or without stimulation (i.e., unstimulated) using SEQ ID NO:225. [Figure 62A] Figures 62A–62E provide graphs showing serum titers or levels of IgG1 (Figure 62A), IgG2b (Figure 62B), and IgA (Figure 62C), as well as urinary levels of IgA (Figure 62D) and IgG(H) (Figure 62E) in C57BL / 6 mice, as determined by standard ELISA, approximately 33 days after the first vaccination (approximately 12 days after the second vaccination) using SEQ ID NO: 225 (groups A, C, E, G, and I) or placebo (groups B, D, F, and H), following the vaccination protocol described elsewhere in this specification (experiment number 11). [Figure 62B] Please refer to the explanation in Figure 62A. [Figure 62C] Please refer to the explanation in Figure 62A. [Figure 62D] Please refer to the explanation in Figure 62A. [Figure 62E] Please refer to the explanation in Figure 62A. [Figure 63A]Figures 63A–63E show wells coated with certain polypeptides (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 no coating) according to the vaccination protocol (experiment number 11) described herein; 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 This document provides bar graphs showing the results of peptide ELISA using serum from C57BL / 6 mice approximately 33 days after the first vaccination (approximately 12 days after the second vaccination) using NO:225 (group G; Figure 63D); placebo (group H; Figure 63E); and SEQ ID NO:225 (group I; group 63E). For Figures 63B-63E, two bars are shown along the x-axis for each coating (e.g., SEQ ID NO:17), where the left and right bars represent data corresponding to groups B and C (Figure 63B), D and E (Figure 63C), F and G (Figure 63D), and H and I (Figure 63E), respectively, of experiment number 11. [Figure 63B] Please refer to the explanation in Figure 63A. [Figure 63C] Please refer to the explanation in Figure 63A. [Figure 63D] Please refer to the explanation in Figure 63A. [Figure 63E] Please refer to the explanation in Figure 63A. [Figure 64]This specification provides bar graphs showing the rates of the activation-inducing markers (AIM) OX40 and PDL1 in splenocytes obtained from C57BL / 6 mice approximately 45 days after initial vaccination 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 (experiment number 11) described herein. Splenocytes from the specified treatment group or placebo group (i.e., groups A-I) were either restimulated (+) or not restimulated (-) with SEQ ID NO:225. [Figure 65] This specification provides bar graphs showing IFN-γ production in the spleen of C57BL / 6 mice immunized with 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) as determined by a cytometric bead array (CBA) Magpix®, according to the vaccination protocol (Experiment No. 11) described herein, and then restimulated with SEQ ID NO:225 approximately 12 days after the second vaccination, or unstimulated (i.e., unstimulated). [Modes for carrying out the invention]

[0043] Detailed description of the invention Herein, specific aspects of the disclosed subject matter are given in detail, some examples of which are shown in the accompanying drawings. The disclosed subject matter is described together with the enumerated claims, but it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0044] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical limits explicitly stated as range boundaries, 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 "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also individual values ​​within the indicated range (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%). The notation "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise specified. Similarly, the notation "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise specified.

[0045] In this text, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly indicates otherwise. The term “or” is used to refer to a non-exclusive “or” unless otherwise specified. The statements “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.” In addition, it should be understood that the usage or terminology used herein is for explanatory purposes only, and not restrictive, unless otherwise defined. Any use of section headings is intended to aid the reading of the document and should not be interpreted as restrictive; information relating to a section heading may be found within or outside that particular section. All publications, patents, and patent documents referenced herein are incorporated herein by reference in whole, as if they were incorporated by reference individually.

[0046] In this text, the term “each” is used to refer to any one of two or more members of a group, and further to refer to a single element when a group has only one member. For example, if group 1 consists of A, B, and C, the term “each” applied to group 1 refers to any one of A, B, and C. Furthermore, if group 2 consists of A, the term “each” applied to group 2 refers to A.

[0047] In the methods described herein, the acts may be performed in any order unless the time or order of operations is explicitly stated. Furthermore, unless the express claims language states that certain acts are performed separately, those acts may be performed simultaneously. For example, the claimed act of performing X and the claimed act of performing Y may be performed simultaneously in a single operation, and the resulting process falls within the literal scope of the claimed process.

[0048] definition As used herein, the term “about” means that some degree of variation in a value or range may be permitted, for example, within 10%, 5%, or 1% of the stated limit of the stated value or range, including the exact stated value or range.

[0049] As used herein, the term “adjuvant” refers to a substance that increases and / or modulates an immune response to a vaccine. In certain embodiments, an adjuvant, when administered in combination with one or more antigens, may act to promote, prolong, and / or enhance an antigen-specific immune response in a subject.

[0050] An infection, disease, or disorder is considered “improving” if the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by the patient, or both, decreases.

[0051] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH (e.g., pH approximately 7.0). These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups linked to neutral lipids.

[0052] As used herein, the term “antigen” refers to a substance that can induce an immune response, for example, by major histocompatibility complex (MHC) cell surface proteins, or that can bind to the antigen-binding domain of an immunoglobulin molecule. A T-cell immune response is achieved by presenting an antigen. As used herein, “antigen” includes, but is not limited to, immunogens, small molecules, carbohydrates, lipids, nucleic acids, or combinations thereof, which may be antigenic determinants, haptens, and peptides. Experienced immunologists, when discussing antigens processed for presentation to T cells, should recognize that the term “antigen” refers to a portion of an antigen that is a T-cell epitope (e.g., a peptide fragment) presented to the T-cell receptor by MHC. When used in the context of a B-cell immune response in the form of an antigen-specific antibody, the complementarity-determining portion (i.e., the binding portion) of the variable domains of the binding antibody (i.e., light and heavy chains) in the antigen may be a linear or three-dimensional epitope.

[0053] As used herein, the term “antigen 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 antigen peptide is a portion of an antigen that is recognized by an antibody, or in the case of MHC, by a T cell receptor.

[0054] As used herein, the term “bacterial infection” refers to any infection directly or indirectly caused by one or more species of Gram-negative, Gram-positive, or atypical bacteria. The term is not limited to infections caused by bacteria. Non-exclusive examples of bacterial infections considered within the scope of this disclosure include urinary tract infections (UTIs), sepsis (e.g., neonatal sepsis), and pneumonia.

[0055] The term “cationic lipid” refers to any of several lipid species that exhibit an effective positive charge at a selected pH, such as physiological pH (e.g., pH approximately 7.0). Cationic lipids containing multiple unsaturated sites, e.g., alkyl chains having at least two or three unsaturated sites, have been found to be particularly useful in forming lipid particles with increased membrane fluidity. Certain cationic lipids and related analogues useful in this disclosure are described in U.S. Patent Applications Publications 20060083780 and 20060240554; U.S. Patents 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for any purpose. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids have a protonable tertiary amine (e.g., pH titrable) head group, C 18 It contains an alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0056] As used herein, the terms “composition” or “pharmaceutical composition” refer to a mixture of at least one polypeptide or compound useful in the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of polypeptides or compounds to a patient or subject. Multiple techniques for administering compounds exist in the art, including but not limited to intravenous, subcutaneous, oral, aerosol, parenteral, intraocular, pulmonary, and topical administration.

[0057] As used herein, the term “conjugated lipid” refers to a lipid conjugated with one or more polymer groups that inhibit the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, e.g., PEG coupled with dialkyloxypropyl, PEG coupled with diacylglycerol, PEG coupled with cholesterol, PEG coupled with phosphatidylethanolamine, PEG conjugated with ceramide (e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in whole for any purpose), cationic PEG lipids, and mixtures thereof. PEG may conjugate directly with lipids or may be linked to lipids via linker moieties. For example, any linker moiety suitable for conjugating PEG to lipids may be used, including non-ester-containing and ester-containing linker moieties. In preferred embodiments, non-ester-containing linker moieties are used.

[0058] The “effective dose” or “therapeutic effective dose” of a compound is the amount of the compound sufficient to provide a beneficial effect to the target to which the polypeptide or compound is administered. The “effective dose” of a delivery vehicle is the amount sufficient to effectively bind to or deliver the compound.

[0059] In particular, in the case of mRNA, the “effective dose” or “therapeutic effective dose” of a therapeutic nucleic acid, when relating to mRNA, is an amount sufficient to produce mRNA-directed expression of the amount of polypeptide or protein in which the polypeptide or protein produces the desired biological effect in the organism in which it is expressed. Suitable assays for measuring mRNA or protein expression include, but are not limited to, dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those skilled in the art.

[0060] As used herein, the term “encodes” refers to the product (e.g., protein and RNA) defined by a given sequence of nucleotides in a nucleic acid (i.e., DNA and / or RNA) during the transcription or translation of DNA or RNA, respectively. In certain embodiments, the term “encodes” refers to an RNA sequence defined by the transcription of a DNA sequence. In certain embodiments, the term “encodes” refers to an amino acid sequence (e.g., polypeptide or protein) defined by the translation of mRNA. In certain embodiments, the term “encodes” refers to an amino acid sequence defined by the transcription of DNA into mRNA and the subsequent translation of the mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may include direct transcription or translation products. In certain embodiments, the encoded product may include post-translational modifications that are understood or reasonably expected by those skilled in the art.

[0061] As used herein, the term “fetus” refers to any prenatal organism (e.g., zygote and embryo) between conception and birth in which it has developed normally in the uterus. This definition also includes prenatal organisms that are first conceived in vitro and subsequently implanted in the uterus. At birth and for approximately 28 days thereafter, the fetus is referred to herein as a “newborn” (e.g., “that newborn”).

[0062] The term "fully encapsulated" indicates that the active or therapeutic agent in the lipid particles is not significantly degraded after exposure to serum or nuclease or protease assays that significantly degrade free DNA, RNA, or proteins. In a fully encapsulated system, in a process that typically degrades 100% of the free active or therapeutic agent, preferably less than about 25% of the active or therapeutic agent in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5%. In relation to nucleoside therapeutic agents, fully encapsulated can be determined by the OLIGREEN® assay. OLIGREEN® is an ultra-sensitive fluorescent nucleic acid dye for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). "Fully encapsulated" also indicates that the lipid particles are stable in serum, i.e., they do not rapidly degrade into their constituent parts upon in vivo administration.

[0063] As used herein, the term “helper lipid” refers to a lipid that can increase the effectiveness of delivery of lipid-based particles, such as cationic lipid-based particles, to a target, preferably into cells. Helper lipids can be neutral, positively charged, or charged. In certain embodiments, helper lipids are neutral or charged. Non-limiting examples of helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0064] As used herein, the term “immune cells” refers to any cells involved in initiating an immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (e.g., dendritic cells and macrophages), monocytes, neutrophils, eosinophils, and basophils.

[0065] As used herein, the term “immunogenic fragment” refers to a portion of a polypeptide sequence that specifically binds to, or is specifically bound to, an antibody produced in an immune response.

[0066] As used herein, the term “more independently selected” refers to the group of referenced items that are the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Therefore, under this definition, “X 1 , X 2 , and X 3 The phrase "is selected independently of noble gases" is, for example, X 1 , X 2 , and X 3 X 1 , X 2 , and X 3 All are different, X 1 and X 2 Although they are the same, X 3 This includes scenarios where the sorting is different and other similar sortings.

[0067] As used herein, the term “ionizable lipid” refers to a lipid having at least one protonable or deprotonable group (e.g., a cationic lipid) that is positively charged at a pH below the physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above the physiological pH. It is understood by those skilled in the art that the addition or removal of protons depending on pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of the dominant species, and that not all lipids need to exist in charged or neutral forms. Generally, ionizable lipids have a pK of protonable groups in the range of about 4 to about 7. aIt holds.

[0068] "Isolated" means modified or removed from its natural state. Isolated nucleic acids can exist in a substantially pure form or in a non-natural environment, such as a host cell. "Isolated" nucleic acids include nucleic acid segments or fragments separated from sequences adjacent to them in their natural state, for example, DNA fragments removed from sequences that are normally adjacent to fragments in a naturally occurring genome. The term also applies to nucleic acids that are substantially purified from nucleic acids and other naturally associated components (e.g., RNA or DNA or proteins that naturally accompany nucleic acids in cells). Thus, the term includes recombinant DNA, for example, incorporated into mRNA or vectors, in self-replicating plasmids or viruses, or in prokaryotic or eukaryotic genomic DNA, or existing as a separate molecule independently of other sequences (e.g., as cDNA or genomic or cDNA fragments produced by PCR or restriction enzyme digestion). The isolated substance does not need to be absolutely pure and may include at least 50% isolated proteins, peptides, nucleic acids, or viral molecules, for example, at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0069] The term "lipids" refers to a group of organic compounds that include, but are not limited to, fatty acid esters, and are characterized by being insoluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" which include not only fats and oils but also waxes; (2) "compound lipids" which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0070] As used herein, “encapsulated in lipids” may refer to lipid particles that provide complete or partial encapsulation of an active or therapeutic agent, such as a nucleic acid (e.g., mRNA cargo). In a preferred embodiment, the nucleic acid is completely encapsulated within the lipid particle.

[0071] The term "lipid nanoparticles" refers to particles having at least one dimension on the nanometer scale (e.g., 1 to 1,000 nm) that contain one or more lipids and / or additional active substances.

[0072] The term “lipid particles” is used herein to refer to lipid formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites of interest. In the lipid particles of this disclosure, which 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 complex lipids that prevent particle aggregation, the active or therapeutic agent may be encapsulated within the lipids, thereby protecting the agent from enzymatic degradation.

[0073] As used herein, the terms “mRNA” or “messenger RNA” refer to a ribonucleic acid sequence that codes for a peptide or protein. In certain embodiments, mRNA may include a “transcript” that codes for a peptide or protein, produced by using a DNA template. 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 transcription kits are commercially available. In accordance with the present invention, mRNA can be modified by further stabilization modifications and cap formation in addition to the modifications according to the present invention.

[0074] The term "neutral amino acid" refers to any of several amino acids that have a side chain containing a substituent (e.g., H, methyl, isopropyl, isobutyl, hydroxyl, and thiol, among others) that is uncharged at a physiologically significant pH. Non-limiting examples of neutral amino acids and / or amino acids with a neutral side chain include glycine, alanine, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, proline, glutamine, phenylalanine, tyrosine, tryptophan, asparagine, and glutamine.

[0075] As used herein, the term “neonatal” refers to an infant (e.g., a human infant) having an age of 0 to approximately 28 days.

[0076] The term "neutral lipids" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0077] The term "noncationic lipid" refers not only to any amphiphilic lipid, but also to any other neutral or anionic lipid.

[0078] As used herein, the term “nucleic acid” refers to polymers comprising at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA and RNA. DNA may 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 may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include known nucleotide analogs or nucleic acids comprising modified skeletal residues or ligatures, which are synthetic, natural, and unnatural, and which have similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids, including known analogues of native nucleotides that have similar binding properties to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes explicitly indicated sequences, as well as its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (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)).

[0079] As used herein, the term “nucleic acid” includes any oligonucleotide or polynucleotide having fragments containing up to 60 nucleotides, which are generally called oligonucleotides, and longer fragments, which are called polynucleotides. In certain embodiments, the oligonucleotides of this disclosure are about 15 to about 60 nucleotides long. Nucleic acids may be administered alone in the form of lipid particles of this disclosure, or in combination with (e.g., co-administered) lipid particles of this disclosure that contain small molecules such as peptides, polypeptides, or conventional drugs. In other embodiments, nucleic acids may be administered in the form of a viral vector.

[0080] A “nucleotide” comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups. A “base” includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, as well as natural analogs and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides. Unless otherwise specified, a particular nucleic acid sequence also implicitly includes the explicitly indicated sequence, as well as its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (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)).

[0081] As used herein, the term “oil-in-water emulsion adjuvant” refers to a biocompatible formulation comprising fine droplets (e.g., micro and / or nanodroplets) of an oil (e.g., squalene) dispersed in an aqueous phase, stabilized by a surfactant or emulsifier (e.g., polysorbate 80). In certain embodiments, the oil phase comprises a metabolizable oil (e.g., squalene and / or α-tocopherol). In certain embodiments, the aqueous phase comprises water or a buffered saline solution (e.g., phosphate-buffered saline). In certain embodiments, the surfactant comprises a nonionic, cationic, or anionic active agent suitable for stabilizing the emulsion and ensuring a uniform distribution of the oil droplets. Non-limiting examples of oil-in-water emulsion adjuvants considered for use in the present invention include AddaS03® and AS03®. In certain embodiments, AddaS03® and / or AS03® contain a nanoemulsion of DL-α-tocopherol (i.e., racemic α-tocopherol) (5% v / v) in squalene oil (5% v / v) and Tween® 80 (1.8% v / v) in phosphate-buffered saline (PBS) (pH 6.8). In certain embodiments, the nanoemulsion is produced using a microfluidizer and filtered through a 0.22 μm filter to substantially reduce or remove large droplets from the final product, sterilize the final product, and / or substantially reduce or remove endotoxins from the final product.

[0082] The term "operably linked" or "operationally linked" refers to the functional linkage between a regulatory sequence and a heterogeneous nucleic acid sequence, enabling them to function as intended (e.g., resulting in the expression of the latter). This term encompasses the positioning of the regulatory region and the sequence to be transcribed within the nucleic acid in a way that influences the transcription or translation of such sequence. For example, to place a coding sequence under the control of a promoter, the translation start site of the polypeptide's translational reading frame is typically positioned 1 to about 50 nucleotides downstream of the promoter. However, the promoter can be positioned about 5,000 nucleotides upstream of the translation start site or about 2,000 nucleotides upstream of the transcription start site.

[0083] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells to which the methods described herein can be applied, whether in vitro or in situ. In certain non-limiting aspects, patient, subject, or individual is human.

[0084] 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 constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, such as oligopeptides and oligomers, which are commonly referred to in the art as peptides, and long chains, of which there are many types, which are commonly referred to in the art as proteins. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0085] As used herein, the term “pharmaceutically acceptable” means a substance such as a carrier or diluent that does not negate the biological activity or properties of a compound and is relatively non-toxic; that is, the substance can be administered to an individual without causing undesirable biological effects or adversely interacting with any of the components of the composition in which it is contained.

[0086] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in transporting or delivering a compound useful within the scope of the invention into or to a patient, so that the compound may perform its intended function. Typically, such a construct is transported or delivered from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation, including polypeptides or compounds useful within the scope of the invention, and is not harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository wax; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffering agents, e.g., magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free substances; isotonic salines; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic, suitable substances used in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any coating agent, antibacterial and antifungal agent, and absorption retarder, etc., that are physiologically acceptable to the patient and have the activity and compatibility of polypeptides or compounds useful within the scope of the present invention. Supplementary active compounds may also be incorporated into the composition. “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of polypeptides or compounds useful within the scope of the present invention.Other additional components that may be included in pharmaceutical compositions used in the implementation 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.

[0087] As used herein, the term “pneumonia” refers to an inflammatory condition of the lungs that primarily affects the small air sacs known as alveoli. Symptoms typically include any combination of a wet or dry cough, chest pain, fever, and difficulty breathing. Pneumonia is usually caused by a bacterial or viral infection. Examples of non-limited bacteria commonly isolated from subjects with pneumonia include Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, and Legionella pneumophila.

[0088] The term "polymer complex lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer complex lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are well 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, DSPE-PEG-carboxy-NHS, DOPE-PEG-carboxylic acid, and DSPE-PEG-carboxylic acid.

[0089] As used herein, the terms “prevent,” “prevent,” or “prevention” mean to avoid or delay the onset or recurrence of symptoms associated with an infection, disease, or condition in a subject who does not exhibit such symptoms at the time administration of the active substance or compound begins. Infections, diseases, conditions, and / or disorders may be used interchangeably herein.

[0090] As used herein, the term “sepsis” refers to a life-threatening condition that arises when the body’s response to an infection causes damage to its own tissues and organs. Common symptoms include, among others, fever, increased heart rate, escalating respiratory rate, confusion, cough, and painful urination. Infections leading to sepsis generally include bacterial infections, but fungal, parasitic, and / or viral infections may also lead to sepsis. In certain aspects, bacterial infections that cause and / or contribute to the development of sepsis may include Gram-positive or Gram-negative bacteria. Non-exclusive exemplary bacteria that may cause and / or contribute to the development of sepsis include, among others, Staphylococcus species, Klebsiella species, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa. As used herein, the term “neonatal sepsis” specifically refers to the occurrence of sepsis in a newborn infant (i.e., a neonatal).

[0091] In the context of amino acid sequences, the terms “sequence homology,” “identity rate (%),” “sequence identity,” “sequence identity rate,” or “identity rate” refer to a quantitative measure of the similarity between two amino acid sequences across their aligned regions.

[0092] As used herein, the term "specifically bind" or "specifically binds" means that a first molecule (e.g., an antibody) preferentially binds to a second molecule (e.g., an antigen and / or immunogenic fragment), but does not necessarily bind only to that second molecule.

[0093] As used herein, the term “substantially” means 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%, the majority or most. As used herein, the term “substantially absent” means that the composition contains about 0 wt% to about 5 wt% of a substance, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or 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 wt%, or less than about 0.001 wt%, or about 4.5 wt%. This can mean having none or a small amount, such as 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 wt%, or equal to about 0.001 wt%, or more than or less 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 wt%, or about 0.001 wt%, or less. The term "substantially contained" means that the composition contains about 0 wt% to about 5 wt% of the substance, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or 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 less than about 0.001 wt%, or about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0 This can mean having a small amount, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or equal to about 0.001 wt%, or 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 greater than or less than about 0.001 wt%, or about 0 wt%.

[0094] As used herein, the terms “to treat,” “to treat,” and “to treat” mean reducing the frequency or severity of symptoms of a disease or condition experienced by the subject by administering an active substance (e.g., a polypeptide or compound) to the subject.

[0095] As used herein, the terms "UPEC" or "UPEC25" refer to "urinary tract pathogenic Escherichia coli," which is a pathogenic strain of Escherichia coli with a diameter of approximately 25 μm to 250 μm and is commonly considered to be the cause of urinary tract infections (UTIs).

[0096] As used herein, the term “urinary tract infection” or “UTI” refers to a bacterial infection affecting the parts of the body that produce and / or transport urine (i.e., the urinary tract), including the kidneys, ureters, bladder, and / or urethra. When the lower urinary tract is affected, it is also known as a bladder infection (cystitis), and when the upper urinary tract is infected, it is also known as a kidney infection (pyelonephritis). Symptoms from a lower UTI may include painful urination, frequent urination, and the urge to urinate even when the bladder is empty, while symptoms of a kidney infection may include fever and flank pain, usually combined with the symptoms of a lower UTI. UTIs can also lead to life-threatening invasive E. coli diseases (e.g., bacteremia, sepsis, or urinary tract sepsis). The most common cause of UTIs is E. coli. However, UTIs can also be caused by other Gram-negative bacteria (e.g., Klebsiella pneumoniae and Proteus mirabilis, among others). Risk factors include female anatomical morphology, sexual activity, diabetes, obesity, and family history. UTI is more common in women than in men and occurs frequently between the ages of 16 and 35. UTI also occurs frequently in older men and women.

[0097] A “vector” is a composition of substances comprising isolated nucleic acids and / or polypeptides that can be used to deliver isolated nucleic acids and / or polypeptides into the intracellular space. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. This term is also interpreted to include non-plasmid compounds and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors. An “expression vector” refers to a vector comprising polynucleotides having an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), phagemids, BACs, YACs, and viral vectors (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0098] explanation As described elsewhere in this specification, bacterial infections, including urinary tract infections (UTIs), sepsis (e.g., neonatal sepsis), and pneumonia, infect millions of individuals each year, posing a substantial public health risk. The lack of preventive agents against such infections, often caused by Escherichia coli, Klebsiella pneumoniae, and / or P. mirabilis, thereby creates an unmet need for compositions and / or methods to treat, prevent, and / or improve bacterial infections (e.g., urinary tract infections (UTIs), sepsis, and / or neonatal sepsis, as well as pneumonia), and / or to generate broad immunity against infection by one or more pathogenic bacteria.

[0099] Accordingly, in one aspect, this disclosure relates to compositions and / or methods for treating, preventing, and / or improving bacterial infections in subjects, and / or for generating immunity against infection by one or more bacterial pathogens. In another aspect, this disclosure relates to vaccine compositions for addressing this unmet need, and methods for using the same.

[0100] In certain embodiments, the compositions and / or methods described herein are suitable for maternal immunity (i.e., use in pregnant subjects). In certain embodiments, the compositions and / or methods described herein are useful for treating, preventing, and / or improving bacterial infections in subjects and / or fetuses, or corresponding neonates, and for generating immunity against infection by one or more bacterial pathogens. In one aspect, maternal immunity utilizing the compositions and / or methods described herein is useful for reducing infant morbidity and / or mortality associated with bacterial infections (e.g., neonatal sepsis).

[0101] Vaccination has been widely used as a method to prevent viral infections (e.g., SARS-CoV-2 virus and COVID-19 infection), but to a lesser extent for the prevention of bacterial infections (e.g., Mycobacterium tuberculosis and tuberculosis infection). However, vaccination functions similarly in both cases.

[0102] In short, vaccination involves exposing a subject to one or more antigens typically present on the surface of foreign particles (e.g., bacteria), thereby stimulating an immune response. The subject's initial exposure to one or more such antigens results in a primary immune response in which B cells produce antigen-specific antibodies, ultimately leading to the destruction of the foreign particle by the host immune system (e.g., T cells). Additionally, B cells produce memory cells that facilitate a more rapid response upon repeated exposure to one or more antigens.

[0103] The surface of *E. coli* contains certain proteins that can be detected by the host immune system, including iron receptor proteins, flagellar proteins, and non-flagellar proteins (e.g., pillars, curli, and / or fimbriaes), which can result in an immune response and the removal of infectious bacteria. Non-limiting examples of proteins that can be detected by the host 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, and IatA. This includes IatB, IatC, IatD, Iha, intimin, IroN, IutA, MrpH, NaIP, OmpA, OmpT, OmpX, PapG, PapC, partactin, PNGA, SfaS, SsIE, S pili, TolC, TosA, UpaB, UpaC, YadC, Yad pili, YeeJ, YghA, YghJ, YgiL, Ygi pili, and YncE.

[0104] Accordingly, in one aspect, the Disclosure relates to compositions comprising bacterial surface proteins and / or immunogenic fragments thereof, which are suitable for inducing an immune response in a subject, thereby generating immunity in the subject against infection by one or more pathogenic bacteria, and / or for treating, preventing and / or improving bacterial infections (e.g., urinary tract infections, sepsis, or pneumonia, in particular). In certain embodiments, the Disclosure relates to maternal immunity. In other embodiments, the Disclosure relates to compositions encoding bacterial surface proteins and / or immunogenic fragments thereof, which are suitable for inducing an immune response in a subject upon delivery and translation, thereby generating immunity against infection by one or more pathogenic bacteria, and / or for treating, preventing and / or improving bacterial infections (e.g., urinary tract infections) in the subject.

[0105] 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 considered for use in lipid nanoparticles of this disclosure include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), and 2,2-dilinoleyl-4-(3-45-dimethylaminopropyl)-[1,3]-di Oxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DL (in-KDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylaminoacetoxypropane (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-dilinole Iloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distea Lyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleo Xy-N-[2(spermine-carboxamidoethyl)]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3 The formulation includes '-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or a mixture thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA ("XTC2"), or a mixture thereof.

[0106] The synthesis of cationic lipids, including DLin-K-C2-DMA ("XTC2"), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U.S. Provisional Patent 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, including DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT application number 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, including CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Application Publication 20060240554, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0107] The range of ionizable and / or cationic lipids considered 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.

[0108] Noncationic lipids In the lipid nanoparticles of this disclosure, the noncationic lipid may include, for example, one or more anionic lipids, helper lipids and / or neutral lipids. In some embodiments, the noncationic lipid includes 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.

[0109] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is known to those skilled in the art and is described in U.S. Patents 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378, all of which are incorporated herein by reference in their entirety for all purposes.

[0110] Non-cationic lipids or helper lipids include, but are not limited to, lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl This product contains sphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dierydoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), phospholipids such as lysophosphatidylcholine and dilinoleoylphosphatidylcholine, and mixtures thereof.

[0111] Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is, for example, C 10 -C 24The acyl group may be derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional examples of noncationic lipids include sterols such as cholesterol and their derivatives, such as cholestanol, cholestanone, cholestane, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0112] complex lipid In the lipid nanoparticles of this disclosure, the complex lipid that inhibits particle aggregation may include, for example, one or more of the following: polyethylene glycol (PEG) lipid conjugate, polyamide (ATTA) lipid conjugate, cationic polymer lipid conjugate (CPL), or a mixture thereof. In some embodiments, the nucleic acid-lipid particles include either a PEG lipid conjugate or an ATTA lipid conjugate.

[0113] PEG is a linear, water-soluble polymer of ethylene-PEG repeating units having two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG2000 has an average molecular weight of about 2,000 daltons, and PEG5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example: monomethoxypolyethylene glycol (MePEGOH), monomethoxypolyethylene glycol succinate (MePEGS), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycolamine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM). Other PEGs (e.g., mPEG(20kDa)amine), such as those described in U.S. Patents 6,774,180 and 7,053,150, are also useful for preparing the PEG-lipid conjugates of this disclosure. The disclosures of these patents are incorporated herein by reference in their entirety for any purpose. In addition, monomethoxypolyethylene glycol acetate (MePEG-CH2COOH) is particularly useful, for example, for preparing PEG-lipid conjugates, including PEG-DAA conjugates.

[0114] In certain embodiments, PEG-lipid conjugates or ATTA-lipid conjugates are used in conjunction with CPL. The complex lipids that inhibit particle aggregation may include, for example, PEG-lipids comprising PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEGDAA conjugates include PEG-dilauryloxypropyl (C) 12 ), PEG-Dimyristyloxypropyl (C 14 ), PEG-Dipalmityloxypropyl (C 16 ), PEG-distearyloxypropyl (C 18 ), or a mixture thereof.

[0115] Additional PEG-lipid conjugates suitable for use in this disclosure include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoyl glyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application number PCT / US08 / 88676, filed on 31 December 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Further additional PEG-lipid conjugates suitable for use in this disclosure include, but are not limited to, 1-[8'-(1,2-dimiristoyl-3-propanoxy)-carboxamide-3',6'-dioxaoctanyl]carbamoyl-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is incorporated herein by reference in its entirety for any purpose.

[0116] The PEG portion of the PEG-lipid conjugates described herein may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain examples, the PEG portion has an average molecular weight of 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 to about 2,000 daltons, etc.). In some embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0117] In addition to those mentioned above, it will be readily apparent to those skilled in the art that other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose.

[0118] In addition to the aforementioned components, the particles of this disclosure (e.g., LNPs) may further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). SPLPs and SPLP-CPLs suitable for use in this disclosure, as well as methods for producing and using SPLPs and SPLP-CPLs, are disclosed, for example, in U.S. Patent No. 6,852,334 and PCT Publication No. WO00 / 62813, the disclosure of which is incorporated herein by reference in its entirety for any purpose.

[0119] In certain examples, the complex lipids that inhibit the aggregation of particles (e.g., PEG-lipid conjugates) may constitute approximately 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 1 mol% to 1.8 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, 1.4 mol% to 1.5 mol%, or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any proportion or range thereof) of the total lipids present in the particles.

[0120] In the lipid nanoparticles of this disclosure, the active or therapeutic agent may be completely encapsulated within the lipid portion of the particle, thereby protecting the active 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 certain examples, the nucleic acid in the nucleic acid-lipid particle remains substantially undegraded for at least about 20, 30, 45, or 60 minutes after the particle is exposed to a nuclease at 37°C. In certain other examples, the nucleic acid in the nucleic acid-lipid particle remains substantially undegraded after the particle is incubated 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 or therapeutic agent (e.g., nucleic acid such as siRNA) forms a complex with the lipid portion of the particle. One of the advantages of the formulations of this disclosure is that the lipid particle composition is substantially non-toxic to mammals such as humans.

[0121] Lipid nanoparticles (LNPs) In another aspect, the Disclosure provides lipid nanoparticle (LNP) compositions comprising isolated nucleic acids of the Disclosure. In certain embodiments, the isolated nucleic acid of the Disclosure is isolated mRNA. In certain embodiments, the isolated nucleic acid of the Disclosure is isolated DNA. In certain embodiments, the LNP has a lipid-to-isolated mRNA ratio in the range of about 5:1 to about 25:1. In certain embodiments, the isolated mRNA is at least partially encapsulated within the LNP. In certain embodiments, the LNP has a lipid-to-isolated DNA ratio in the range of about 5:1 to about 25:1. In certain embodiments, the isolated DNA is at least partially encapsulated within the LNP.

[0122] In certain embodiments, the isolated polynucleotide is at least partially encapsulated within the LNP. In certain embodiments, the isolated polynucleotide is completely encapsulated within the LNP.

[0123] In another aspect, the Disclosure provides lipid nanoparticle (LNP) compositions comprising isolated polynucleotides of the Disclosure. In certain embodiments, the LNPs have a lipid-to-isolated polynucleotide ratio in the range of about 5:1 to about 25:1. In certain embodiments, isolated mRNA is completely encapsulated within the LNPs. In certain embodiments, isolated DNA is completely encapsulated within the LNPs.

[0124] In certain embodiments, LNP is (a) at least one ionizable lipid; (b) at least one helper lipid; (c) Cholesterol or modified derivatives thereof, and any combination thereof; (d) at least one complex lipid Includes.

[0125] In a particular embodiment, the ionizable lipid is at least one selected from the group consisting of 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.

[0126] In a particular embodiment, at least one ionizable lipid is 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, 48 It constitutes 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%.

[0127] In a particular embodiment, at least one ionizable lipid is 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, 48, Consists of less than 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 less than 99 mol%.

[0128] In a particular embodiment, at least one ionizable lipid is 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, 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 more than approximately 99 mol%.

[0129] In a particular embodiment, the helper lipid is at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0130] In a particular embodiment, at least one helper lipid constitutes 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.

[0131] In certain embodiments, at least one helper 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, or about 25 mol% of the LNP.

[0132] In certain embodiments, at least one helper 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.

[0133] In certain embodiments, cholesterol constitutes 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.

[0134] In certain embodiments, cholesterol constitutes less than 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 about 60 mol% of LNP.

[0135] In certain embodiments, cholesterol constitutes 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 more than approximately 60 mol% of LNP.

[0136] In a particular embodiment, the complex lipid is at least one selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DPG), mPEG-OH, mPEG-AA (mPEG-CM), mPEG-CH2CH2CH2-NH2, mPEG-DMG, mPEG-N,N-ditetradecylacetamide (ALC-0159), mPEG-DSPE, and mPEG-DPPE.

[0137] In a particular embodiment, at least one complex lipid constitutes 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.

[0138] In certain embodiments, at least one complex lipid constitutes 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 less than about 5.0 mol% of LNP.

[0139] In certain embodiments, at least one complex lipid constitutes approximately 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 more than approximately 5.0 mol% of the LNP.

[0140] nucleic acid Messenger RNA (mRNA) In one aspect, the present disclosure provides isolated messenger ribonucleic acid (mRNA) encoding the polypeptide of the present disclosure. In certain embodiments, the mRNA is codon-optimized for expression in mammals. In certain embodiments, the mammal is human. In certain embodiments, the mRNA is codon-optimized for expression in prokaryotes. In certain embodiments, the prokaryote is Escherichia coli.

[0141] In another aspect, the present disclosure provides isolated polynucleotides encoding mRNA of the present disclosure, wherein the polynucleotide comprises one or more promoter and / or polyadenylation signals functionally linked to the mRNA-coding sequence.

[0142] In certain aspects, the disclosed nucleic acid is or includes ribonucleic acid. A non-limiting ribonucleic acid is messenger RNA (mRNA). The term messenger RNA (mRNA) can refer to any ribonucleic acid that directly codes for a polypeptide of interest. Thus, the disclosed mRNA can be translated to produce one or more coded polypeptides of interest. In certain non-limiting aspects, the mRNA is produced by in vitro transcription.

[0143] mRNA can be of any appropriate length. For example, this length can vary depending on the size of the encoded polypeptide. mRNA molecules are typically 200 to 10,000 nucleotides long. In certain non-limiting embodiments, mRNA may have or not have a poly(A) tail, 5'UTR, and / or 3'UTR, and may be approximately 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, Includes 1,400, 1,500, 1,600, 1,700, 1,800, 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.

[0144] mRNA can be codon-optimized. For example, mRNA can be codon-optimized for expression in eukaryotic or prokaryotic cells (e.g., E. coli). Eukaryotic cells are cells of or derived from certain organisms, including but not limited to humans, non-human eukaryotes, animals, or mammals, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates, such as plants or mammals. Codon optimization is a genetic engineering approach that uses changes from rare codons to synonymous codons more frequently used in the cell type of interest, with the aim of increasing protein production. Generally, codon optimization involves modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon in the native sequence (e.g., approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more) with a codon more frequently or most frequently used in the host cell's gene, while maintaining the native amino acid sequence. Different species exhibit specific biases to certain codons of particular amino acids. Codon bias (differences in codon usage frequency between organisms) often correlates with messenger RNA translation efficiency, which is thought to depend, in turn, on the nature of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be specialized based on codon optimization for optimal gene expression in a given organism. Codon usage frequency tables are readily available, for example, from the "Codon Usage Database" at www.kazusa.orjp / codon / , and these tables can be adapted in several ways. See, for example, Nakamura, Y., et al., Nucl. Acids Res., 28:292 (2000).Computer algorithms for codon-optimizing specific sequences for expression in specific host cells, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In certain non-limiting embodiments, one or more codons in mRNA (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all of them) correspond to the most frequently used codons for a particular amino acid.

[0145] Typically, a disclosed, isolated messenger ribonucleic acid (mRNA) comprises a 5' untranslated region (UTR), a 3'UTR, and an open reading frame (also referred to as the coding region). In certain non-limiting embodiments, the mRNA further comprises a 5' cap or an analogue thereof, a poly(A) tail, one or more modified nucleotides, or a combination thereof. In certain embodiments, the mRNA comprises at least a 5' cap or an analogue thereof, a 5'UTR, a 3'UTR, one or more open reading frames, and a poly(A) tail. In certain embodiments, the mRNA comprises at least a 5' cap or an analogue thereof, a 5'UTR, a 3'UTR, one or more open reading frames, a poly(A) tail, and one or more modified nucleotides.

[0146] mRNA can contain different caps or cap analogues (e.g., ARCA). The mRNA body can use modified nucleosides. One or more coding sequences or open reading frames can contain various elements such as signal peptides, localization signals (e.g., NLS), inteins, and others. The mRNA structure can be manipulated to optimize GC motifs, folding, cyclization signals, and / or structured UTR elements.

[0147] 5' Cap Typically, the 5' cap of mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding proteins (CBPs), which, in turn, are responsible for mRNA stability and translational readiness in cells by associating with poly(A)-binding proteins to form mature cyclic mRNA species. Endogenous mRNA molecules may be 5'-capped, generating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to produce an N7-methyl-guanylate residue. In certain non-limiting embodiments, mRNA may contain a non-hydrolyzable cap, which can inhibit or interfere with cap removal, thereby increasing the mRNA half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, the 5' cap may contain modified nucleotides to inhibit such hydrolysis.

[0148] The 5' cap may be a single nucleotide or a series of nucleotides. For example, the cap may contain nucleotide lengths of 1 to 10, e.g., 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 1 or 2, or 10 or less. In certain non-limiting embodiments, the cap is absent.

[0149] Cap analogs have a different chemical structure from natural (e.g., endogenous, wild-type, or physiological) 5'-caps, but retain cap function. Cap analogs may be synthesized chemically (e.g., non-enzymatically) or enzymatically and / or linked to nucleic acid molecules. For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, where one guanine contains not only an N7 methyl group but also a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine(m)). 7G-3'mppp-G; sometimes equivalently referred to as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is then linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA). N7- and 3'-O-methylated guanines provide the terminal portions of the capped nucleic acid molecule. Another exemplary cap is mCAP, similar to ARCA but with a 2'-O-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G).

[0150] In certain non-limiting embodiments, the 5' cap may include an endogenous cap or cap analogue. For example, the 5' cap may include a guanine analogue. Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0151] Suitable 5' caps or analogues that can be included in mRNA are known in the art and include, but are 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, beta-S-ARCA, m7G, mCAP, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, tri-methylgranosine (TMG), nicotinamide adenine dinucleotide (NAD), cap AG, cap AU, cap GG, and 2-azido-guanosine.

[0152] Deoxyribonucleic acid (DNA) In one aspect, the disclosure provides isolated deoxyribonucleic acid (DNA) encoding the polypeptide of the disclosure. In certain embodiments, the DNA is codon-optimized for expression in mammals. In certain embodiments, the mammal is a human.

[0153] DNA can be of any appropriate length. For example, the length can vary depending on the size of the polypeptide it encodes. DNA molecules are typically 200 to 10,000 nucleotides long. In certain non-limiting embodiments, DNA may have or not have a poly(A) tail, 5'UTR, and / or 3'UTR, and may be approximately 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 , including 400, 1,500, 1,600, 1,700, 1,800, 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.

[0154] DNA can be codon-optimized. For example, DNA can be codon-optimized for expression in prokaryotic cells. In certain embodiments, prokaryotic cells are E. coli. In other embodiments, DNA can be codon-optimized for expression in eukaryotic cells. Eukaryotic cells are cells of or derived from certain organisms, including but not limited to humans, non-human eukaryotes, animals, or mammals, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates, such as plants or mammals.

[0155] In certain embodiments, DNA (e.g., codon-optimized DNA for expression in mammals) can be administered to mammals as a vector, where expression may occur in mammals (e.g., humans) to produce a therapeutic effect. In other embodiments, DNA (e.g., codon-optimized DNA for expression in prokaryotes) can be administered and / or delivered to prokaryotic cells (e.g., E. coli) for the production and isolation of its protein products.

[0156] Non-translated areas The untranslated region (UTR) is a gene region that is transcribed but not translated. Generally, the 5' UTR begins at the transcription start site and continues to the start codon, but does not contain the start codon; on the other hand, the 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. The 5' UTR may contain specific regions, such as the Kozak sequence, which is involved in the initiation of translation by ribosomes. The 5' UTR is also known to form secondary structures involved in the binding of elongation factors. UTRs can have important regulatory effects on associated mRNA, for example, affecting mRNA stability and / or translation. Generally, the translation efficiency of mRNA (including activation or inhibition of translation) can be controlled by UTRs. In certain non-limiting embodiments, the regulatory features of UTRs can be incorporated into disclosed mRNA to enhance molecular stability. In certain non-limiting embodiments, mRNA is engineered to include UTRs found in richly expressed genes to enhance stability and protein production from the mRNA. For example, mRNA expression can be enhanced by introducing 5'UTRs of mRNAs expressed in the liver, such as albumin, serum amyloid A, apolipoproteins A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII. Similarly, 5'UTRs from other tissue-specific mRNAs can be used to improve expression in muscle (MyoD, myosin, myoglobin, myogenin, 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).

[0157] Poly A tail During RNA processing, to increase stability, a long chain of adenine nucleotides called a poly(A) tail may be added to a polynucleotide, such as mRNA. Immediately after transcription, the 3' end of the transcript may be cleaved to release the 3' hydroxyl. Then, poly-A polymerase adds the adenine nucleotide chain to the RNA. This process, called polyadenylation, adds a poly(A) tail, which can be approximately 100-250 residues long.

[0158] In certain non-limiting embodiments, the poly(A) tail contains approximately 10–100, approximately 100–300, approximately 100–250, or approximately 100–200 adenines. In certain non-limiting embodiments, the poly(A) tail contains approximately 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.

[0159] Polypeptides encoded by Open Reading Frames (ORFs) 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 (e.g., proteins or peptides) from pathogenic microorganisms, such as bacteria, fungi, protozoa, or viruses. In certain non-limiting embodiments, open reading frames encode one or more proteins from a virus, or an immune response-inducing fragment or variant thereof.

[0160] A suitable variant may contain at least one point mutation or substitution (e.g., 1, 2, 3, 4, 5 or more mutations) in any amino acid residue compared to the reference. Amino acid substitutions include conserved amino acid substitutions in certain non-limiting embodiments, but non-conservative substitutions may also be used. Examples of conserved amino acid substitutions include those in which the substitution falls within one of the following five groups: (1) small aliphatic, nonpolar or micropolar residues (Ala, Ser, Thr, Pro, Gly); (2) polar, charged residues and their amides (Asp, Asn, Glu, Gln); polar, positively charged residues (His, Arg, Lys); large aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and large aromatic residues (Phe, Tyr, Trp). Examples of non-conservative amino acid substitutions include: (1) a hydrophilic residue, e.g., ceryl or threonyl, being substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (2) cysteine ​​or proline being substituted for (or by) any other residue; (3) a residue with an electronegative side chain, e.g., lysyl, arginyl, or histidyl, being substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (4) a residue with a bulky side chain, e.g., phenylalanine, being substituted for (or by) a residue without a side chain, e.g., glycine.

[0161] Other polynucleotides The polynucleotides of this disclosure may further include functional polynucleotide regions (e.g., non-coding polynucleotides). The polynucleotides may include one or more promoter and / or polyadenylation signals functionally linked to an mRNA-coding sequence. In certain non-limiting embodiments, the polynucleotide is or is contained within a plasmid. In certain non-limiting embodiments, the polynucleotide is or is contained within a vector, such as an expression vector.

[0162] Expression vectors include all known in the art that incorporate polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes), phagemids, artificial chromosomes (e.g., BAC, YAC), and viral vectors (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0163] In certain non-limiting embodiments, a polynucleotide (e.g., the portion encoding mRNA) is functionally linked to regulatory elements, such as transcriptional regulatory elements, or promoters. These transcriptional regulatory elements may be functional in either eukaryotic cells, e.g., mammalian cells, or prokaryotic cells (e.g., bacterial or archaeal cells). In certain non-limiting embodiments, a polynucleotide (e.g., the portion encoding mRNA) is functionally linked to multiple regulatory elements that enable the expression of a polynucleotide sequence encoding mRNA in either prokaryotic or eukaryotic cells. Depending on the host / vector system used, any of certain suitable transcriptional and translational regulatory elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., may be used in an expression vector (e.g., U6 promoter, HI promoter, CMV promoter, T7 promoter, SV40 promoter, bGH poly(A) signal, SV40 poly(A) signal, etc.).

[0164] Numerous vectors and expression systems are commercially available from distributors 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 based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and human immunodeficiency virus, and retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and retroviral vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, 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).

[0165] Numerous suitable expression vectors are known to those skilled in the art, and many, including pET29 (Novagen), pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pCDNA3.1, and pSVLSV40 (Pharmacia), are commercially available. However, any other vector may be used, as long as it is compatible with the host cells.

[0166] Any cell may be used in accordance with the foregoing. In certain non-limiting aspects, the cell is a prokaryotic cell (e.g., an archaeal or bacterial cell). In certain non-limiting aspects, the cell is Escherichia coli. In other forms, the cell is a eukaryotic cell. For example, the cell may be a cell of a unicellular eukaryote, a plant cell, an algal cell, or a fungal cell (e.g., a yeast cell). The cell may be a mammalian cell. Mammalian cells may be human or non-human mammalian cells, such as primates, cattle, sheep, pigs, dogs, rodents, monkeys, rats, or mouse cells.

[0167] The generation of polynucleotides can be achieved using any suitable genetic engineering technique well known in the art, including, but not limited to, standard techniques such as restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, as described, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)).

[0168] vector In another aspect, the Disclosure provides a vector comprising an isolated nucleic acid of the Disclosure. In certain embodiments, the isolated nucleic acid comprises RNA. In certain embodiments, the nucleic acid comprises DNA. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV). In certain embodiments, the AAV is AAV9.

[0169] In another aspect, the Disclosure provides a vector comprising an isolated polynucleotide of the Disclosure. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV). In certain embodiments, the AAV is AAV9.

[0170] In certain non-limiting embodiments, a vector encoding a vaccine antigen (e.g., mRNA and / or polypeptide) is a viral vector. In certain non-limiting embodiments, a viral vector is an adeno-associated virus (AAV) vector. In certain embodiments, an ITR sequence derived from AAV2, or its deletion form (ΔITR), is used for convenience and for rapid regulatory approval. However, ITRs from other AAV sources may be selected. When the ITR source is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as a pseudoform.

[0171] AAV is a non-pathogenic single-stranded DNA virus that has been actively used for several years to deliver therapeutic genes in both in vitro and in vivo systems (Choi, et al., Curr. Gene Ther., 5:299-310, (2005)). AAV belongs to the Parvoviridae family and relies on co-infection with other viruses, mainly adenoviruses, for replication. Each end of the single-stranded DNA genome contains an inverted end repeat (ITR), which is the only cis-acting element necessary 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 start sites, and differential splicing. These coding sequences are adjacent to the ITR. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), but cap expression not only produces viral capsid proteins (VP; VP1 / VP2 / VP3) that form an outer capsid shell protecting the viral genome, but also actively participates in cell binding and internalization. The viral coat is presumed to consist of 60 proteins arranged in an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3).

[0172] Recombinant AAV vectors lacking the Rep and / or Cap genes can be non-integrated. In the absence of the Rep protein, the ITR flanking transgene encoded within the rAAV can form a circular concatema that persists as an episome within the nucleus of the transduced cell. Since the recombinant episomal DNA is not integrated into the host genome, it is eventually diluted over time as the cell undergoes repeated rounds of replication. This ultimately results in the absence of the transgene and its expression.

[0173] The sequences placed between ITRs typically include a promoter, the gene of interest (e.g., encoding the disclosed mRNA), and a terminator. The promoter can be native or non-native. Often, a potent constitutively active promoter is desirable for high levels of expression of the gene of interest. Examples of promoters include, but are not limited to, viral promoters, plant promoters, and mammalian promoters. Commonly used promoters include the CMV (cytomegalovirus) promoter / enhancer, EF1a (elongation factor 1a), SV40 (monkey 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 are subject to silencing in certain cell types, so this consideration can be evaluated on a case-by-case basis.

[0174] 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 beta-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof.

[0175] Viral vectors (e.g., AAV vectors) may also have one or more restriction sites located near the promoter sequence to provide insertion of a nucleic acid sequence encoding the mRNA / protein of interest.

[0176] The AAV vectors used in the disclosed compositions and methods may include, but are not limited to, natural serotypes of AAV such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, as well as artificial variants such as AAV.rhlO, AAV.rh32 / 33, AAV.rh43, AAV.rh64Rl, rAAV2-retro, AAV-DJ, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, or other operational versions of AAV. In a 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 transduction into specific cell types in certain aspects. Typically, AAV vectors have a packaging limit of approximately 4.7 kb. AAV itself can be immunogenic, and it can be used in some situations due to its adjuvant effect.

[0177] Expression cassettes for AAV vectors typically include an AAV 5'-ITR, a coding sequence and an optional control sequence, as well as an AAV 3'-ITR. However, other arrangements of these elements may be appropriate. A shortened version of the 5'-ITR, called a ΔITR, lacking the D sequence and terminal dissociation sites (trs), has been described. In other embodiments, full-length AAV 5'-ITR and 3'-ITR are used.

[0178] An expression cassette typically includes a promoter sequence as part of an expression regulatory sequence located between, for example, a selected 5'-ITR sequence and a coding sequence.

[0179] In addition to the promoter, the expression cassette and / or vector contains one or more other suitable transcription start, termination, and enhancer sequences, efficient RNA processing signals, such as splicing and polyadenylation (poly-A) signals. These may include sequences that stabilize mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of coding products.

[0180] Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA.

[0181] Examples of suitable enhancers include, for example, α-fetoprotein enhancers, TTR minimal promoters / enhancers, and LSPs (TH-binding globulin promoters / α-microglobulin / bicinine enhancers). In certain embodiments, the expression cassette includes one or more expression enhancers. In certain embodiments, the expression cassette includes two or more expression enhancers. These enhancers may be the same or different from one another. Enhancers may exist in two copies located adjacent to each other. Alternatively, duplicate copies of an enhancer may be separated by one or more sequences. In other embodiments, the expression cassette further includes introns such as the Promega intron. Other suitable introns include those known in the art, such as those described in International Publication No. 2011 / 126808, which is incorporated herein by reference in whole for all purposes.

[0182] Recombinant AAV viral vectors are well-suited for the delivery of the coding sequences described herein. Such AAV vectors are ITRs derived from the same AAV source as the capsid. Alternatively, the AAV ITR may be derived from a different AAV source than that which supplies the capsid.

[0183] Other promoters may be selected, including tissue-specific promoters. Methods for producing and isolating AAV virus vectors suitable for delivery to a target are known in the art, for example, U.S. Patent Application Publication No. 2007 / 0036760 (February 15, 2007), U.S. Patents No. 7,790,449; No. 7,282,199; No. 7,588,772; and International Publication Nos. 2003 / 042397; No. 2005 / 033321; No. 2006 / 11689, all of which are incorporated herein by reference in their entirety. Methods for producing vectors based on the sequence of AAV8 and the AAV8 capsid are described in U.S. Patents No. 7,282,199; No. 7,790,449; and No. 8,318,480, all of which are incorporated herein by reference in their entirety. In certain embodiments, the vector is based on the AAV9 capsid.

[0184] In certain embodiments, the vector is a bacterial expression vector. In certain embodiments, the bacterial expression vector contains Escherichia coli. In certain embodiments, the bacterial expression vector contains a pET E. coli T7 expression vector. In certain embodiments, the pET E. coli T7 expression vector contains pET29b(Novagen).

[0185] composition In one aspect, this disclosure provides a polypeptide comprising o instances of B, p instances of T, and q instances of L, where, Each occurrence of B, if present, independently contains immunogenic fragments of bacterial surface proteins. Here, each example in B has a C-terminus and an N-terminus; Each appearance of T, if present, independently contains an immunogenic fragment of the iron receptor protein. Here, each instance of T has a C-terminus and an N-terminus; Each occurrence of L independently contains a polypeptide of 1 to 40 amino acids. Here, 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 by covalent peptide bonds 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 by covalent peptide bonds 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 is an integer in the range of 0 to 15; p is an integer in the range of 0 to 15; q is an integer in the range of 5 to 30; Here, o+p≧6.

[0186] In certain embodiments, the C-terminus of a polypeptide may contain one or more amino acids (e.g., a poly-His tag or HHHHHH) related to the synthesis and / or purification of the polypeptide. It is understood by those skilled in the art that the design of a poly-His tag (i.e., terminal and relatively short in length of 6-10 residues) does not confer any biological activity (e.g., immunogenicity) to the polypeptide to which they are covalently linked. It is further understood by those skilled in the art that the design of a poly-His tag minimizes and / or eliminates interference with the biological activity of the polypeptide to which they are covalently linked. As will be recognized by those skilled in the art, particularly in consideration of the disclosure of this application, this disclosure is not limited to the exemplary polypeptides described herein that include a poly-His tag (e.g., SEQ ID NO: 127-145), but further encompasses their analogues lacking poly-His functionalization (e.g., SEQ ID NO: 223-241).

[0187] In certain embodiments, the N-terminus of the polypeptides of the Disclosure may include one or more amino acids (e.g., methionine) related to the synthesis and / or purification of the polypeptide. In certain embodiments, certain exemplary polypeptides of the Disclosure include an N-terminal methionine residue. As those skilled in the art will recognize, the N-terminal methionine residue present in certain exemplary polypeptides is an artifact of the method by which the polypeptide is synthesized, where the AUG start codon (i.e., adenine-uracil-guanine) of the mRNA transcript encodes methionine. Thus, as those skilled in the art will recognize, particularly in consideration of the disclosure of this application, the Disclosure is not limited to the exemplary polypeptides described herein that include an N-terminal methionine residue (e.g., SEQ ID NO: 127-145 and SEQ ID NO: 223-241), but further includes analogues thereof lacking an N-terminal methionine residue (e.g., SEQ ID NO: 242-260). Furthermore, those skilled in the art will know of methods by which an N-terminal methionine residue is posttranslationally removed (i.e., excision or cleavage of N-terminal methionine). In certain embodiments, the excision and / or cleavage of the N-terminal methionine is catalyzed by methionine aminopeptidase (MetAP).

[0188] In certain embodiments, each occurrence of L independently comprises a polypeptide of 1 to 10 amino acids. In certain embodiments, each occurrence of L independently comprises a polypeptide of 3 to 10 amino acids. In certain embodiments, each occurrence of L independently comprises a polypeptide of 3 to 25 amino acids. In certain embodiments, multiple occurrences of L may be located adjacent to each other.

[0189] In a particular mode, each occurrence of L is The molecule independently comprises a polypeptide having an amino acid sequence selected from the group consisting of TIFF2026521465000002.tif36157. In certain embodiments, each L occurrence may be cleavable by a protease.

[0190] In certain embodiments, B does not exist. In certain embodiments, the 1 to 15 cases of B are, respectively, B 1B 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 15 It is expressed as follows. In a particular manner, each case of B is independent of 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 It is expressed as follows.

[0191] In certain embodiments, T does not exist. In certain embodiments, the 1 to 15 cases of T are, respectively, 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 It is expressed as follows. In a particular manner, each case of T is independent of T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , T13 、T 14 、 or T 15 is represented as.

[0192] In a particular embodiment, 5 to 30 instances of L are each 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 is represented as. In a particular embodiment, each instance of L is independently 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 、L28 、L 29 、 or L 30 and is represented as

[0193] In a particular embodiment, the polypeptide is 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<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0194] In a particular embodiment, 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 -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 30As shown, a polypeptide can contain one instance of L that is not adjacent to either of the two instances of B, either of the two instances of T, or any single instance of B or T (i.e., a terminal L).

[0195] This disclosure encompasses any instance of B and any instance of T in any configuration.

[0196] In certain aspects, the following non-limiting aspects: 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 -T14 -L 27 -B 14 -L 28 -T 15 -L 29 -B 15 As shown, polypeptides can be initiated by a single instance of T (i.e., the N-terminus of the polypeptide).

[0197] In certain embodiments, 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 -T13 -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, as shown in many other embodiments (all of which are considered herein) in which the arrangement of cases B and T is swapped, two cases of B and / or two cases of T may be separated by one case of L (for example, B 1 -L 1 -B 2 and / or T 1 -L 2 -T 2 ).

[0198] In a particular manner, 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 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 , L20 , L 21 , L 22 , L 23 , L 24 , L 25 , L 26 , L 27 , L 28 , L 29 , or L 30 Either of these may or may not exist, resulting in the following non-limiting aspect: 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 -L5 -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, as shown in many other embodiments of cleavage polypeptides (all of which are considered herein), there are at least five instances of L and a total of six instances of B and / or T.

[0199] 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 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 15In a particular aspect of which each identity of B is defined, any two identical or non-identical cases of B (for example, B) are represented in the following pairs of non-restrictive aspects. 1 and B 2 ) and / or any two identical or non-identical cases of T (for example, T 1 and T 2 The position of ) can be replaced: 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 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 -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 (For example, B 1 and B 2 The position has been 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 -T6 -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 およびB 1 -L 1 -T 2 -L 2 -B 2 -L 3 -T 1 -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 (For example, T 1 and T 2 (The position has been replaced.)

[0200] In a particular embodiment, the polypeptide has 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 It is a polypeptide.

[0201] In a particular embodiment, the polypeptide has 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 It is a polypeptide.

[0202] In a particular embodiment, the polypeptide has structure 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 It is a polypeptide.

[0203] In a particular embodiment, the polypeptide has structure 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 It is a polypeptide.

[0204] In a particular embodiment, the polypeptide has 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 -L24 -B 13 -L 25 -T 13 It is a polypeptide.

[0205] In a particular embodiment, the polypeptide has structure 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 It is a polypeptide.

[0206] In a particular embodiment, the polypeptide has 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 -B8 -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 It is a polypeptide.

[0207] In a particular embodiment, the polypeptide has 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 -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 -L21 -T 10 -L 22 -T 11 -L 23 -T 12 It is a polypeptide.

[0208] In a particular embodiment, the polypeptide has structure 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 It is a polypeptide.

[0209] Those skilled in the art will recognize that this disclosure is not limited to the embodiments expressly disclosed herein.

[0210] In certain embodiments, the polypeptide includes the polypeptide of formula (I), or a salt or solvate thereof: (B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 ) n (I) During the ceremony: 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 may or may not exist; B 1 B 2 , and B 3 Each occurrence, if present, independently contains immunogenic fragments of bacterial surface proteins; T 1 , T 2 , and T 3 Each occurrence, if present, independently contains an immunogenic fragment of the iron receptor protein. Here, T 1 , T 2 , and T 3 At least two of these exist, or T 1 , T 2 and T 3 There exists at least one of the above, and n is at least 2; L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each occurrence, if present, independently contains a polypeptide of 1 to 10 amino acids. Here, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 At least one of these exists, Here, B 1 B 2 B 3 , T 1 , T 2 , and T 3 Each occurrence is L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 They are separated from each other by at least one of the following; n is an integer selected from the group consisting of 1, 2, 3, 4, and 5.

[0211] In a particular embodiment, the polypeptide of formula (I) is B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 It includes n independent occurrences (i.e., 1, 2, 3, 4, or 5). In a certain non-restrictive form where n is 2, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 The first independent appearance of B is via an amide (peptide) bond. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T2 -L 4 -B 3 -L 5 -T 3 -L 6 It is covalently linked to the second independent appearance of B. In a particular aspect, the covalent linkage is B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 This includes a linear arrangement of each occurrence of B. For example, in a particular embodiment, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 Terminal B of the first appearance 1 B 2 B 3 , T 1 , T 2 , or T 3 B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 Terminal L of the second appearance 1 , L 2 , L 3 , L 4 , L 5 , or L 6 It is covalently connected to it. For example, if n is 2, then B 1 B 2 B 3 , L 1 , L 2 , L3 , L 4 , L 5 , L 6 , T 1 , T 2 , and T 3 Each of them is B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 In one non-restrictive aspect present in both occurrences, the polypeptide of formula (I) has the following structure (for example, terminal L in the first occurrence). 6 This is the terminal B of the second appearance. 1 (It is covalently bonded to it.) 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 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6

[0212] However, B is defined by the number of occurrences n. 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L6 Given the appearance of 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 Whether any one of them exists or does not is B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 The presence or absence of each base in any other occurrence of is irrelevant. For example, in one non-restrictive aspect where n is 2, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 The first appearance of B 1 , L 1 , T 1 , and L 2 only (B 1 -L 1 -T 1 -L 2 ) may include, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 The second appearance of T 1 , L 5 , and T3 only (T 1 -L 5 -T 3 ) may include. In the non-limiting embodiments described above, the polypeptide of formula (I) has the following structure: B 1 -L 1 -T 1 -L 2 -T 1 -L 5 -T 3 Therefore, in the aforementioned non-limiting embodiment, B 1 , L 1 , and L 2 B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 It exists only in the first appearance of L 5 and T 3 B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 It exists only in the second appearance of T 1 B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 It is present in both the first and second appearances.

[0213] Furthermore, the number of occurrences is defined by n in B 1 -L 1 -T1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 B in each occurrence 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 identity is independent of the others. For example, if n is 2, then T 1 B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 In the aforementioned non-limiting aspects present in both occurrences, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 T in the first appearance 1 This may contain immunogenic fragments of the iron receptor protein defined by SEQ ID NO:10, B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L6 T in the second appearance 1 This may include immunogenic fragments of the iron receptor protein, as defined by SEQ ID NO:12.

[0214] In the polypeptide of formula (I), B 1 B 2 , and B 3 Each occurrence, if present, independently includes an immunogenic fragment of a bacterial surface protein. In certain embodiments, B 1 B 2 , and / or B 3 Each occurrence may or may not exist independently. In a particular mode, B 1 B 2 , and B 3 Each occurrence may contain the same immunogenic fragment of the bacterial surface protein. In certain embodiments, B 1 B 2 , and B 3 Each appearance may contain different immunogenic fragments of bacterial surface proteins. Therefore, B 1 and B 2 Each of these has one occurrence, B 1 and B 2 In certain non-limiting aspects, both bacterial surface proteins may correspond to SEQ ID NO:1, B 1 The immunogenic fragment of bacterial surface protein in B may contain SEQ ID NO:16. 2 The immunogenic fragment of the bacterial surface protein in this case may contain SEQ ID NO:17. Additionally, the identity of the bacterial surface protein is B 1 B 2 , and B 3 It does not need to be the same in each occurrence. In a particular manner, B 1 B 2 , and B 3 Each appearance may contain immunogenic fragments of different bacterial surface proteins. For example, B 1 and B 2 In a certain non-limiting mode in which each of the single occurrences of exists, B 1The bacterial surface protein may contain SEQ ID NO:1, B 2 The bacterial surface protein may contain SEQ ID NO:2. Similarly, the polypeptide of formula (I) may contain B 1 In a certain non-limiting aspect that includes two occurrences of B, 1 The first appearance may include bacterial surface protein SEQ ID NO:1, B 1 The second appearance may involve bacterial surface protein SEQ ID NO:2. Alternatively, the polypeptide of formula (I) may be B 1 In a certain non-limiting aspect that includes two occurrences of B, 1 Both the first and second appearances may include bacterial surface protein SEQ ID NO:1.

[0215] In the polypeptide of formula (I), T 1 , T 2 , and T 3 Each occurrence, if present, independently contains an immunogenic fragment of the iron receptor protein. In certain embodiments, T 1 , T 2 , and / or T 3 Each occurrence may or may not exist independently. In a particular mode, T 1 , T 2 , and T 3 Each occurrence may contain the same immunogenic fragment of the iron receptor protein. In certain embodiments, T 1 , T 2 , and T 3 Each appearance may contain different immunogenic fragments of the iron receptor protein. Therefore, T 1 and T 2 Each of these has one occurrence, T 1 and T 2 In a certain non-limiting aspect, both iron receptor proteins can correspond to SEQ ID NO:10, T 1 The immunogenic fragment of the iron receptor protein in T may contain SEQ ID NO:42. 2The immunogenic fragment of the iron receptor protein in this case may contain SEQ ID NO:43. Additionally, the identity of the iron receptor protein is T 1 , T 2 , and T 3 It does not need to be the same in each occurrence. In a particular mode, T 1 , T 2 , and T 3 Each appearance may contain immunogenic fragments of different iron receptor proteins. For example, T 1 and T 2 In a certain non-limiting mode in which each of the single occurrences of exists, T 1 The iron receptor protein may contain SEQ ID NO:10, T 2 The iron receptor protein may contain SEQ ID NO:12. Similarly, the polypeptide of formula (I) may contain T 1 In a certain non-limiting aspect that includes two occurrences of T, 1 The first appearance may include the iron receptor protein SEQ ID NO:10, T 1 The second appearance may involve the iron receptor protein SEQ ID NO:12. Alternatively, the polypeptide of formula (I) may be T 1 In a certain non-limiting aspect that includes two occurrences of T, 1 Both the first and second appearances may involve the iron receptor protein SEQ ID NO:10.

[0216] In the polypeptide of formula (I), L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each occurrence, if present, independently comprises a polypeptide of 1 to 10 amino acids. In certain embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and / or L 6 Each occurrence of may or may not exist independently. In a particular mode, L 1 , L 2, L 3 , L 4 , L 5 , and / or L 6 Each occurrence may contain a different 1-10 amino acid polypeptide. Therefore, L 1 and L 2 In a certain non-limiting mode in which each of the single occurrences of exists, L 1 This may include SEQ ID NO:124, L 2 This may include SEQ ID NO:125. Or, L 1 and L 2 In a certain non-limiting mode in which each of the single occurrences of exists, L 1 and L 2 Both can contain SEQ ID NO:124. Similarly, if n is 2 and the polypeptide of formula (I) is L 1 In a certain non-limiting aspect that includes two occurrences of L, 1 The first occurrence may include SEQ ID NO:124, L 1 The second occurrence may include SEQ ID NO:125. Alternatively, n is 2 and the polypeptide of formula (I) is L 1 In a certain non-limiting aspect that includes two occurrences of L, 1 Both the first and second occurrences may include SEQ ID NO:124.

[0217] In a particular manner, B 1 B 2 , and B 3 At least one of these exists.

[0218] In a particular embodiment, each bacterial surface protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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.

[0219] In a particular embodiment, each immunogenic fragment of the bacterial surface protein is 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 It shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of NO:41.

[0220] In a particular embodiment, each immunogenic fragment of a bacterial surface protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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: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.

[0221] In a particular manner, B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B10 B 11 B 12 B 13 B 14 , and B 15 Each of these, if present, is 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 It shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of NO:41.

[0222] In a particular embodiment, each iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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.

[0223] In a particular embodiment, each immunogenic fragment of the iron receptor protein is 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, SEQIt shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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.

[0224] In a particular embodiment, each immunogenic fragment of the iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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.

[0225] In a particular manner, 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 these, if present, is 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 IDIt shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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.

[0226] In a particular manner, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these independently comprises a polypeptide in which, if present, the amino acid residue side chain contains a neutral (i.e., uncharged) substituent.

[0227] In a particular manner, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these independently comprises a 4-amino acid polypeptide, if present. In a particular embodiment, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these independently comprises a 5-amino acid polypeptide, if present. In a particular embodiment, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these independently contains a 6-amino acid polypeptide, if present.

[0228] In a particular manner, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these, if present, It is independently selected from the group consisting of TIFF2026521465000003.tif28157.

[0229] In a particular mode, n is 1. In a particular mode, n is 2. In a particular mode, n is 3. In a particular mode, n is 4. In a particular mode, n is 5.

[0230] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide selected from the group consisting of SEQ ID NO:130 and SEQ ID NO:129.

[0231] In a particular aspect, the polypeptides are 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, 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 polypeptides selected from the group consisting of NO:260 share at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

[0232] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:135.

[0233] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0234] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0235] In certain embodiments, polypeptides are (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 -T4 -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 -T2 -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 -L6 -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 ;および (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 -L15 -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 comprising a polypeptide selected from the group consisting of the following, or a salt or solvate thereof; During the ceremony: Each occurrence of B, if present, independently contains immunogenic fragments of bacterial surface proteins. Here, each example in B has a C-terminus and an N-terminus; Each appearance of T independently contains an immunogenic fragment of the iron receptor protein. Here, each instance of T has a C-terminus and an N-terminus; Each occurrence of L independently contains a polypeptide of 1 to 10 amino acids. Here, 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 by covalent peptide bonds 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 by covalent peptide bonds 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.

[0236] In a particular embodiment, each immunogenic fragment of the bacterial surface protein is 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 It shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of NO:41.

[0237] In a particular embodiment, each immunogenic fragment of a bacterial surface protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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: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.

[0238] In a particular embodiment, each iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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.

[0239] In a particular embodiment, each immunogenic fragment of the iron receptor protein is 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 IDIt shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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.

[0240] In a particular embodiment, each immunogenic fragment of the iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides independently selected from the group consisting of 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.

[0241] In a particular manner, 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 25Each of these independently comprises a polypeptide in which, if present, each amino acid residue side chain contains a neutral (i.e., uncharged) substituent.

[0242] In a particular manner, 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 independently contains a polypeptide of 4-6 amino acids, if present.

[0243] In a particular manner, 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 25Each of these, if present, is GSGS (SEQ ID NO: 124), GPGP (SEQ ID NO: 125), LLSVGG (SEQ ID NO: 126), (SGSG) 1-2 It is independently selected from the group consisting of (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).

[0244] In a particular aspect, the polypeptides are 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: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 The polypeptides share at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides selected from the group consisting of NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, and SEQ ID NO:260.

[0245] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0246] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0247] method In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering to a subject at least one polypeptide of the Disclosure.

[0248] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering to a subject at least one isolated mRNA of the Disclosure.

[0249] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering to a subject at least one isolated DNA of the Disclosure.

[0250] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In certain embodiments, the method includes the step of administering to a subject at least one isolated polynucleotide of the Disclosure.

[0251] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering at least one vector of the Disclosure to a subject.

[0252] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering at least one LNP of the Disclosure to a subject.

[0253] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In certain embodiments, the method includes the step of administering to a subject at least one pharmaceutical composition of the Disclosure.

[0254] In another aspect, the Disclosure provides a method for treating, preventing, and / or improving a bacterial infection in a subject where such treatment is needed. In a particular embodiment, the method includes the step of administering to a subject at least one vaccine composition of the Disclosure.

[0255] In certain aspects, a bacterial infection is a urinary tract infection. In certain aspects, a bacterial infection includes a urinary tract infection and sepsis. In certain aspects, a bacterial infection is sepsis. In certain aspects, sepsis is neonatal sepsis. In certain aspects, a bacterial infection is pneumonia.

[0256] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes the step of administering to a subject at least one polypeptide of the Disclosure.

[0257] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In certain embodiments, the method includes administering to a subject at least one isolated mRNA of the Disclosure. In certain embodiments, the method includes administering to a subject at least one isolated DNA of the Disclosure.

[0258] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes administering to a subject at least one isolated polynucleotide of the Disclosure.

[0259] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes the step of administering at least one vector of the Disclosure to the subject.

[0260] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes administering at least one LNP of the Disclosure to the subject.

[0261] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes the step of administering to a subject at least one pharmaceutical composition of the Disclosure.

[0262] In another aspect, the Disclosure provides a method for generating immunity in a subject to infection by one or more pathogenic bacteria. In a particular embodiment, the method includes the step of administering to a subject at least one vaccine composition of the Disclosure.

[0263] In certain aspects, one or more pathogenic bacteria include Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Shigella dysenteriae, Salmonella enterica, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, and Legionella pneumophila, and Streptococcus pyogenes. It includes at least one selected from the group consisting of Pseudomonas aeruginosa and Salmonella bongori.

[0264] In certain aspects, immunity prevents bacterial infections.

[0265] In certain aspects, a bacterial infection is a urinary tract infection (UTI). In certain aspects, a bacterial infection includes urinary tract infections and sepsis. In certain aspects, a bacterial infection is sepsis. In certain aspects, sepsis is neonatal sepsis. In certain aspects, a bacterial infection is pneumonia.

[0266] In certain embodiments, the subject is administered one or more additional doses of the polypeptides disclosed therein after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the polypeptides disclosed therein after the initial dose.

[0267] In certain embodiments, a subject is administered one or more additional doses of the isolated mRNA of the Disclosure after an initial dose. In certain embodiments, a subject is administered a second, third, or fourth dose of the isolated mRNA of the Disclosure after an initial dose.

[0268] In certain embodiments, the subject is administered one or more additional doses of the isolated polynucleotides of the Disclosure after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the isolated polynucleotides of the Disclosure after the initial dose.

[0269] In certain embodiments, the subject is administered one or more additional doses of the vector disclosed therein after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the vector disclosed therein after the initial dose.

[0270] In certain embodiments, the subject is administered one or more additional doses of the LNP disclosed herein after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the LNP disclosed herein after the initial dose.

[0271] In certain embodiments, the subject is administered one or more additional doses of the pharmaceutical composition of the Disclosure after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the pharmaceutical composition of the Disclosure after the initial dose.

[0272] In certain embodiments, the subject is administered one or more additional doses of the vaccine composition of the Disclosure after the initial dose. In certain embodiments, the subject is administered a second, third, or fourth dose of the vaccine composition of the Disclosure after the initial dose.

[0273] In certain embodiments, each additional dose is administered at intervals ranging from approximately 1 to approximately 365 days. In a particular embodiment, the interval between doses is 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 ,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,1 42, 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, 204, 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, 315, 3 The group is selected from intervals of 16, 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.

[0274] In certain contexts, the subject is a mammal. In certain contexts, the mammal is a human.

[0275] In certain aspects, administration includes maternal immunity.

[0276] In certain circumstances, the subject is pregnant with a fetus.

[0277] In certain embodiments, bacterial infections are treated, prevented, and / or improved in at least one of the subject and the fetus, or in the newborn. In certain embodiments, bacterial infections are treated, prevented, and / or improved in both the subject and the fetus, or in the newborn. In certain embodiments, bacterial infections are treated, prevented, and / or improved in the newborn or infant after the birth of the fetus.

[0278] In certain embodiments, immunity to infection by one or more pathogenic bacteria is generated in at least one of the subject and the fetus, or in the newborn. In certain embodiments, immunity to infection by one or more pathogenic bacteria is generated in both the subject and the fetus, or in the newborn. In certain embodiments, immunity to infection by one or more pathogenic bacteria is generated in the newborn or infant after the birth of the fetus.

[0279] Pharmaceutical compositions and formulations In another aspect, the Disclosure provides a vaccine composition comprising the polypeptide of the Disclosure and at least one pharmaceutically acceptable excipient.

[0280] In another aspect, the Disclosure provides a vaccine composition comprising at least one LNP of the Disclosure and a pharmaceutically acceptable excipient.

[0281] In a particular aspect, the polypeptides are 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: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 The polypeptides share at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with polypeptides selected from the group consisting of NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, and SEQ ID NO:260.

[0282] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225.

[0283] In certain embodiments, the polypeptide shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226.

[0284] In certain embodiments, the vaccine composition further comprises an adjuvant. In certain embodiments, the adjuvant is aluminum hydroxide (AlOH), double mutant thermolabic toxin (dmLt), CpG, AddaS03 (trademark), AddaVax (trademark), MF59 (registered trademark), W 20 5EC and / or W 805EC (NanoBio®), Matrix-M®, Quil-A®, Monophosphoryl Lipid A (MPLA; e.g., PHAD® (synthetic) or E. coli-derived), Monophosphoryl Lipid A Salmonella Minnesota (MPLA-SM; e.g., MPLA-SM VacciGrade®), Alhydrogel®, Aluminum Phosphate (ALPO4; e.g., Imject® 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 Compositions comprising cCpG, one or more cationic liposomes and / or one or more immunomodulatory agents (e.g., CAF01), AS01b, gardisil adjuvant, AS02, AS03®, AS04, GLA-SE (i.e., glucopyranosyllipid adjuvant);Synthetic TLR4 agonist), IC31 (registered trademark), Montanide (trademark) ISA-51 VG, Montamide (trademark) ISA-70 VG, immunostimulatory complex (ISCOM) (e.g., H5N1), cholera toxin (CT), cholera toxin subunit B (CTB), thermolabile enterotoxin subunit B (LTB), imiquimod, reciquimod (R848), double-stranded RNA (dsRNA) hairpin (e.g., polyinosine polycytidic acid (i.e., poly-I:C)), Riboxxim®, rintatolimod (Ampligen®), poly-ICLC (e.g., Hiltonol®), AF03, polyphosphazene (PCEP and / or PCPP), cyclic diguanylic acid monophosphate (c-di-GMP or CDG), CAF01, CAF05, CAF09, trehalose dibehenate (TDB), furfurman, curdlan, retinoic acid, protrin, proteosome (ID At least one selected from the group consisting of Biomedical, Endocine (trademark) (L3B), N30A, N30ASq, NanoVax (NanoBio (registered trademark)), inulin, delta-inulin (e.g., Advax (trademark)), Advax-CpG55.2, polyactin and / or alpha-polyactin, mannatide, β-glucosylceramide, α-galactosylceramide, cholesterol, IFN-α, flagellin, MALT chemokines (e.g., CCL25, CCL27, and CCL28), glucosylpyranosyllipid adjuvants, ADP-ribosylated toxins, trehalose, and hydroxypropyl methacrylamide (HPMA) copolymers.

[0285] In certain embodiments, the adjuvant contains aluminum hydroxide. In certain embodiments, the adjuvant contains dmLT. In certain embodiments, the adjuvant contains CpG. In certain embodiments, the adjuvant contains AddaS03 (trademark). In certain embodiments, the adjuvant contains AS03 (registered trademark).

[0286] In certain embodiments, the adjuvant includes AlOH and CpG. In certain embodiments, the adjuvant includes AddaS03® and / or AS03® and CpG. In certain embodiments, the adjuvant includes AddaS03® and CpG. In certain embodiments, the adjuvant includes AS03® and CpG.

[0287] In certain embodiments, the adjuvant in the composition of the Disclosure suitable for the treatment and / or prevention of urinary tract infections in a subject includes AddaS03® and / or AS03® and CpG. In certain embodiments, the adjuvant in the composition of the Disclosure suitable for the treatment and / or prevention of urinary tract infections in a subject includes AddaS03® and CpG. In certain embodiments, the adjuvant in the composition of the Disclosure suitable for the treatment and / or prevention of urinary tract infections in a subject includes AS03® and CpG.

[0288] In certain embodiments, the adjuvant in the composition of the disclosure suitable for the treatment and / or prevention of neonatal sepsis in a subject comprises AlOH. In certain embodiments, the adjuvant in the composition of the disclosure suitable for the treatment and / or prevention of neonatal sepsis in a subject comprises AlOH and CpG.

[0289] In certain embodiments, the compositions of the present disclosure are approximately 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, Contains 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 100 μg of polypeptide.

[0290] In certain embodiments, the compositions of the Disclosure contain approximately 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 approximately 500 μg of polypeptide.

[0291] In certain embodiments, the vaccine composition has a polypeptide concentration of 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.

[0292] In certain embodiments, the vaccine composition is administered in a volume selected from the group consisting of approximately 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 approximately 500 μL.

[0293] In certain embodiments, the compositions of the Disclosure are approximately 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, 3000, 3 Contains 100, 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 of CpG.

[0294] In certain embodiments, the compositions of the present disclosure contain about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or about 750 μg of AlOH.

[0295] In certain embodiments, the compositions of the Disclosure contain 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 of dmLT.

[0296] In certain embodiments, polypeptides and CpGs are present in pharmaceutical or vaccine compositions in approximately 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, 50:5750, 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, 25:5 500, 30:5500, 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, 85:5 250, 90:5250, 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, 50:4 750, 55:4750, 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, 30:4500,35:4500、40:4500、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: 1000, 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, 65:250, 70: It has mass ratios of 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 (polypeptide:CpG).

[0297] In certain embodiments, the pharmaceutical composition contains about 0.4% (w / w%) AlOH. In certain embodiments, the polypeptide and AlOH are present in the pharmaceutical or vaccine composition in proportions of 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, 5 5:700, 60:700, 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, 40:600, 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, 20:500, 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, 10:450, 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, 95:450, 100:450, 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, 95:4 00, 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, 10:250, 15:250 It has a mass ratio of 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 (polypeptide:AlOH).

[0298] In certain embodiments, the pharmaceutical composition contains approximately 50% AddaS03™.

[0299] In certain embodiments, polypeptides and dmLTs are present in pharmaceutical or vaccine compositions in ratios of approximately 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, 30:3, 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, 85:1 5, 90:15, 95:15, 100:15, 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,Alternatively, it has a mass ratio of approximately 100:25 (polypeptide:dmLT).

[0300] In certain embodiments, the oil-in-water emulsion adjuvant contains 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 certain embodiments, the oil-in-water emulsion adjuvant contains about 2.43 mg of polysorbate 80. In certain embodiments, the oil-in-water emulsion adjuvant contains about 4.86 mg of polysorbate 80. In certain embodiments, the oil-in-water emulsion adjuvant contains about 9.72 mg of polysorbate 80.

[0301] In certain embodiments, the oil-in-water emulsion adjuvant is approximately 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 Contains 0.0, 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 certain embodiments, the oil-in-water emulsion adjuvant contains about 5.35 mg of squalene. In certain embodiments, the oil-in-water emulsion adjuvant contains about 10.69 mg of squalene. In certain embodiments, the oil-in-water emulsion adjuvant contains about 21.38 mg of squalene.

[0302] In certain embodiments, the oil-in-water emulsion adjuvant is approximately 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. Contains 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 certain embodiments, the oil-in-water emulsion adjuvant contains about 5.93 mg of α-tocopherol. In certain embodiments, the oil-in-water emulsion adjuvant contains about 11.86 mg of α-tocopherol. In certain embodiments, the oil-in-water emulsion adjuvant contains about 23.72 mg of α-tocopherol.

[0303] In certain embodiments, the oil-in-water emulsion adjuvant constitutes 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 of the Disclosure.

[0304] In certain aspects, this disclosure is, (a) A polypeptide that shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225, wherein the vaccine composition contains about 10 μg to about 100 μg of the polypeptide; (b) α-tocopherol, which constitutes approximately 2.5% (v / v) of the vaccine composition; (c) Squalene, which constitutes approximately 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, which makes up about 0.9% (v / v) of the vaccine composition Provide a vaccine composition containing; Here, the polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0305] In certain aspects, this disclosure is, (a) A polypeptide that shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:225, wherein the vaccine composition contains about 10 μg to about 100 μg of the polypeptide; (b) α-tocopherol, which constitutes approximately 2.5% (v / v) of the vaccine composition; (c) Squalene, which makes up about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80, which constitutes approximately 0.9% (v / v) of the vaccine composition; (e) The vaccine contains approximately 100 μg to 6000 μg of CpG. Provide a vaccine composition containing; Here, polypeptides, α-tocopherol, squalene, polysorbate, and CpG are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0306] In certain aspects, this disclosure is, (a) A polypeptide that shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226, wherein the vaccine composition contains about 10 μg to about 100 μg of the polypeptide; (b) α-tocopherol, which constitutes approximately 2.5% (v / v) of the vaccine composition; (c) Squalene, which constitutes approximately 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, which makes up about 0.9% (v / v) of the vaccine composition Provide a vaccine composition containing; Here, the polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0307] In certain aspects, this disclosure is, (a) A polypeptide that shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with SEQ ID NO:226, wherein the vaccine composition contains about 10 μg to about 100 μg of the polypeptide; (b) α-tocopherol, which constitutes approximately 2.5% (v / v) of the vaccine composition; (c) Squalene, which makes up about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80, which constitutes approximately 0.9% (v / v) of the vaccine composition; (e) The vaccine contains approximately 100 μg to 6000 μg of CpG. Provide a vaccine composition containing; Here, polypeptides, α-tocopherol, squalene, polysorbate, and CpG are dissolved or suspended in phosphate-buffered saline (PBS) solution.

[0308] In another aspect, the Disclosure provides a pharmaceutical composition comprising at least one LNP of the Disclosure and a pharmaceutically acceptable excipient.

[0309] The present invention provides a pharmaceutical composition comprising at least one polypeptide or compound thereof, or a salt or solvate thereof, useful for carrying out the methods of the present invention. Such a pharmaceutical composition may consist of at least one polypeptide or compound thereof, or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one polypeptide or compound thereof, or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional components, or any combination thereof. At least one polypeptide or compound thereof may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, for example, in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0310] In certain embodiments, a pharmaceutical composition useful for carrying 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, a pharmaceutical composition useful for carrying out the present invention may be administered to deliver a dose of 100 ng / kg / day to 1 mg / kg / day.

[0311] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical composition of the present invention vary depending on the identity, size, and condition of the target being treated, as well as the route through which the composition is to be administered. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0312] Pharmaceutical compositions useful in the methods of the present invention can be appropriately developed for nasal, inhalation, oral, rectal, vaginal, thoracic, abdominal, parenteral, topical, transdermal, pulmonary, intranasal, oral, ocular, epidural, subarachnoid, intravenous, or other routes of administration. Compositions useful in the methods of the present invention can be administered directly to the brain, brainstem, or any other part of the central nervous system of mammals or birds. Other formulations to be considered include projected nanoparticles, microspheres, liposome preparations, coated particles, polymer conjugates, resealed erythrocytes containing active ingredients, and immunology-based formulations.

[0313] In certain embodiments, the compositions of the present invention are part of a pharmaceutical matrix that enables the manipulation of insoluble substances and the improvement, control, or development of sustained-release products and the production of homogeneous compositions. For example, the pharmaceutical matrix may be prepared using thermal melt extrusion, solid solutions, solid dispersions, grinding techniques, molecular complexes (e.g., cyclodextrins, etc.), microparticles, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

[0314] The route of administration is readily apparent to those skilled in the art and depends on any number of factors, including the type and severity of the disease being treated, and the type and age of the animal or human patient being treated.

[0315] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or to be developed in the art of pharmacology and pharmaceuticals. Generally, such preparation methods include the step of associating the active ingredient with a carrier or one or more other minor components, and then, if necessary or desirable, forming or packaging the product into desired single-dose or multi-dose units.

[0316] As used herein, “unit dose” refers to an individual amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the drug dose of the active ingredient administered to the subject, or a convenient fraction of such a drug dose, such as half or one-third of that dose.

[0317] The descriptions of pharmaceutical compositions provided herein are primarily directed toward pharmaceutical compositions suitable for ethical administration to humans, but it is understood by those skilled in the art that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and skilled veterinary pharmacologists can design and perform such modifications, if any, simply in routine experiments. The subjects for which administration of the pharmaceutical compositions of the present invention is considered include, but are not limited to, humans and other primates, and mammals including commercially significant mammals such as cattle, pigs, horses, sheep, cats, dogs, and rodents.

[0318] In certain embodiments, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain 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, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatin (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions, 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).

[0319] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), recombinant human albumin, solubilized gelatin, a suitable mixture thereof, and vegetable oil. Adequate fluidity may be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, isotonic agents, such as sugars, sodium chloride, or polyalcohols, such as mannitol and sorbitol, are included in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate or gelatin.

[0320] The formulations may be used in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, intravenous, subcutaneous, transdermal, enteral, or any other suitable mode of administration known in the art. The pharmaceuticals may be sterilized and, if desired, mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavoring agents, and / or fragrance agents. They may also be combined with other activators, such as other analgesics, anxiolytics, or hypnotics, if desired. As used herein, “additional components” include, but are not limited to, one or more components that can be used as pharmaceutically acceptable carriers.

[0321] The compositions of the present invention may contain a preservative in an amount of about 0.005% to 2.0% of the total weight of the composition. The preservative is used to prevent deterioration when exposed to contaminants in the environment. Examples of preservatives useful in the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, and any combination thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0322] The composition may contain antioxidants and chelating agents that inhibit the degradation of polypeptides or compounds. For some polypeptides or compounds, the antioxidants are BHT, BHA, alpha-tocopherol, and ascorbic acid in an exemplary range of about 0.01% to 0.3% by weight relative to the total weight of the composition, or BHT in an amount ranging from 0.03% to 0.1% by weight. Chelating agents may be present in an amount ranging from 0.01% to 0.5% by weight relative to the total weight of the composition. Exemplary chelating agents include EDTA salts (e.g., disodium EDTA) and citric acid in a weight range of about 0.01% to 0.20% by weight, or 0.02% to 0.10% by weight relative to the total weight of the composition. Chelating agents are useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. BHT and disodium edetate are exemplary antioxidants and chelating agents, respectively, but for some polypeptides or compounds, other suitable and equivalent antioxidants and chelating agents may be substituted, as is known to those skilled in the art.

[0323] Liquid suspensions may be prepared using conventional methods to achieve the suspension of active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as pea, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further include, but are not limited to, one or more additional components, such as suspending agents, dispersing or wetting agents, emulsifiers, lubricants, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions may further include 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 carboxymethylcellulose, methylcellulose, and hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, natural phosphatides, such as lecithin, condensation products of alkylene oxide fatty acids and long-chain aliphatic alcohols and partial esters derived from fatty acids and hexitol, or partial esters derived from fatty acids and anhydrous hexitol (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene 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, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, or n-propyl parahydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0324] Solutions of active ingredients in aqueous or oily solvents may be prepared substantially similarly to liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, “oily” liquids contain carbon-containing liquid molecules and exhibit properties of lower polarity than water. Solutions of the pharmaceutical compositions of the present invention may contain each of the components described in relation to suspensions, and it is understood that suspending agents do not necessarily aid in the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic salines. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as pea, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0325] Powder and granular formulations of the pharmaceuticals of the present invention may be prepared using known methods. Such formulations may be administered directly to a subject or used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding an aqueous or oily vehicle thereto. Each of these formulations may further contain one or more of the following: dispersing or wetting agents, suspending agents, ionic and nonionic surfactants, and preservatives. Additional excipients, such as bulking agents and sweeteners, flavoring agents, or coloring agents, may also be included in these formulations.

[0326] The pharmaceutical compositions of the present invention may also be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase may be a vegetable oil, e.g., olive or pea oil, a mineral oil, e.g., liquid paraffin, or a combination thereof. Such compositions may further contain one or more emulsifiers, e.g., natural gums such as gum arabic or tragacanth gum, natural phosphatides, e.g., soy or lecithin phosphatides, esters or partial esters derived from a combination of fatty acids and hexitol anhydride, e.g., sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, e.g., polyoxyethylene sorbitan monooleate. These emulsions may also contain additional components, e.g., sweeteners or flavorings.

[0327] Methods for impregnating or coating substances with chemical compositions are known in the art and include, but are not limited to, methods for depositing or bonding chemical compositions to surfaces, methods for incorporating chemical compositions into the structure of a substance during its synthesis (i.e., using, for example, physiologically degradable substances), and methods for absorbing aqueous or oily solutions or suspensions into absorbent substances, followed by drying or not drying. Methods for mixing components include physical grinding, the use of pellets in solid and suspension formulations, and mixing in transdermal patches, as known to those skilled in the art.

[0328] Administration / Medication In clinical settings, the delivery systems for the compositions described herein can be introduced to the target by any of several methods, each well known in the art. For example, the pharmaceutical formulations of the compositions can be administered by intravenous injection.

[0329] The administration regimen may affect what constitutes the effective dose. Therapeutic formulations may be administered to a subject either before or after the onset of symptoms associated with a disease or condition (i.e., to prevent, treat, and / or improve an infection or its symptoms in the subject, and / or to prevent a recurrence of an infection or its symptoms in the subject).

[0330] The administration of the compositions of the present invention to subjects, preferably mammals, more preferably humans and / or laboratory mammals (e.g., rodents), may be carried out using known procedures in doses and durations effective to treat, prevent, and / or improve a disease or condition in the subject. The effective amount of the composition required to achieve a therapeutic effect may vary depending on the time of administration; the duration of administration; other drugs, polypeptides, compounds, or substances used in combination with the composition; the condition of the disease or disorder; factors such as the age, sex, weight, condition, overall health, and medical history of the subject being treated; and similar factors well known in the medical field. The dosing regimen may be adjusted to provide an optimal therapeutic response. For example, the dose may be annual or semi-annually, or the dose may be proportionally reduced if indicated by an emergency in the treatment situation. Those skilled in the art can study the relevant factors and determine the effective amount of the composition without excessive experimentation. The formulations may be used in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. Pharmaceuticals are sterilized and, if desired, may be mixed with adjuvants such as lubricants, preservatives, stabilizers, humectants, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances. They may also be combined with other active ingredients, such as other analgesics, if desired.

[0331] Any route of administration of the compositions of the present invention may include oral, nasal, rectal, vaginal, parenteral, oral, sublingual, or topical administration. Polypeptides for use in the present invention may be formulated for administration by any suitable route, e.g., oral or parenteral, percutaneous, transmucosal (e.g., sublingual, tongue, (trans)oral, (trans)urethral, ​​intravaginal (e.g., transvaginal and perival), nasal (intra) and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, subarachnoid, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0332] In a particular preferred embodiment, the composition of the present invention is administered intramuscularly.

[0333] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma, lozenges, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, and compositions and formulations for intravesical administration. It should be understood that formulations and compositions useful for the present invention are not limited to the specific formulations and compositions described herein.

[0334] Oral administration Tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable for oral administration. Compositions intended for oral use may be prepared by any method known in the art, and such compositions may contain one or more active ingredients selected from the group consisting of inert and non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as corn starch; binders such as starch; and lubricants such as magnesium stearate. Tablets may be uncoated, or they may be coated by known techniques for appearance or to delay the release of the active ingredient. Formulations for oral use may also be presented as gelatin hard capsules, in which the active ingredient is mixed with an inert diluent.

[0335] For oral administration, the polypeptides or compounds of the present invention may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as binders (e.g., polyvinylpyrrolidone, hydroxypropylcellulose, or hydroxypropylmethylcellulose); fillers (e.g., corn starch, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets may be coated using appropriate methods and coating materials such as the OPADRY® film coating system available from Colorcon, West Point, Pa. (e.g., OPADRY® OY type, OYC type, Organic Enteric OY-P type, Aqueous Enteric OY-A type, OY-PM type, and OPADRY® White, 32K18400). Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fat); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily ester, or ethyl alcohol); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, or sorbic acid).

[0336] Parenteral administration For parenteral administration, the polypeptides or compounds of the present invention may be formulated for injection or infusion, for example, for intravenous, intramuscular, or subcutaneous injection or infusion, or for administration in bolus doses and / or continuous infusions. Optionally, suspensions, solutions, or emulsions in oily or aqueous vehicles containing other formulation agents such as suspending agents, stabilizers, and / or dispersants may be used.

[0337] Controlled-release formulations and drug delivery systems In certain embodiments, the formulations of the present invention may be not only short-acting and rapidly-offset, but also controlled, such as sustained-release, delayed-release, and pulsed-release formulations.

[0338] The term "sustained-release" is used in its conventional sense to refer to a drug formulation that provides a gradual release of the drug over a long period, resulting in a substantially constant blood level of the drug for an extended period, even if it is not necessary. The duration can be longer than one month and should be longer than the release of the same amount of active ingredient administered in bolus form.

[0339] For sustained release, polypeptides or compounds may be formulated with a suitable polymer or hydrophobic substance that provides sustained release properties to the polypeptide or compound. Thus, polypeptides or compounds for use in the method of the present invention may be administered in the form of microparticles, for example by injection, or by implantation in the form of wafers or disks.

[0340] In certain embodiments, the polypeptide or compound of the present invention is administered to a patient, either alone or in combination with another pharmaceutically active substance, using a sustained-release formulation.

[0341] The term delayed release is used herein to refer to a drug formulation that provides an initial release of the drug some time after administration, which may include delays of approximately 10 minutes to up to approximately 12 hours.

[0342] The term "pulsed release" is used herein to refer, in its conventional sense, to a drug formulation that provides drug release in a manner that produces a pulsed plasma profile of the drug after administration.

[0343] The term "immediate release" is used in its traditional sense to refer to drug formulations that provide drug release immediately after administration.

[0344] As used herein, short-term means any period of any whole or partial interval after drug administration, including up to 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 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.

[0345] As used herein, rapid-offset refers to any period of time in any whole or partial increment after drug administration, including up to 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 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.

[0346] dosage The therapeutically effective amount or dose of the polypeptides, 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 considered herein in the patient being treated. Those skilled in the art can determine an appropriate dosage depending on these and other factors.

[0347] Suitable doses of the polypeptides, compounds, and / or compositions of the present invention may be in the range of about 1 ng to about 1 mg / dose, for example, about 100 ng to about 1 mg, for example, about 100 ng to about 500 ng / dose. In certain embodiments, suitable doses of the polypeptides, compounds, and / or compositions of the present invention may be in the range of about 10 μg to about 100 μg. Doses may be administered as single doses or in multiple doses, for example, 1 to 4 or more times. When multiple doses are used, the amount of each dose may be the same or different.

[0348] In certain embodiments, the composition may be administered to a subject in 1 to 4 doses. In certain embodiments, the subject is administered a single dose. In certain embodiments, the subject is administered two doses. In certain embodiments, the subject is administered three doses. In certain embodiments, the subject is administered four doses. In certain embodiments, the subject is administered more than four doses.

[0349] In certain embodiments, each dose is administered with an intervening period of approximately one week. In certain embodiments, each dose is administered with an intervening period of approximately one week or less. In certain embodiments, each dose is administered with an intervening period of approximately one week or more. In certain embodiments, each dose is administered with an intervening period of approximately two weeks. In certain embodiments, each dose is administered with an intervening period of approximately two weeks or less. In certain embodiments, each dose is administered with an intervening period of approximately two weeks or more. In certain embodiments, each dose is administered with an intervening period of approximately three weeks. In certain embodiments, each dose is administered with an intervening period of approximately three weeks or less. In certain embodiments, each dose is administered with an intervening period of approximately three weeks or more. In certain embodiments, each dose is administered with an intervening period of approximately four weeks. In certain embodiments, each dose is administered with an intervening period of approximately four weeks or less. In certain embodiments, each dose is administered with an intervening period of approximately four weeks or more.

[0350] In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately two weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately three weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately four weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately five weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately six weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately seven weeks between doses. In certain embodiments, the subject is administered two doses, with an inter-dose interval of approximately eight weeks between doses.

[0351] In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately two weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately three weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately four weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately five weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately six weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately seven weeks. In certain embodiments, the subject is administered three doses, with each dose administered over a dose interval of approximately eight weeks.

[0352] In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately two weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately three weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately four weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately five weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately six weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately seven weeks. In certain embodiments, the subject is administered four doses, with each dose administered over a dose interval of approximately eight weeks.

[0353] In a certain preferred, non-limiting embodiment, the subject receives four doses with an inter-dose interval of approximately two weeks (i.e., the first dose on day 0, the second dose on approximately day 14, the third dose on approximately day 28, and the fourth dose on approximately day 42).

[0354] In certain preferred non-limiting embodiments, subjects are administered two doses with an inter-dose interval of approximately 3 weeks (i.e., the first dose on day 0 and the second dose on approximately day 21). In certain preferred non-limiting embodiments, subjects are administered two doses with an inter-dose interval of approximately 4 weeks (i.e., the first dose on day 0 and the second dose on approximately day 28). In certain preferred non-limiting embodiments, subjects are administered two doses with an inter-dose interval of approximately 8 weeks (i.e., the first dose on day 0 and the second dose on approximately day 56).

[0355] The actual dose level of cells in the pharmaceutical formulation of the present invention may be varied to obtain an amount of the composition that is effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration, and that is not toxic to the subject.

[0356] The toxicity and therapeutic efficacy of such treatment regimens are LD 50 (A lethal dose in 50% of the population) and ED 50 The dose (a dose therapeutically effective in 50% of the population) is determined arbitrarily in cell cultures or research animals, with the exception of the determination of the dose therapeutically effective in 50% of the population. The dose-to-toxicity ratio is the LD50. 50 and ED 50 This is a therapeutic index expressed as a ratio to . Data obtained from cell culture assays and animal experiments are optionally used to determine the dosage range for use in humans. The dosage of such polypeptides, compounds and / or compositions is preferably ED 50 It is within the circulating concentration range that contains the substance with minimal toxicity. The dosage is arbitrary and varies within this range depending on the dosage form used and the route of administration utilized. [Examples]

[0357] Examples Various aspects of this application can be better understood by referring to the following examples provided as illustrations. The scope of this application is not limited to the examples given herein.

[0358] Antigen preparation In certain embodiments, the polypeptides described herein were prepared using one or more bacterial expression vectors (e.g., Escherichia coli) by 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 (a) producing a single-stranded DNA copy (cDNA) of purified messenger RNA (mRNA) as a template for the desired protein; (b) converting the cDNA to double-stranded DNA; (c) binding the DNA to a suitable point in a suitable clonal carrier to form a recombinant DNA molecule; and (d) transforming a suitable host using this recombinant DNA molecule. Such transformation causes the host to produce the desired protein.

[0359] Expression vectors are characterized by the fact that any vector may have sites suitable for gene insertion, such as an origin of replication, a selection marker, and a multiple cloning site. Cloned genes may be transferred from a specialized cloning vector to an expression vector, but they can also be cloned directly into an expression vector. The cloning process can be carried out in E. coli. Vectors used for protein production in organisms other than E. coli may have elements that allow them to be maintained in another organism, in addition to an origin of replication suitable for their growth in E. coli (i.e., shuttle vectors).

[0360] HLA-DR4 Mouse The HLA-DR4 allele is associated with the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. To provide a mouse model for these diseases, we engineered a hybrid MHC class II molecule between the peptide-binding domains of human HLA-DRA and HLA-DRB*0401 and the membrane-bound domain of mouse IE(H2-E), and co-injected it into C57BL / 6 fertilized eggs. Transgenic offspring were then reared into mice unable to express other MHC class II molecules (Abb knockout with a B6 background). By preserving the alpha-2 and beta-2 domains of the mouse MHC class II, interaction with the CD4 coreceptor on T cells was preserved. These mice are healthy and develop normally. Immunotherapy using peptides from proteolipid proteins known to bind to HLA-DR4 induced a potent T-cell proliferation response, leading to inflammatory lesions and symptoms of experimental allergic encephalomyelitis in the CNS white matter (Ito K, et al. J. Exp. Med. 1996, 183(6):2635-2644).

[0361] Immunotherapy of HLA-DR4 mice The experiments described herein used HLA-DR4 transgenic mice, each of which was within one week of the age of all other mice in the study. The HLA-DR4 mice were divided into a treatment group and a control group (e.g., naive and / or placebo). On specific days, the mice were immunized twice with a vaccine composition having a total volume of 50 μL or 100 μL via the route of administration (e.g., subcutaneous or intramuscular) as shown elsewhere herein. Here, the vaccine composition contained 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 AddaS03®), diluted to a total volume in phosphate-buffered saline (PBS) solution. In certain embodiments, the HLA-DR4 mice were immunized as described in Tables 1–7 (i.e., experiment numbers 1–7). Serum and urine samples were obtained from mice at specific points in time, as described elsewhere in this specification.

[0362] (Table 1) Immunotherapy of HLA-DR4 mice a (Experiment number 1) TIFF2026521465000004.tif48161 a n=3 mice / group, 100 μg of antigen in a total volume of 100 μL of PBS diluent was administered subcutaneously twice (vaccination interval was approximately 3 weeks); b Naive control group; c Aluminum hydroxide; d Double mutant thermolabic toxin (dmLT); e CpG ODN2395.

[0363] (Table 2) Immunotherapy of HLA-DR4 mice a (Experiment number 2) TIFF2026521465000005.tif56161 a n=5 mice / group, 100 μL total volume, PBS diluent, 100 μg of antigen administered subcutaneously twice, mice 9-10 months old at the time of the first vaccination (vaccination interval approximately 2 weeks); bPlacebo-controlled group; c Aluminum hydroxide; d CpG ODN2395.

[0364] (Table 3) Immunotherapy of HLA-DR4 mice a (Experiment number 3) TIFF2026521465000006.tif24161 a n=5 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 8-10 weeks old at the time of the first vaccination (vaccination interval approximately 3 weeks); b CpG ODN2395.

[0365] (Table 4) Immunotherapy of HLA-DR4 mice a (Experiment number 4) TIFF2026521465000007.tif56161 a (A) consisted of 5 mice / group, (B-F) consisted of 4 mice / group, with a total volume of 50 μL, PBS diluent, and 100 μg of antigen administered intramuscularly twice. Mice were 6-7 weeks old at the time of the first vaccination (vaccination interval was approximately 3 weeks). b Placebo-controlled group; c CpG ODN2395.

[0366] (Table 5) Immunotherapy of HLA-DR4 mice a (Experiment number 5) TIFF2026521465000008.tif57161 a n=5 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 9-10 weeks old at the time of the first vaccination (vaccination interval approximately 3 weeks); b Placebo-controlled group; c Aluminum hydroxide; d CpG ODN2395.

[0367] (Table 6) Immunotherapy of HLA-DR4 mice a (Experiment number 6) TIFF2026521465000009.tif57161 an=3 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 6-8 weeks old at the time of the first vaccination (vaccination interval is approximately 3 weeks); b Placebo-controlled group; c CpG ODN2395.

[0368] (Table 7) Immunotherapy of HLA-DR4 mice a (Experiment number 7) TIFF2026521465000010.tif64161 a n=3 mice / group (A-B and D-G), n=4 mice / group (C), 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice (vaccination interval approximately 3 weeks), mice were 7-9 weeks old at the time of first vaccination, all mice received one induction using UPEC25; b Placebo-controlled group; c CpG ODN2395; d Aluminum hydroxide; e Buffer conditions (20mM Na2PO4, 300mM urea, 300mM NaCl, pH9.5); f Buffer conditions (20mM Na2PO, 300mM NaCl, pH9.5).

[0369] CD1 Mouse CD1 IGS (i.e., Crl:CD1(ICR)) is a mouse model of albino non-inbred strains often used in toxicological and pharmacological studies. A notable feature of CD1 IGS mice is their large genetic diversity, similar to that observed within and between human populations. CD1 mice were immunized in a manner similar to that described for HLA-DR4 mice. A detailed description of the experiments performed using CD1 mice is provided herein (Table 8).

[0370] (Table 8) Immunotherapy of CD1 mice a (Experiment number 8) TIFF2026521465000011.tif33161 aA total volume of 100 μL of PBS diluent and 100 μg of antigen were administered subcutaneously twice. Mice were 11 weeks old at the time of the first vaccination (the interval between vaccinations was approximately 2 weeks). b n=3 mice; c n = 4 mice; d Placebo-controlled group; e Aluminum hydroxide.

[0371] C57BL / 6 Mouse The C57BL / 6 mouse is the most commonly used inbred strain in research and is typically used as a model for human diseases. C57BL / 6 mice were immunized using a method similar to that described for HLA-DR4 mice. A detailed description of the experiments performed using C57BL / 6 mice is provided herein (Tables 9-11).

[0372] (Table 9) Immunotherapy of C57BL / 6 a (Experiment number 9) TIFF2026521465000012.tif49161 a n=15 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 6-7 weeks old at the time of initial vaccination (vaccination interval approximately 3 weeks), one induction using UPEC25, immunological samples collected approximately 7 days after injection; b Placebo-controlled group; c CpG ODN2395.

[0373] (Table 10) Immunotherapy of C57BL / 6 mice a (Experiment number 10) TIFF2026521465000013.tif40161 a n=20 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 6-7 weeks old at the time of the first vaccination (vaccination interval approximately 3 weeks), immunological samples collected approximately 14 days after the second vaccination; b Placebo-controlled group; c CpG ODN2395; d Aluminum hydroxide.

[0374] (Table 11) Immunotherapy of C57BL / 6 mice a (Experiment number 11) TIFF2026521465000014.tif80161 a n=4 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice were 6 weeks old at the time of the first vaccination (vaccination interval was approximately 3 weeks), immunological samples were collected approximately 12 days after the second vaccination; b Placebo-controlled group; c CpG ODN2395; d Aluminum hydroxide.

[0375] C3H / HeN mice C3H / HeN mice are homozygous for the Pde6brd1 allele and have premature severe retinal degeneration that causes blindness at weaning. Despite an atherosclerotic diet, C3H / HeN mice do not develop atherosclerosis of the aorta, in contrast to C57BL / 6J mice. There are genetically mediated differences between C3H / HeN and C3H / HeJ regarding their response to bacterial endotoxin (LPS). This response is linked to the TL4 protein (Toll-like receptor 4). C3H / HeN is Tlr4lps-n (Toll-like receptor 4; normal LPS response). This strain has a normal response to LPS induction and is endotoxin sensitive. In contrast, the C3H / HeJ strain is Tlr4lps-d and is said to be endotoxin resistant. C3H / HeN mice were immunized using a method similar to that described for HLA-DR4 mice. A detailed description of the experiments performed using C3H / HeN mice is provided herein (Table 12).

[0376] (Table 12) Immunotherapy of C3H / HeN mice a (Experiment number 12) TIFF2026521465000015.tif41161 an=10 mice / group, 50 μL total volume, PBS diluent, 100 μg of antigen administered intramuscularly twice, mice 6-7 weeks old at the time of the first vaccination (vaccination interval approximately 12 days), immunological samples collected approximately 14 days after the second vaccination; b Placebo-controlled group; c CpG ODN2395; d Aluminum hydroxide.

[0377] Cytometric bead array (CBA) The Cytometric Bead Array (CBA) assay (BD Biosciences) provides a method for capturing a soluble analyte or set of analytes using beads of known size and fluorescence, enabling detection of the analyte using flow cytometry. Each capture bead in the CBA kit has distinct fluorescence and is coated with an antibody specific to the soluble protein. The detection reagent is a mixture of phycoerythrin (PE)-conjugate antibodies that provide a fluorescent signal proportional to the amount of bound analyte.

[0378] When capture beads and detection reagents are incubated with a standard or unknown sample containing the analyte to be recognized, a sandwich complex is formed (e.g., capture beads + analyte + detection reagent). These complexes are measured using flow cytometry to identify particles with fluorescence properties in both the beads and the detector, enabling analyte identification.

[0379] The immunological response to the compositions described herein was 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.

[0380] Enzyme-linked immunoassay (ELISA) The immunological response to the compositions described herein was determined by measuring the optical density of the sample and / or the titers of antibodies, antigens, proteins, and / or glycoproteins (e.g., IgG1, IgG2b, IgA, and / or GTxMS IgG) by standard ELISA and / or peptide-based ELISA. Experimental protocols for standard ELISA and / or peptide ELISA are known to those skilled in the art. Examples are provided below. It is understood that the scope of this application is not limited to the examples provided herein.

[0381] In short, 100 μL of 2 pg / mL polypeptide dissolved in 5M urea was added to each well of a 96-well EIA / RIA plate (Coming / Costar 3590) and incubated overnight at 4°C. All remaining steps were performed at room temperature. The plate was washed three times with PBS washing buffer (PBS containing 0.05% Tween 20), followed by the addition of 200 μL / well of sample buffer consisting of PBS containing 0.05% Tween 20 and 1% bovine serum albumin. After 90 minutes, the sample buffer was replaced with 100 μL / well of PBS sample buffer. In the plate, a sequential 1:3 dilution of the primary antiserum was performed by adding 50 μL to the first column, mixing 10 times, and transferring 50 μL to the next column. The plate was incubated for 90 minutes, followed by three washes, and only 100 μL / well of the antibody, antigen, protein, and / or glycoprotein to be measured was added. After a 90-minute incubation period, the plates were washed four times, followed by the addition of 100 pl / well of TMB (BioFx; Surmodics, Eden Prairie, MN).

[0382] The reaction was stopped by adding 100 pl of stop reagent (BioFx) after spreading for 30 minutes. Absorbance was measured at a wavelength of 450 nm, and titer was calculated as the reciprocal of the dilution corresponding to an absorbance of 1.0. Controls included standardized primary serum placed in each plate to monitor assay variability, and uncoated wells to subtract background. The limit of detection for the assay was the reciprocal of the initial serum dilution.

[0383] Evaluation of secreted cytokines using MILLIPLEX® multiplex immunoassay. The Millipore Sigma multi-immunoassay, based on the Luminex® xMAP® bead-based multi-assay platform, provides a method for capturing a soluble analyte or set of analytes with magnetic microspheres of known size and fluorescence, enabling detection of the analyte using a Luminex® detection system. In short, each magnetic MagPlex® microsphere bead is fluorescently encoded in one of 500 specificity ratios of two different fluorescence and coated with an analyte-specific capture antibody. These microspheres are incubated with a sample, washed, and then incubated with a detection reagent consisting of a mixture of a biotinylated analyte-specific detection antibody and an R-phycoerythrin-conjugate streptavidin reagent, which provides a fluorescent signal proportional to the amount of bound analyte. When the captured microspheres and detection reagent are incubated with a standard or unknown sample containing the recognized analyte, a sandwich complex is formed (e.g., captured microsphere + analyte + detection reagent). By measuring these complexes using the Luminex MAGPIX® detection system, it becomes possible to identify particles with fluorescence properties in both the beads and the detector, thereby enabling analyte identification.

[0384] The immunological response to the compositions described herein was determined by measuring cytokine levels (e.g., IL-6, IL-17A, IL-2, TNF-α, IFN-γ, IL-4, IL-22, and / or IL-10) using the MILLIPLEX® multiplex immunoassay.

[0385] For the assays described herein, splenic lymphocytes (1 × 10⁶ per 0.2 mL) 6 (individual cells) and bladder lymphocytes (2 × 10 in 0.2 mL) 5 Cells were cultured 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 pellet the cells, and then the culture supernatant was removed. Samples were acquired using a Luminex MAGPIX® analyzer with Luminex® acquisition software, and the data were analyzed using Belysa® immunoassay curve fitting software.

[0386] splenocyte research Mice used for T-cell research were euthanized, their spleens were removed and processed to lyse red blood cells. Splenic lymphocytes (1 × 10⁶ cells in 0.2 mL of RPMI-1640 containing 10% fetal bovine serum) 6 Cells were cultured with 12.5 pg of antigen for 48 hours, and cytokine release into the cell supernatant was measured according to the manufacturer's protocol (BD Cytometric Bead Array (CBA), BD Biosciences, San Jose, CA).

[0387] Dissociation of bladder and spleen tissue Bladder cells were dissected, pooled into groups (3-5 bladders / group), cut into small pieces, and digested at 37°C for 1 hour using Multi Tissue Dissociation Kit 1 (Miltenyi) according to the manufacturer's protocol, with vigorous hand-mixing every 15 minutes. Digestion was stopped by adding an equal volume of RPMI-1640 containing 10% fetal bovine serum. The remaining tissue was destroyed using a plunger from a 3cc syringe and passed through a 70μm filter (Greiner Bio-One). Cells obtained from the pooled bladder preparations were added to RPMI-1640 containing 10% fetal bovine serum to a final concentration of 2 × 10⁶ cells. 6 The cells were resuspended at individual cells / mL.

[0388] Individual spleens were dissected, destroyed using a plunger from a 3cc syringe, passed through a 70μm filter, processed to remove red blood cells, and resuspended in RPMI-1640 containing 10% fetal bovine serum. Cells obtained from individual spleen preparations were then added to RPMI-1640 containing 10% fetal bovine serum to a final concentration of 1 × 10⁶ 7 The cells were resuspended at individual cells / mL.

[0389] Evaluation of activation-inducing markers (AIMs) on restirized lymphocytes by flow cytometry Activation-inducing marker (AIM) proteins are a group of proteins that become expressed on immune cells when they come into contact with and / or bind to their congener antigens and become activated. AIM proteins are not expressed, or are expressed at very low levels, on resting, inactivated immune cells. In the experiments described herein, a combination of CD49d and CD11a was used to evaluate the effector / memory CD4 induced by vaccination. + and CD8 + T cells were identified. Effector / memory B cells induced by vaccination were identified using the combination of CD49d and CD80. Subsequently, CD4 cells activated in vitro by protein antigens were identified using the combination of AIM protein OX40 and PD-L1. +Identify T cells; then, use the combination of AIM proteins CD69 and PD-L1 to identify CD8 cells that have been in vitro activated by protein antigens. + T cells were identified; subsequently, B cells activated in vitro by protein antigens were identified using combinations of AIM proteins CD69 and PD-L1 or CD69 and CD86.

[0390] Splenic lymphocytes (1 x 10⁶ per 0.2 mL) 6 (individual cells) and bladder lymphocytes (2 × 10 in 0.2 mL) 5 Cells were cultured with 12.5 pg of antigen at 37°C for 24–48 hours. At the end of the culture period, the cells were washed in PBS, resuspended in amine-reactive live / dead cell staining solution to remove dead cells, and Fc receptors were blocked using anti-mouse CD16 / CD32 antibody. The cells were incubated in the dark at 4°C for 25–30 minutes. The cells were then washed in FACS buffer (PBS supplemented with 2% fetal bovine serum and 0.095% sodium azide), 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 was determined 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 concentrations. Cells were stained with fluorescent antibodies at 4°C for 25–30 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 on living single cells was evaluated.

[0391] Example 1: Immunotherapy mice exhibit an innate immune response. Experiment number 1 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 1 as described herein. Serum samples were obtained from the mice 24 hours after the second vaccination and analyzed for serum IL-6 concentrations. Similar levels of IL-6 were observed across all treatment groups (i.e., groups 2–5 in Table 1), while serum IL-6 levels in naive mice were below the lower limit of quantification (Figure 1). Therefore, the data indicate that immunization induces an innate immune response.

[0392] Example 2: Immunotherapy mice exhibit an adaptive immune response (B cell-mediated antibody production). Experiment number 1 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 1, as described herein.

[0393] Approximately 14 days after the second vaccination (i.e., around day 35), serum samples were obtained from mice in groups 1-2 and 4 (i.e., SEQ ID NO:127 with either AlOH+dmLT or AlOH+CpG adjuvant) and analyzed by ELISA for serum IgG1 (Figure 2A), serum IgG2b (Figure 2B), serum IgA (Figure 2C), and urinary IgG(H) (Figure 2D). The results indicate that immunization with SEQ ID NO:127 induces the production of both IgG1 and IgG2b in HLA-DR4 mice.

[0394] Approximately 14 days after the second vaccination (i.e., around day 35), serum samples were obtained from mice in groups 1, 3, and 5 (i.e., SEQ ID NO:128 with either AlOH+dmLT or AlOH+CpG adjuvant) and analyzed for serum IgG1 (Figure 3A) and serum IgG2b (Figure 3B) by ELISA. The results indicate that immunization with SEQ ID NO:128 induces the production of both IgG1 and IgG2b in HLA-DR4 mice.

[0395] Approximately 14 days after the second vaccination (i.e., around day 35), serum and urine samples were obtained from mice in groups 1, 3, and 5 (i.e., SEQ ID NO:128 with either AlOH+dmLT or AlOH+CpG adjuvant) and analyzed by ELISA for serum IgA (Figure 4A), urine IgA (Figure 4B), and urine IgG(H) (Figure 4C). The results, as demonstrated by the urine and / or serum samples, indicate that immunization with SEQ ID NO:128 induces IgA and IgG(H) production in HLA-DR4 mice.

[0396] Approximately 14 days after the second vaccination (i.e., around day 35), serum samples were obtained from mice in groups 1, 2, and 4 (i.e., SEQ ID NO: 127 with either AlOH+dmLT or AlOH+CpG adjuvant) and analyzed by peptide ELISA. Here, 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 no coating). In unvaccinated subjects, antibody production was virtually nonexistent (i.e., the signal was less than or minimally greater than the control group 1 serum albumin only), while significant peptide-specific antibody production was observed for many of the antigens or antigenic fragments evaluated in this study (Figure 5).

[0397] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as non-limiting examples show by elevated levels of several antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0398] Experiment number 2 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 2, as described herein.

[0399] Approximately 14 days after the second vaccination (i.e., day 28), serum samples were obtained from mice in groups A and D-F, and serum IgG1 (Figures 9A, 10A, 11A) and serum IgG2b (Figures 9B, 10B, and 11B) were analyzed by ELISA. The results indicate that immunization with SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:135 induces the production of both IgG1 and IgG2b in HLA-DR4 mice.

[0400] Approximately 14 days after the second vaccination (i.e., day 28), serum samples were obtained from mice in groups A (i.e., placebo) and D (i.e., SEQ ID NO: 132) and analyzed by peptide ELISA. Here, 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 (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 no coating). In the placebo-treated subjects, virtually no antibody production was observed (i.e., the signal was less than or minimally greater than the control group A serum albumin only), while significant peptide-specific antibody production was observed for many of the antigens or antigenic fragments evaluated in this study (Figure 12).

[0401] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as non-limiting examples show by elevated levels of several antibodies, including IgG1 and IgG2b.

[0402] Experiment number 3 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 3, as described herein.

[0403] Serum samples were obtained from mice in the treatment group (i.e., group A) on day 0 (i.e., the day of the first vaccination; samples were collected before vaccination), day 21 (i.e., the day of the second vaccination), day 28, and day 41 (i.e., approximately 20 days after the second vaccination), and analyzed for serum IgG1 (Figure 16A) and IgG2b (Figure 16B) by ELISA. The results indicate that immunization using SEQ ID NO:132 induces the production of both IgG1 and IgG2b in HLA-DR4 mice.

[0404] Serum and urine samples were obtained from mice from the treatment group (i.e., group A) at day 0 (i.e., the day of the first vaccination; samples were collected before vaccination), and at approximately day 21 (i.e., the day of the second vaccination), day 28, and day 41 (i.e., 20 days after the second vaccination), and were analyzed by ELISA for serum IgA (Figure 17A), urine IgA (Figure 17B), and urine IgG(H) (Figure 17C). As demonstrated by the urine and / or serum samples, the results indicate that immunization with SEQ ID NO:132 induces IgA and IgG(H) production in HLA-DR4 mice.

[0405] Serum samples were obtained from mice in the treatment group (i.e., group A) at approximately day 21 and day 41 and analyzed by peptide ELISA. Here, 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 (i.e., SEQ ID NO: 132), and controls (i.e., tetanus toxin, serum albumin only, and uncoated). Generally, antibody production was greater at approximately day 41 than at approximately day 21 (Figure 18).

[0406] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as non-limiting examples show by elevated levels of several antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0407] Experiment number 4 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 4, as described herein.

[0408] Serum and urine samples were obtained from mice in each treatment group (i.e., groups B-F) and the control group (i.e., group A) at several time points after the initial vaccination, and were analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urinary IgA, and urinary IgG(H) in wells coated with SEQ ID NO:136 (Figures 28A-28E), SEQ ID NO:137 (Figures 29A-29E), SEQ ID NO:138 (Figures 30A-30E), SEQ ID NO:139 (Figures 31A-31E), and SEQ ID NO:140 (Figures 32A-32E).

[0409] In certain embodiments, the results show that all evaluated immunizations induce the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice compared to controls, approximately 5 weeks after the first vaccination. In certain embodiments, the results show that most evaluated immunizations induce the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice compared to controls, approximately 2 weeks after the second vaccination. In certain embodiments, the results show that most evaluated immunizations induce the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice compared to controls, approximately 2 weeks after the first vaccination.

[0410] In certain aspects, the results show that all evaluated immunization treatments induce IgA and IgG(H) production in the urine of HLA-DR4 mice compared to controls approximately 5 weeks after initial vaccination.

[0411] Serum samples were obtained from mice 36 days after the initial vaccination (approximately two weeks after the second vaccination) from each of the control group (i.e., group A) and treatment groups B (Figure 33A), D (Figure 33B), and F (Figure 33C), and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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). The data represent antibody production in wells containing each antigen and certain exemplary antigen fragments used in groups B-F.

[0412] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as a non-limiting example is shown by elevated levels of certain antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0413] Experiment number 5 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 5, as described herein.

[0414] 33 days after the first vaccination (approximately 2 weeks after the second vaccination), serum and urine samples were obtained from mice in each treatment group (i.e., groups B-C and E-F) and the control group (i.e., groups A and D). These samples were analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urinary IgA, and urinary IgG(H) in wells coated with SEQ ID NO: 136 (Figures 36A-36E) or SEQ ID NO: 138 (Figures 37A-37E).

[0415] In certain embodiments, the results show that all evaluated immunization treatments induce the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice compared to controls 33 days after the first vaccination (approximately 2 weeks after the second vaccination). In certain embodiments, the results show that most evaluated immunization treatments induce the production of IgA and IgG(H) in the urine of HLA-DR4 mice compared to controls 33 days after the first vaccination (approximately 2 weeks after the second vaccination).

[0416] Serum samples were obtained from mice in treatment groups B and D (Figure 38A) and E and F (Figure 38B) 33 days after the initial vaccination (approximately 2 weeks after the second vaccination) and analyzed by peptide ELISA. Here, wells were coated with specific polypeptides 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 no coating). The data represent antibody production in wells containing each antigen utilized for the exemplary vaccine antigens and / or fragments.

[0417] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as a non-limiting example is shown by elevated levels of certain antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0418] Experiment number 6 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 6, as described herein.

[0419] Thirty-four days after the first vaccination (approximately two weeks after the second vaccination), serum and urine samples were obtained from mice in each treatment group (i.e., groups B-F) and the control group (i.e., group A), and analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urinary IgA, and urinary IgG(H) (Figures 41A-41E).

[0420] In certain embodiments, the results show that the evaluated immunization induces the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice compared to controls 34 days after the first vaccination (approximately 2 weeks after the second vaccination). In certain embodiments, the results show that the evaluated immunization induces the production of IgA and IgG(H) in the urine of HLA-DR4 mice compared to controls 34 days after the first vaccination (approximately 2 weeks after the second vaccination).

[0421] Serum samples were obtained from mice 34 days after the initial vaccination (approximately two weeks after the second vaccination) from each of the treatment groups B (Figure 42A), C (Figure 42B), D (Figure 42C), and E (Figure 42D) and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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) as well as controls (i.e., tetanus toxin, serum albumin only, and no coating). The data represent antibody production in wells containing each antigen utilized for the exemplary vaccine antigens and / or fragments.

[0422] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as a non-limiting example is shown by elevated levels of certain antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0423] Experiment number 7 HLA-DR4 mice were immunized using the vaccine compositions shown in Table 7 as described herein.

[0424] Thirty-five days after the initial vaccination (approximately two weeks after the second vaccination), serum and urine samples were obtained from mice in each treatment group (i.e., groups B-C, E, and G) and each control group (i.e., groups A, D, and F), and analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urinary IgA, and urinary IgG(H) in wells coated with SEQ ID NO: 138(1) or (2). Here, (1) and (2) differ in terms of buffer conditions as shown in Table 7 (Figures 45A-45E).

[0425] In certain embodiments, the results show that certain evaluated immunization treatments induce the production of IgG1, IgG2b, and IgA in the serum of HLA-DR4 mice 35 days after the first vaccination (approximately 2 weeks after the second vaccination) compared to controls. In certain embodiments, the results show that certain evaluated immunization treatments induce the production of IgA and IgG(H) in the urine of HLA-DR4 mice 35 days after the first vaccination (approximately 2 weeks after the second vaccination).

[0426] Serum samples were obtained from mice 35 days after the initial vaccination (approximately two weeks after the second vaccination) from each of the control (group A) and treatment groups B-C (Figure 46) and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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) differ with respect to buffer conditions as described in Table 7) and controls (i.e., tetanus toxin, serum albumin only, and no coating). The data represent antibody production in wells containing each antigen utilized for the exemplary vaccine antigen and / or fragment.

[0427] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as a non-limiting example is shown by elevated levels of certain antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0428] Experiment number 8 CD1 mice were immunized using the vaccine compositions shown in Table 8 as described herein.

[0429] Approximately 14 days after the second vaccination (i.e., around day 28), serum samples were obtained from mice in each of groups A-C, and serum IgG1 (Figure 19A and Figure 21A) and serum IgG2b (Figure 19B and Figure 21B) were analyzed by ELISA. The results show that immunization using SEQ ID NO:132 and SEQ ID NO:135, respectively, induces the production of both IgG1 and IgG2b in CB1 mice.

[0430] Approximately 14 days after the second vaccination (i.e., around day 28), serum samples were obtained from mice in each of groups A-C and analyzed by ELISA for serum IgA (Figures 20A and 22A), urinary IgA (Figures 20B and 22B), and urinary IgG(H) (Figures 20C and 22C). As demonstrated by the urine and / or serum samples, the results indicate that immunization with SEQ ID NO:132 induces IgA and IgG(H) production in CB1 mice.

[0431] On day 28 (i.e., approximately 14 days after the second vaccination), serum samples were obtained from mice in groups A (i.e., placebo) and B (i.e., SEQ ID NO: 132) and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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 no coating). Substantially no antibody production was observed in the placebo-treated subjects (i.e., the signal was less than or minimally greater than the control in serum albumin only from group A), while significant peptide-specific antibody production was observed for many of the antigens or antigen fragments evaluated in this study (Figure 23).

[0432] On day 28 (i.e., approximately 14 days after the second vaccination), serum samples were obtained from mice in groups A (i.e., placebo) and C (i.e., SEQ ID NO: 135) and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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 no coating). In the placebo-treated subjects, virtually no antibody production was observed (i.e., the signal was less than or minimally greater than the control in serum albumin only from group A), while significant peptide-specific antibody production was observed for many of the antigens or antigen fragments evaluated in this study (Figure 24).

[0433] Experiment number 9 C57BL / 6 mice were immunized with the vaccine compositions shown in Table 9 as described herein.

[0434] Thirty-four days after the first vaccination (approximately two weeks after the second vaccination), serum and urine samples were obtained from mice in each treatment group (i.e., groups B-E) and the control group (i.e., group A), and analyzed by ELISA for serum IgG1, serum IgG2b, serum IgA, urinary IgA, and urinary IgG(H) (Figures 51A-51E).

[0435] In certain embodiments, the results show that certain evaluated immunization treatments induce the production of IgG1, IgG2b, and IgA in the serum of C57BL / 6 mice 34 days after the first vaccination (approximately two weeks after the second vaccination) compared to controls. In certain embodiments, the results show that certain evaluated immunization treatments induce the production of IgA and IgG(H) in the urine of HLA-DR4 mice 34 days after the first vaccination (approximately two weeks after the second vaccination).

[0436] Serum samples were obtained from mice 34 days after the initial vaccination (approximately two weeks after the second vaccination) from each of the control (group A), treatment groups B and D (Figure 52A), and treatment groups C and E (Figure 52B), and analyzed by peptide ELISA. Here, wells were coated with certain polypeptides 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 no coating). The data represent antibody production in wells containing each antigen utilized for the exemplary vaccine antigens and / or fragments.

[0437] Therefore, in certain embodiments, the antigens and / or antigenic fragments of this disclosure induce an adaptive immune response mediated by B cell activation, as a non-limiting example is shown by elevated levels of certain antibodies including IgG1, IgG2b, IgA, and IgG(H).

[0438] Experiment number 10 C57BL / 6 mice were immunized using the vaccine compositions shown in Table 10 as described herein.

[0439] 33 days after the first vaccina...

Claims

1. Polypeptides containing formula (I) or their salts or solvates: (B 1 -L 1 -T 1 -L 2 -B 2 -L 3 -T 2 -L 4 -B 3 -L 5 -T 3 -L 6 ) n (I) During the ceremony: 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 may or may not exist; B 1 B 2 , and B 3 Each occurrence, if present, independently contains immunogenic fragments of bacterial surface proteins; T 1 , T 2 , and T 3 Each occurrence, if present, independently contains an immunogenic fragment of the iron receptor protein. Here, T 1 , T 2 , and T 3 At least two of these exist, or T 1 , T 2 and T 3 There exists at least one of the above, and n is at least 2; L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each occurrence, if present, independently contains a polypeptide of 1 to 10 amino acids. Here, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 At least one of the following exists, Here, B 1 B 2 B 3 , T 1 , T 2 , and T 3 Each occurrence is L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 They are separated from each other by at least one of the following; and n is an integer selected from the group consisting of 1, 2, 3, 4, and 5.

2. B 1 B 2 , or B 3 The polypeptide according to claim 1, wherein at least one of the following 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 to a polypeptide independently selected from the group consisting of 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. Each of the immunogenic fragments of the aforementioned bacterial surface protein is 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 The polypeptide according to claim 3, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of NO:

41.

5. The polypeptide according to claim 3 or 4, wherein each immunogenic fragment of the bacterial surface protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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: 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 to 5, wherein each iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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. Each of the immunogenic fragments of the aforementioned iron receptor protein is 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 polypeptide according to claim 6, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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.

8. The polypeptide according to claim 6 or 7, wherein each immunogenic fragment of the iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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. L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of the following independently comprises a polypeptide, where each amino acid residue side chain comprises a neutral (i.e., uncharged) substituent, according to any one of claims 1 to 8.

10. L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 The polypeptide according to any one of claims 1 to 9, wherein each of the present molecules independently comprises a polypeptide of 4 to 6 amino acids.

11. L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 If each of these exists, A polypeptide according to any one of claims 1 to 10, independently selected from the group consisting of the following.

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

13. A polypeptide according to any one of claims 1 to 12, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide selected from the group consisting of SEQ ID NO: 130 and SEQ ID NO:

129.

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

15. 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, 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 according to any one of claims 1 to 11 and 14, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide selected from the group consisting of NO:

260.

16. (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 ;および (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 Polypeptides or their salts or solvates selected from the group consisting of: During the ceremony, Each occurrence of B, if present, independently contains immunogenic fragments of bacterial surface proteins. Here, each instance of B has a C-terminus and an N-terminus; Each appearance of T independently contains an immunogenic fragment of the iron receptor protein. Here, each instance of T has a C-terminus and an N-terminus; Each occurrence of L independently contains a polypeptide of 1 to 10 amino acids. Here, 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 by 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 by covalent peptide bonds 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 according to claim 16, wherein each bacterial surface protein has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a polypeptide independently selected from the group consisting of 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. Each of the immunogenic fragments of the aforementioned bacterial surface protein is 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 The polypeptide according to claim 17, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of NO:

41.

19. The polypeptide according to claim 17 or 18, wherein each immunogenic fragment of the bacterial surface protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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: 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 to 19, wherein each iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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. Each of the immunogenic fragments of the aforementioned iron receptor protein is 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 polypeptide according to claim 20, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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.

22. The polypeptide according to claim 20 or 21, wherein each immunogenic fragment of the iron receptor protein shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide independently selected from the group consisting of 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. 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 the following independently comprises a polypeptide, where present, wherein each amino acid residue side chain contains a neutral (i.e., uncharged) substituent, according to any one of claims 16 to 22.

24. 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 The polypeptide according to any one of claims 16 to 23, wherein each of the present molecules independently comprises a polypeptide of 4 to 6 amino acids.

25. 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 If each of these exists, A polypeptide according to any one of claims 16 to 24, independently selected from the group consisting of the following.

26. 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: 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 The polypeptide according to any one of claims 16 to 25, which shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptide selected from the group consisting of NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO:

260.

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

226.

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

30. The aforementioned mRNA (a) Prokaryotes, or (b) Mammals The isolated mRNA according to claim 29, which is codon-optimized for expression in [location].

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

32. Isolated deoxyribonucleic acid (DNA) encoding a polypeptide according to any one of claims 1 to 28.

33. The aforementioned DNA, (a) Prokaryotes, or (b) Mammals The isolated DNA according to claim 32, which is codon-optimized for expression in [location].

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

35. An isolated polynucleotide encoding mRNA according to any one of claims 29 to 31, wherein the polynucleotide comprises one or more promoter and / or polyadenylation signals functionally linked to the mRNA-coding sequence.

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

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

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

39. The vector according to claim 36, which 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 according to any one of claims 29 to 31, isolated DNA according to any one of claims 32 to 34, and / or isolated polynucleotide according to claim 35.

42. below: (a) The LNP has a lipid-to-isolated mRNA or DNA ratio in the range of about 5:1 to about 25:1; and (b) The LNP has a lipid-to-isolated polynucleotide ratio in the range of about 5:1 to about 25:1 The LNP according to claim 41, wherein at least one of the following applies.

43. (a) at least one ionizable lipid; (b) at least one helper lipid; (c) Cholesterol or modified derivatives thereof, and any combination thereof; (d) at least one conjugated lipid The LNP according to claim 41 or 42, including the LNP.

44. The LNP according to claim 43, wherein the ionizable lipid is at least one selected from the group consisting of 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 constitutes about 50 mol% to about 90 mol% of the LNP.

46. The LNP according to any one of claims 43 to 45, wherein the helper lipid is at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

47. The LNP according to any one of claims 43 to 46, wherein the at least one helper lipid constitutes about 1 to about 25 mol% of the LNP.

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

49. The aforementioned complex lipids include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DPG), mPEG-OH, mPEG-AA (mPEG-CM), and mPEG-CH 2 CH 2 CH 2 -NH 2 The LNP according to any one of claims 43 to 48, which is at least one selected from the group consisting of mPEG-DMG, mPEG-N,N-ditetradecylacetamide (ALC-0159), mPEG-DSPE, and mPEG-DPPE.

50. The LNP according to any one of claims 43 to 49, wherein the at least one composite lipid constitutes about 0.1 to about 5 mol% of the LNP.

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

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

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

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

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

226.

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

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

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

60. (a) A polypeptide that shares 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 constituting about 2.5% (v / v) of the vaccine composition; (c) Squalene constituting about 2.5% (v / v) of the vaccine composition; and (d) Polysorbate 80, which constitutes about 0.9% (v / v) of the vaccine composition A vaccine composition comprising, The polypeptide, α-tocopherol, squalene, and polysorbate are dissolved or suspended in a phosphate-buffered saline (PBS) solution. The vaccine composition.

61. (a) A polypeptide that shares 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 constituting about 2.5% (v / v) of the vaccine composition; (c) Squalene constituting about 2.5% (v / v) of the vaccine composition; (d) Polysorbate 80 constituting about 0.9% (v / v) of the vaccine composition; and (e) The vaccine contains approximately 100 μg to approximately 6000 μg of CpG. A vaccine composition comprising, The polypeptide, α-tocopherol, squalene, polysorbate, and CpG are dissolved or suspended in a phosphate-buffered saline (PBS) solution. The vaccine composition.

62. A method for treating, preventing, and / or improving bacterial infections in a subject that requires such treatment, (a) The polypeptide according to any one of claims 1 to 28; (b) Isolated mRNA according to any one of claims 29 to 31; (c) Isolated DNA according to any one of claims 32 to 34; (d) an isolated polynucleotide according to claim 35; (e) The vector according to any one of claims 36 to 40; (f) LNP according to any one of claims 41 to 51; (g) the pharmaceutical composition according to claim 52; and (h) The vaccine composition according to any one of claims 53 to 61 The method comprising the step of administering at least one selected from the group consisting of the following.

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

64. The method according to claim 62, wherein the bacterial infection includes urinary tract infections (UTIs) and sepsis.

65. The method according to 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 according to claim 62, wherein the bacterial infection is pneumonia.

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

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

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

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

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

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

74. The one or more pathogenic bacteria mentioned above include Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Shigella dysenteriae, Salmonella enterica, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Streptococcus pyogenes, and Pseudomonas aeruginosa. The method according to claim 73, comprising at least one selected from the group consisting of *Aeruginosa* and *Salmonella bongori*.

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

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

77. The method according to claim 75, wherein the bacterial infection includes urinary tract infections (UTIs) and sepsis.

78. The method according to 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 according to claim 75, wherein the bacterial infection is pneumonia.

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

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

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

84. The method according to claim 83, wherein the immunity to infection by one or more pathogenic bacteria is generated in at least one of the subject and the fetus, or in the newborn thereof.

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

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