Products and methods for inducing immune response against bacteria

Abstract

The present invention relates to peptides, fusion proteins and compositions for inducing an immune response against bacteria. The invention also relates to the treatment or prevention of infections by sepsis-inducing bacteria, and to the treatment and prevention of pathological conditions caused by such bacteria. Such pathological conditions include sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infection, soft tissue infection, gastrointestinal tract infection, bloodstream infection, encephalitis, premature delivery and dead delivery.

Description

Invention Field

[0001] This invention relates to peptides, fusion proteins, and compositions for inducing immune responses against bacteria. The invention also relates to the treatment or prevention of infections caused by bacteria that induce sepsis, and the treatment and prevention of pathological conditions caused by such bacteria. Such pathological conditions include sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, encephalitis, premature birth, and stillbirth. Background of the Invention Bacterial infections are a leading cause of death worldwide. Recent studies estimated that 7.7 million people died from complications related to bacterial infections in 2019. Among these, Staphylococcus aureus (S. aureus) is a major cause of death. Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) and agalactococci ( Streptococcus agalactiae (Commonly known as Group B Streptococcus) Streptococcus GBS was responsible for 53.6% of these deaths.

[0003] As previously described by the inventors, the neutralization of extracellular bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a good strategy for preventing or treating infections caused by the bacteria mentioned above (e.g., WO2015 / 189422 A1).

[0004] Extracellular bacterial GAPDH is an IL-10-inducing protein that causes generalized immunosuppression in the host at a very early time point after infection (Madureira, P., et al., PLoS Pathog, 2011. 7(11): p. e1002363). Extracellular bacterial GAPDH exhibits high homology among the aforementioned bacterial species. However, humans also possess GAPDH. Bacterial and human GAPDH can share 20-40% homology. Therefore, the inventors have disclosed a list of peptides that can be used as targets for immunotherapies aimed at neutralizing bacterial GAPDH without any cross-reactivity with human homologs (WO2015 / 189422 A1). These peptides are exposed to bacterial GAPDH but are not present in human GAPDH at all. From each bacterial GAPDH, the inventors selected a list of 7 to 12 peptides ranging in length from 9 to 22 amino acids that meet these criteria.

[0005] The inventors have previously demonstrated that antibody neutralization against IgG-dependent bacterial GAPDH can provide protection against infection (Madureira, P., et al., PLoS Pathog, 2011. 7(11): p. e1002363).

[0006] There is a need for vaccine compositions that are effective against a wide range of bacteria. Invention Overview In the first aspect, a peptide is provided which comprises the amino acid sequence according to SEQ ID NO: 3 (TQTTEITAVGDQLVKTVA).

[0008] In the second aspect, a fusion protein is provided, which contains peptides according to the first aspect.

[0009] In a third aspect, a composition or kit is provided comprising the peptide of the first aspect or the fusion protein of the second aspect. In one embodiment, the composition or kit comprises the peptide of the first aspect or the fusion protein of the second aspect, and a peptide comprising the amino acid sequence according to SEQ ID NO: 13, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 13; and / or comprising the amino acid sequence according to SEQ ID NO: 14, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 14. In one embodiment, a composition or kit is provided comprising a first peptide which is the peptide of the first aspect, a second peptide comprising the amino acid sequence according to SEQ ID NO: 13, and a third peptide comprising the amino acid sequence according to SEQ ID NO: 14.

[0010] In a fourth aspect, a first peptide is provided, comprising the sequence according to SEQ ID NO: 3, or having one or more added, substituted, or deleted peptides according to the sequence according to SEQ ID NO: 3; a second peptide, comprising the sequence according to SEQ ID NO: 13, or having one or more added, substituted, or deleted peptides according to the sequence according to SEQ ID NO: 13; and a third peptide, comprising the sequence according to SEQ ID NO: 14, or having one or more added, substituted, or deleted peptides according to the sequence according to SEQ ID NO: 14.

[0011] In one embodiment, the peptide comprising SEQ ID NO: 3 is according to SEQ ID NO: 12; the peptide comprising SEQ ID NO: 13 is according to SEQ ID NO: 15; and the peptide comprising SEQ ID NO: 14 is according to SEQ ID NO: 16.

[0012] In the fifth aspect, one or more nucleic acids are provided that encode a peptide according to the first aspect, a fusion protein according to the second aspect, or a peptide of a composition or kit according to the fourth or fifth aspect.

[0013] In the sixth aspect, one or more vectors are provided that contain one or more nucleic acids from the fifth aspect.

[0014] In the seventh aspect, a cell is provided that contains one or more nucleic acids as described in the fifth aspect or one or more vectors as described in the sixth aspect.

[0015] In the eighth aspect, one or more pharmaceutical compositions are provided, comprising a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third or fourth aspect, a peptide of a kit according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, or one or more carriers according to the sixth aspect.

[0016] In the ninth aspect, one or more immunogenic compositions are provided, comprising a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third or fourth aspect, a peptide of a kit according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, or one or more vectors according to the sixth aspect.

[0017] In the tenth aspect, the following uses are provided for stimulating an immune response: peptides according to the first aspect, fusion proteins according to the second aspect, compositions according to the third or fourth aspect, peptides of kits according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, one or more carriers according to the sixth aspect, one or more pharmaceutical compositions according to the eighth aspect, or one or more immunogenic compositions according to the ninth aspect.

[0018] In the eleventh aspect, there are provided peptides according to the first aspect, fusion proteins according to the second aspect, compositions according to the third or fourth aspect, peptides for kits according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, one or more carriers according to the sixth aspect, one or more pharmaceutical compositions according to the eighth aspect, or one or more immunogenic compositions according to the ninth aspect, which are used as pharmaceutical agents.

[0019] In one embodiment, a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third or fourth aspect, a peptide of a kit according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, one or more vectors according to the sixth aspect, one or more pharmaceutical compositions according to the eighth aspect, or one or more immunogenic compositions according to the ninth aspect are used to prevent infection by one or more species of bacteria that induce sepsis, reduce the risk of said infection, or reduce the severity of said infection.

[0020] In some implementations, one or more species of bacteria that induce sepsis are GBS, Escherichia coli, or Staphylococcus species. Staphylococcus spp.), Streptococcus pneumoniae and / or Klebsiella pneumoniae.

[0021] In one embodiment, the peptide according to the first aspect, the fusion protein according to the second aspect, the composition according to the third or fourth aspect, the peptide of the kit according to the third or fourth aspect, one or more nucleic acids according to the fifth aspect, one or more carriers according to the sixth aspect, one or more pharmaceutical compositions according to the eighth aspect, or one or more immunogenic compositions according to the ninth aspect are used to: prevent, reduce the risk of, or reduce the severity of any one or more of the following: sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infection, soft tissue infection, gastrointestinal infection, bloodstream infection, and encephalitis, or to prevent or reduce the risk of preterm birth or stillbirth. Brief description of the attached diagram Figure 1 Different conjugated peptide formulations target the immunogenicity of different bacterial GAPDH.

[0023] Figure 2 The immunogenicity of Paragon's novel vaccine (PNV) is higher than the sum of the immunogenicities of all its peptides.

[0024] Figure 3 PNV protects adult mice from bacterial infection.

[0025] Figure 4 : Protect offspring from bacterial infection by using maternal vaccination with PNV. Invention Details The inventors’ prior work provided a mechanism of action that enables peptides derived from bacterial GAPDH to be used in vaccines for the prevention of bacterial infections (see the background section of this document). The inventors have now expanded upon this work and have generated improved peptides suitable for use in such compositions. Furthermore, the inventors provide herein synergistic compositions of peptides suitable for the aforementioned purposes.

[0027] The inventors identified a single peptide, TQTEITAVGDLQLVKTVA (SEQ ID NO: 1), in their previous work, containing a domain common to Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus agalactiae. This domain is TQT-XXXXXXXX-QLVK (where X represents any amino acid – SEQ ID NO: 2). Chemical synthesis of this peptide did not yield a stable product. Interestingly, the random introduction, substitution, and / or deletion of specific amino acids outside the conserved domain allowed for the synthesis and purification of a highly stable, soluble peptide. The final peptide, TQTTEITAVGDQLVKTVA (SEQ ID NO: 3), was obtained.

[0028] The peptide containing SEQ ID NO: 3 was tested in the examples and found to be suitable for use in immunogenic compositions. In the examples, this peptide is referred to as "TQT" and is shown to induce an anti-GAPDH IgG response against *Escherichia coli* GAPDH and *Klebsiella pneumoniae* GAPDH. Figure 2 Furthermore, it was found that the TQT peptide is synergistic with other peptides and is therefore suitable for compositions used to induce a broad-spectrum immune response against sepsis-inducing bacteria. As part of the synergistic formulation, an IgG response was induced against all tested sepsis-inducing bacteria, including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus agalactiae. Figure 1 – Formulation #5 contains TQT).

[0029] Therefore, in the first aspect, a peptide is provided which comprises the amino acid sequence according to SEQ ID NO: 3.

[0030] When administered to a subject, the region of the peptide that is SEQ ID NO: 3 should be able to act as an antigen. Therefore, in some embodiments, the length of the peptide of the first aspect is equal to or less than 150 residues. This increases the likelihood that the peptide does not take on a structure that masks the antigen. In other embodiments, the length of the peptide of the first aspect is equal to or less than 100 residues, 50 residues, or 30 residues. In some instances, the peptide of the first aspect may contain no more than 12, 10, 5, 4, 3, 2, or 1 additional residue at the N-terminus and / or C-terminus of SEQ ID NO: 3. In some embodiments, the peptide contains only SEQ ID NO: 3, and residues or portions of the non-antigenic aspect of the composition, such as those required for labeling and / or conjugation. In one instance, the peptide may contain only SEQ ID NO: 3, β-alanine or other labeling, and cysteine ​​for conjugation with a carrier protein. The peptide may contain β-alanine and cysteine ​​at the N-terminus or C-terminus of the peptide, wherein the cysteine ​​is the terminus of the peptide.

[0031] The peptide of the first aspect may comprise the sequence of SEQ ID NO: 3 side-mounted on any one or both sides. In some instances, these sequences may be derived from bacteria GAPDH, such as bacterial species that induce sepsis. For example, the sequence may be derived from any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 3 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 3 may match the sequence within any of SEQ ID NO: 4 to 8, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions.

[0032] In one particular instance, the N-terminal end of SEQ ID NO: 3 may be attached to a sequence from SEQ ID NO: 5, wherein residue 293 of SEQ ID NO: 5 is attached to the first residue of SEQ ID NO: 3. An example of such an addition is provided below: …IIGSHFGSVFDA TQTTEITAVGDQLVKTVA (SEQ ID NO: 10).

[0033] In another instance (which could be an alternative or supplement to the above), the C-terminal end of SEQ ID NO: 3 may be attached to the sequence from SEQ ID NO: 5, wherein residue 312 of SEQ ID NO: 5 is attached to the last residue of SEQ ID NO: 3. An example of such an addition is provided below: TQTTEITAVGDQLVKTVA WYDNEYGFVTQL… (SEQ ID NO: 11).

[0034] One or more sequences derived from SEQ ID NO: 5 and side-connected to SEQ ID NO: 3 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 5. Sequences side-connected to SEQ ID NO: 3 may match sequences within SEQ ID NO: 5, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions.

[0035] Side-joining one or more sequences of SEQ ID NO: 5 to SEQ ID NO: 3 can result in a peptide of length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues. The peptide may also include non-antigenic aspects of the composition, such as labeled and / or conjugated residues or portions.

[0036] The desired peptide does not contain any autoantigens associated with the subject to be treated. Therefore, in some embodiments, the peptide of the first aspect does not contain human autoantigens. In some instances, this is achieved by avoiding any adjacent sequences comprising the eight amino acids from SEQ ID NO: 9. The peptide of the first aspect may contain fewer than 7, 6, 5, 4, or 3 residues from adjacent sequences within SEQ ID NO: 9.

[0037] The peptide in the first aspect can be an isolated peptide. An isolated peptide can be separated from any other sequence and / or substance associated with it in nature. An isolated peptide can be part of a purified composition in which other essential components are characterized. An isolated peptide can be linked to or fused with other residues or other polypeptide sequences.

[0038] The isolated peptide may optionally be linked to a labeled, identified, or quantified portion or residue of the accessory peptide. For example, a β-alanine residue may be included in the isolated peptide. In some instances, the β-alanine residue is directly attached to the first or last residue of SEQ ID NO: 3.

[0039] The isolated peptide may optionally be linked to a portion or residue of a accessory peptide or conjugated with other parts, residues, or polypeptides. For example, a cysteine ​​residue may be included in the isolated peptide. In a particular instance, the cysteine ​​residue may be an N-terminal or C-terminal residue of the peptide of the first aspect. Cysteine ​​may be suitable for the conjugation of a carrier protein with the peptide of the first aspect.

[0040] In some embodiments, the peptide of the first aspect is linked to a carrier protein. Examples of carrier proteins include keyhole hemocyanin (KLH), cross-reactive material 197 (CRM), and tetanus toxoid (TT). In a particular embodiment, the carrier protein is KLH. KLH can be obtained by purification from a natural source, and purified KLH may contain one or more KLH subunits. Therefore, when acting as a carrier protein, KLH can be or may contain one or more subunits of KLH. A carrier protein can be or may contain multiple subunits of KLH.

[0041] The peptide of the first aspect can be linked to the carrier protein via the aforementioned cysteine ​​residue. In a particular embodiment, the peptide of the first aspect is linked to KLH via a C-terminal cysteine ​​residue.

[0042] The carrier protein can be conjugated to the peptide at a carrier protein:peptide ratio of 1:4, 1:3, 1:2, or 1:1 (w / w), or any range between these values, such as from 1:2 to 1:1. In one particular embodiment, the ratio is 1:1. In one example, the peptide is conjugated to KLH via a terminal cysteine ​​residue at a 1:1 ratio.

[0043] In one particular embodiment, the peptide of the first aspect comprises TQTTEITAVGDQLVKTVA(β-alanine)C (SEQ ID NO: 12). This sequence may constitute the C-terminal end of the peptide of the first aspect, such that cysteine ​​is C-terminus. The peptide of the first aspect may have a sequence according to SEQ ID NO: 12 only. The peptide of the first aspect may have a sequence according to SEQ ID NO: 12, wherein the first residue of SEQ ID NO: 12 is N-terminus and the last residue of SEQ ID NO: 12 is C-terminus. The peptide may be conjugated to a carrier protein such as KLH via cysteine.

[0044] The isolated peptide in the first aspect can be a portion of a fusion protein and can be linked to other antigen sequences. Therefore, in the second aspect, a fusion protein is provided that comprises the amino acid sequence according to SEQ ID NO: 3.

[0045] Fusion proteins may contain any peptides disclosed in relation to the first aspect.

[0046] In some instances, the fusion protein may contain more than one copy of SEQ ID NO: 3. Alternatively or additionally, the fusion protein may contain one or more other peptides designed to induce an immune response against bacteria that induce sepsis. These other one or more peptides may be one or more peptides as defined or described in WO2015 / 189422 A1 (incorporated herein by reference). In some instances, the fusion peptide may contain the amino acid sequence according to EVKEGGFEVNGKFIKVSA (SEQ ID NO: 13) and / or DVTVEQVNEAMKNASNESF (SEQ ID NO: 14).

[0047] In fusion proteins comprising two or more peptides, the bonding can be direct and therefore without amino acids between the peptides to be linked. Alternatively, the bonding can be indirect and therefore may include one or more amino acid residues or other portions between the peptides to be linked. Patterns of one or more of the peptides can be repeated to form larger peptides / small proteins. The repeats can be directly adjacent to each other in so-called tandem repeats, or they can be spaced apart in each case. Alternatively, the linked peptides can appear in a random order.

[0048] The peptide of the first aspect or the fusion protein of the second aspect may be present in a composition containing other peptides or in a kit containing other peptides. Therefore, in a third aspect of the invention, a composition or kit is provided comprising the peptide of the first aspect or the fusion protein of the second aspect.

[0049] The composition or kit may contain other peptides, such as other peptides containing antigens derived from bacterial GAPDH proteins. Other peptides, one or more, may be one or more peptides as defined or described in WO2015 / 189422 A1. In some instances, the composition or kit may include peptides comprising the amino acid sequences according to SEQ ID NO: 13 and / or SEQ ID NO: 14.

[0050] The peptides in the composition or kit may be conjugated to a carrier protein. Examples of carrier proteins include KLH, CRM, and TT. In one particular embodiment, the carrier protein is KLH. The carrier protein may be conjugated to the peptide at a carrier protein:peptide ratio of 1:4, 1:3, 1:2, or 1:1 (w / w), or any range between these values, such as a carrier protein:peptide ratio from 1:2 to 1:1. The carrier protein may be conjugated to the peptide at a 1:1 (w / w) ratio. In one particular embodiment, the peptide is conjugated to KLH at a 1:1 ratio.

[0051] Any details or features disclosed herein regarding the compositions or kits of the fourth aspect may also apply to the compositions or kits of the third aspect.

[0052] The inventors have determined that specific antigen combinations have a synergistic effect. Figure 2 The document presents illustrative data demonstrating that the exemplary formulation (referred to as "PNV") results in a greater response when induced individually by any one of the three antigens within the PNV. For example, each of the three antigens induces only a low IgG titer against Staphylococcus aureus GAPDH; however, in combination, the IgG titer is very high. Figure 2 ).

[0053] Table 1 discloses exemplary peptides containing the relevant antigens. The antigens are SEQ ID NO: 3, 13, and 14 (respectively). The peptides are SEQ ID NO: 12, 15, and 16 (respectively).

[0054] Table 1: Peptide sequences of Paragon novel vaccine (PNV) .

[0055] When compared with other agents containing bacterial GAPDH antigen, PNV was found to induce high titers across a range of bacterial species. Figure 1–See formulation #5). Therefore, this formulation is surprisingly effective compared to other formulations containing bacterial GAPDH antigens.

[0056] The inventors further demonstrated that the exemplary composition containing the above-described antigen can be used to protect adult mice from bacterial infection. Figure 3 It can also be used for maternal vaccination to protect offspring from bacterial infections. Figure 4 ).

[0057] Therefore, in a fourth aspect of the invention, a composition or kit is provided comprising: a first peptide comprising the sequence according to SEQ ID NO: 3, or a peptide having one or more added, substituted, or deleted sequences according to SEQ ID NO: 1; a second peptide comprising the sequence according to SEQ ID NO: 13, or a peptide having one or more added, substituted, or deleted sequences according to SEQ ID NO: 13; and a third peptide comprising the sequence according to SEQ ID NO: 14, or a peptide having one or more added, substituted, or deleted sequences according to SEQ ID NO: 14.

[0058] Compared to the listed sequences, each of the first, second, and third peptides may contain one or more additions, substitutions, or deletions. For example, when based on... Figure 2 When the experiments shown are performed, they can be tolerable if such modifications do not alter the combined immunogenicity of the antigens. In some instances, the peptides contain no more than five, four, three, two, or one addition, substitution, or deletion. In some instances, the deletion is truncated and thus reduces the size of the peptide. In some instances, the peptides contain five, four, three, two, or one deletion at the N-terminal or C-terminal end of the enumerated sequences to result in a truncated peptide. In some instances, the first, second, and / or third peptides contain the enumerated sequences without additions, substitutions, or deletions. In some instances, the first peptide contains the common sequence shown in SEQ ID NO: 2, and therefore retains that sequence even if it includes one or more additions, substitutions, or deletions.

[0059] The first peptide can be any as disclosed in the first aspect of this disclosure. The first peptide can be comprised of any fusion protein as discussed in the second aspect of this disclosure.

[0060] Specifically, the length of the first peptide may be equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues. In some instances, the first peptide may contain no more than 12, 10, 5, 4, 3, 2, or 1 additional residue at the N-terminus and / or C-terminus of SEQ ID NO: 3. In some embodiments, the peptide contains only SEQ ID NO: 3, and residues or portions of non-antigenic aspects such as labeling and / or conjugation required. In one instance, the peptide may contain only SEQ ID NO: 3, β-alanine or other labeling, and cysteine ​​for conjugation with a carrier protein. The peptide may contain β-alanine and cysteine ​​at the N-terminus or C-terminus of the peptide, wherein the cysteine ​​is the terminus of the peptide. The peptide of the first aspect may contain the sequence of SEQ ID NO: 3 side-joined on either side or both sides. These sequences may be derived from bacteria GAPDH, such as bacterial species that induce sepsis. For example, the sequence may be derived from any of SEQ ID NO: 4 to 8. The sequence flanked by SEQ ID NO: 3 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with any of SEQ ID NO: 4 to 8. The sequence flanked by SEQ ID NO: 3 may match any of the sequences within SEQ ID NO: 4 to 8, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions. In one particular example, the N-terminal end of SEQ ID NO: 3 may be attached to the sequence from SEQ ID NO: 5, wherein residue 293 of SEQ ID NO: 5 is attached to the first residue of SEQ ID NO: 3. In another example (which may be a substitution or supplement to the above), the C-terminal end of SEQ ID NO: 3 may be attached to the sequence from SEQ ID NO: 5, wherein residue 312 of SEQ ID NO: 5 is attached to the last residue of SEQ ID NO: 3. One or more sequences of SEQ ID NO: 3 sidegated from SEQ ID NO: 5 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 5. The sequence of SEQ ID NO: 3 sidegated may match the sequence within SEQ ID NO: 5, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions. One or more sequences of SEQ ID NO: 5 sidegated with SEQ ID NO: 3 may result in a peptide of length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues. The peptide may also include non-antigenic aspects, such as residues or portions required for labeling and / or conjugation.

[0061] When administered to a subject, the region of the second peptide that is SEQ ID NO: 13 should be able to act as an antigen. To increase this possibility, in some embodiments, the length of the second peptide is equal to or less than 150 residues. This increases the likelihood that the peptide does not take on a structure that masks the antigen. In other embodiments, the length of the second peptide is equal to or less than 100 residues, 50 residues, or 30 residues. In some instances, the second peptide may contain no more than 12, 10, 5, 4, 3, 2, or 1 additional residue at the N-terminus and / or C-terminus of SEQ ID NO: 13. In some embodiments, the second peptide contains only SEQ ID NO: 13, and residues or portions required for non-antigenic aspects such as labeling and / or conjugation. In one instance, the second peptide may contain only SEQ ID NO: 13, β-alanine or other labeling, and cysteine ​​for conjugation with a carrier protein. The second peptide may contain β-alanine and cysteine ​​at the N-terminus or C-terminus of the peptide, wherein the cysteine ​​is at the end of the peptide.

[0062] When administered to a subject, the region of the third peptide that is SEQ ID NO: 14 should be able to act as an antigen. To increase this possibility, in some embodiments, the length of the third peptide is equal to or less than 150 residues. This increases the likelihood that the peptide does not take on a structure that masks the antigen. In other embodiments, the length of the third peptide is equal to or less than 100 residues, 50 residues, or 30 residues. In some instances, the third peptide may contain no more than 12, 10, 5, 4, 3, 2, or 1 additional residue at the N-terminus and / or C-terminus of SEQ ID NO: 14. In some embodiments, the third peptide contains only SEQ ID NO: 14, and residues or portions required for non-antigenic aspects such as labeling and / or conjugation. In one instance, the third peptide may contain only SEQ ID NO: 14, β-alanine or other labeling, and cysteine ​​for conjugation with a carrier protein. The third peptide may contain β-alanine and cysteine ​​at the N-terminus or C-terminal end of the peptide, wherein the cysteine ​​is at the end of the peptide.

[0063] The second peptide may comprise the sequence of SEQ ID NO: 13 side-mounted on any one or both sides. In some instances, these sequences are derived from bacteria GAPDH, such as bacterial species that induce sepsis. For example, the sequence may be derived from any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 13 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 13 may match the sequence within any of SEQ ID NO: 4 to 8, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions.

[0064] In one particular instance, the N-terminal end of SEQ ID NO: 13 may be attached to a sequence from SEQ ID NO: 7, wherein residue 58 of SEQ ID NO: 7 is attached to the first residue of SEQ ID NO: 13. An example of such an addition is provided below: …YDTTQGRFDGTV EVKEGGFEVNGKFIKVSA (SEQ ID NO: 17).

[0065] In another instance (which could be a supplement to the above), the C-terminal end of SEQ ID NO: 13 may be attached to the sequence from SEQ ID NO: 7, wherein residue 77 of SEQ ID NO: 7 is attached to the last residue of SEQ ID NO: 13. An example of such an addition is provided below: EVKEGGFEVNGKFIKVSA ERDPEQIDWATD… (SEQ ID NO: 18).

[0066] One or more sequences derived from SEQ ID NO: 7 and side-joined with SEQ ID NO: 13 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 7. The sequence side-joined with SEQ ID NO: 13 may match the sequence within SEQ ID NO: 7, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions. One or more sequences of SEQ ID NO: 7 side-joined with SEQ ID NO: 13 may result in a peptide of length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues. The peptide may additionally include non-antigenic aspects, such as residues or portions required for labeling and / or conjugation.

[0067] The third peptide may comprise the sequence of SEQ ID NO: 14 side-mounted on any one or both sides. In some instances, these sequences are derived from bacteria GAPDH, such as bacterial species that induce sepsis. For example, the sequence may be derived from any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 14 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with any of SEQ ID NO: 4 to 8. The sequence side-mounted with SEQ ID NO: 14 may match the sequence within any of SEQ ID NO: 4 to 8, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions.

[0068] In one particular instance, the N-terminal end of SEQ ID NO: 14 may be attached to a sequence from SEQ ID NO: 6, wherein residue 252 of SEQ ID NO: 6 is attached to the first residue of SEQ ID NO: 14. An example of such an addition is provided below: …LTELTVVLEKQ DVTVEQVNEAMKNASNESF (SEQ ID NO: 19).

[0069] In another instance (which could be a supplement to the above), the C-terminal end of SEQ ID NO: 14 can be attached to the sequence from SEQ ID NO: 6, wherein residue 272 of SEQ ID NO: 6 is attached to the last residue of SEQ ID NO: 14. An example of such an addition is provided below: DVTVEQVNEAMKNASNESF GYTEDEIVSS… (SEQ ID NO: 20).

[0070] One or more sequences derived from SEQ ID NO: 6 and side-joined with SEQ ID NO: 14 may have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 6. The sequence side-joined with SEQ ID NO: 14 may match the sequence within SEQ ID NO: 6, accompanied by 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 or no substitutions, insertions, or deletions. One or more sequences of SEQ ID NO: 6 side-joined with SEQ ID NO: 14 may result in a peptide of length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues. The peptide may additionally include non-antigenic aspects, such as residues or portions required for labeling and / or conjugation.

[0071] The desired peptide does not contain any self-antigens associated with the subject to be treated. Therefore, in some embodiments, the first, second, and / or third peptides of the fourth aspect do not contain human self-antigens. In some instances, this is achieved by avoiding any adjacent sequences comprising the eight amino acids from SEQ ID NO: 9. The peptide may contain fewer than 7, 6, 5, 4, or 3 residues from adjacent sequences within SEQ ID NO: 9.

[0072] The first, second, and / or third peptides can be isolated peptides. Isolated peptides can be separated from any sequence and / or substance associated with them in nature. Isolated peptides can be part of a purified composition in which other essential components are characterized. Isolated peptides can be linked to or fused with other residues or other polypeptide sequences.

[0073] The isolated first, second, and / or third peptides may optionally be attached to a labeled, identified, or quantified portion or residue of the accessory peptide. For example, a β-alanine residue may be included in each of the isolated peptides. In some instances, the β-alanine residue is directly attached to the first or last residue of SEQ ID NO: 3, SEQ ID NO: 13, and / or SEQ ID NO: 14.

[0074] The isolated first, second, and / or third peptides may optionally be linked to portions or residues of the accessory peptide or conjugated with other parts, residues, or peptides. For example, cysteine ​​residues may be included in the isolated peptides. In specific instances, cysteine ​​residues may be N-terminal or C-terminal residues of the first, second, and / or third peptides. Cysteine ​​residues may be suitable for conjugating carrier proteins with peptides in a fourth aspect.

[0075] In some embodiments, the peptide within the composition or kit of the fourth aspect is linked to a carrier protein. Examples of carrier proteins include KLH, CRM, and TT. In a particular embodiment, the carrier protein is KLH. As discussed, KLH can be or may contain one or more subunits of KLH. The carrier protein can be or may contain multiple subunits of KLH.

[0076] The first peptide, second peptide, and / or third peptide may be linked to the carrier protein via the aforementioned cysteine ​​residues. In one particular embodiment, the first peptide, second peptide, and / or third peptide are linked to KLH via a C-terminal cysteine ​​residue.

[0077] The carrier protein can be conjugated to the peptide at a ratio of 1:4, 1:3, 1:2, or 1:1 (w / w), or any range between these values, such as a carrier protein:peptide ratio from 1:2 to 1:1. The carrier protein can be conjugated to the peptide at a 1:1 (w / w) ratio. In one particular embodiment, the peptide is conjugated to KLH at a 1:1 ratio.

[0078] In one embodiment, the first peptide comprises SEQ ID NO: 12. This sequence may form the C-terminal end of the first peptide, such that cysteine ​​is C-terminus. The first peptide may have a sequence according to SEQ ID NO: 12 only. The first peptide may have a sequence according to SEQ ID NO: 12, wherein the first residue of SEQ ID NO: 12 is N-terminus and the last residue of SEQ ID NO: 12 is C-terminus. The first peptide may be conjugated to a carrier protein such as KLH via cysteine.

[0079] In one embodiment, the second peptide comprises EVKEGGFEVNGKFIKVSA(bALA)C (SEQ ID NO: 15). This sequence may form the C-terminal end of the second peptide, such that cysteine ​​is C-terminus. The second peptide may have a sequence according to SEQ ID NO: 15 only. The second peptide may have a sequence according to SEQ ID NO: 15, wherein the first residue of SEQ ID NO: 15 is N-terminus and the last residue of SEQ ID NO: 15 is C-terminus. The second peptide may be conjugated to a carrier protein such as KLH via cysteine.

[0080] In one embodiment, the third peptide comprises DVTVEQVNEAMKNASNESF(bALA)C (SEQ ID NO:16). This sequence may form the C-terminal end of the third peptide, such that cysteine ​​is C-terminus. The third peptide may have a sequence according to SEQ ID NO:16 only. The third peptide may have a sequence according to SEQ ID NO:16, wherein the first residue of SEQ ID NO:16 is N-terminus and the last residue of SEQ ID NO:16 is C-terminus. The third peptide may be conjugated to a carrier protein such as KLH via cysteine.

[0081] In some embodiments, the compositions and kits of the third and fourth aspects include only the listed peptides and exclude other peptides.

[0082] In one particular embodiment, a composition or kit is provided that comprises three types of peptides and does not contain other types of peptides, wherein the three types of peptides are: Contains the first peptide of SEQ ID NO: 3; Contains a second peptide of SEQ ID NO: 13; and Contains the third peptide of SEQ ID NO: 14.

[0083] In one particular embodiment, a composition is provided comprising: A first peptide comprising SEQ ID NO: 3 and conjugated to a carrier protein such as KLH; A second peptide comprising SEQ ID NO: 13 and conjugated to a carrier protein such as KLH; and A third peptide containing SEQ ID NO: 14 and conjugated to a carrier protein such as KLH.

[0084] In one particular embodiment, a composition is provided comprising: The first peptide containing SEQ ID NO: 3 and a terminal cysteine ​​residue, and conjugated to a carrier protein such as KLH; A second peptide containing SEQ ID NO: 13 and a terminal cysteine ​​residue, and conjugated to a carrier protein such as KLH; and A third peptide containing SEQ ID NO: 14 and a terminal cysteine ​​residue, and conjugated to a carrier protein such as KLH.

[0085] In one particular embodiment, a composition is provided comprising: According to the first peptide of SEQ ID NO: 12, the β-alanine listed therein is optional; According to the second peptide of SEQ ID NO: 15, the β-alanine listed therein is optional; and According to the third peptide of SEQ ID NO: 16, the β-alanine listed therein is optional.

[0086] In one particular embodiment, a composition is provided comprising: According to the first peptide of SEQ ID NO: 12, wherein the listed β-alanine is optional, wherein the first peptide is conjugated to a carrier protein such as KLH via the listed cysteine. According to the second peptide of SEQ ID NO: 15, wherein the listed β-alanine is optional, wherein the second peptide is conjugated to a carrier protein such as KLH via the listed cysteine; and According to the third peptide of SEQ ID NO: 16, wherein the listed β-alanine is optional, wherein the third peptide is conjugated to a carrier protein such as KLH via the listed cysteine.

[0087] In one particular embodiment, a composition is provided comprising: A first peptide consisting of the amino acid sequence according to SEQ ID NO: 12, wherein the first peptide is conjugated to KLH via the listed cysteine ​​residues; A second peptide comprising the amino acid sequence according to SEQ ID NO: 15, wherein the second peptide is conjugated to KLH via the listed cysteine ​​residues; and A third peptide consisting of the amino acid sequence according to SEQ ID NO: 16, wherein the third peptide is conjugated to KLH via the listed cysteine ​​residues.

[0088] The amino acid sequence of GAPDH from GBS (UniProt accession number Q8E3E8) is as follows: - MVVKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPNMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGQFVKVSAEREPANIDWATDGVEIVLEATGFFASKEKAEQHIHENGAKKVVITAPGGNDVKTVVFNTNHDILDGTETVISGASCTTNCLAPMAKALQDNF GVKQGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVIPELNGKLDGAAQRVPVPTGSVTELVATLEKDVTVEEVNAAMKAAANDSYGYTEDPIVSSDIVGISYGSLFDATQTKVQTVDGNQLVKVVSWYDNEMSYTSQLVRTLEYFAKIAK (SEQ ID NO: 4).

[0089] The amino acid sequence of GAPDH from *Escherichia coli* (UniProt accession number D5D2F1) is as follows: - MSKVGINGFGRIGRLVLRRLLEVKSNIDVVAINDLTSPKILAYLLKHDSNYGPFPWSVDYTEDSLIVNGKSIAVYAEKEAKNIPWKAKGAEIIVECTGFYTSAEKSQAHLDAGAKKVLISAPAGEMKTIVYNVNDDTLDGNDTIVSVASCTTNCLAPMAKALHDSF GIEVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENIIPHTTGAAKAIGLVIPELSGKLKGHAQRVPVKTGSVTELVSILGKKVTAEEVNNALKKATNNNESFGYTDEEIVSSDIIGSHFGSVFDATQTEITAVGDLQLVKTVAWYDNEYGFVTQLIRTLEKFAKL (SEQ ID NO: 5).

[0090] The amino acid sequence of GAPDH from Staphylococcus aureus (UniProt accession number A6QF81) is as follows: MAVKVAINGFGRIGRLAFRRIQEVEGLEVVAVNDLTDDDMLAHLLKYDTMQGRFTGEVEVVDGGFRVNGKEVKSFSEPDASKLPWKDLNIDVVLECTGFYTDKDKAQAHIEAGAKKVLISAPATGDLKTIVFNTNHQELDGSETVVSGASCTTNSLAPVAKVLNDDFG LVEGLMTTIHAYTGDQNTQDAPHRKGDKRRARAAAENIIPNSTGAAKAIGKVIPEIDGKLDGGAQRVPVATGSLTELTVVLEKQDVTVEQVNEAMKNASNESFGYTEDEIVSSDVVGMTYGSLFDATQTRVMSVGDRQLVKVAAWYDNEMSYTAQLVRTLAYLAELSK (SEQ ID NO: 6).

[0091] Although only the GAPDH sequence from Staphylococcus aureus is provided here, all available GAPDH sequences from Staphylococcus species have more than 98% sequence similarity.

[0092] The amino acid sequence of GAPDH from Streptococcus pneumoniae (UniProt accession number Q97NL1) is as follows: - MVVKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPVMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGKFIKVSAERDPEQIDWATDGVEIVLEATGFFAKKEAAEKHLKGGAKKVVITAPGGNDVKTVVFNTNHDVLDGTETVISGASCTTNCLAPMAKALQDNF GVVEGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVIPELNGKLDGSAQRVPTPTGSVTELVAVLEKNVTVDEVNAAMKAASNESYGYTEDPIVSSDIVGMSYGSLFDATQTKVLDVDGKQLVKVVSWYDNEMSYTAQLVRTLEYFAKIAK (SEQ ID NO: 7).

[0093] The amino acid sequence of GAPDH from Klebsiella pneumoniae (UniProt accession number B5XRG0) is as follows: - MSKLGINGFGRIGRLVLRRLLEVDSSLEVVAINDLTSPKVLAYLLKHDSNYGPFPWSVDFTEDALIVNGKTITVYAEKEAQHIPWQAAGAEVIVECTGFYTSAEKSQAHIQAGARKVLISAPAGEMKTIVYNVNDDTLTPDDTIISVASCTTNCLAPMAKVLQDAF GITVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENVIPHTTGAAKAIGLVIPALSGKLKGHAQRVPTKTGSVTELVSVLEKKVTADEVNQAMKQAAEGNESFGYTEEEIVSSDIIGSHFGSIYDATQLEIVEAGGVQLVKTVAWYDNEYGFVTQLIRVLEKFAR (SEQ ID NO: 8).

[0094] The amino acid sequence of human GAPDH (UniProt accession number P04406) is as follows: MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDN FGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE (SEQ ID NO: 9).

[0095] This disclosure extends to nucleic acid molecules encoding peptides, fusion proteins, and peptides in the compositions and kits of the present invention.

[0096] Therefore, according to the fifth aspect, one or more nucleic acids are provided that encode a peptide according to the first aspect, a fusion protein according to the second aspect, a peptide and / or fusion protein contained in a composition or kit of the third aspect, or a peptide and / or fusion protein contained in a composition or kit of the fourth aspect.

[0097] The nucleic acid encoding a peptide based on the first aspect or a fusion protein based on the second aspect can be a single nucleic acid molecule.

[0098] The fifth aspect, one or more nucleic acids, can be a nucleic acid molecule encoding all peptides or fusion proteins within the composition or kit of the third or fourth aspect. Peptides and / or fusion proteins can be encoded in such a manner that they are translated into separate molecules. Peptides and / or fusion proteins can be separated by cleavable sequences. A single nucleic acid can be cleaved, or a polypeptide that is a precursor to a peptide and / or fusion protein can be cleaved.

[0099] The fifth aspect, one or more nucleic acids, can be multiple nucleic acids, wherein each individual nucleic acid molecule encodes one of the peptides or fusion proteins within the composition or kit of the third or fourth aspect. Thus, one or more nucleic acids may comprise a first nucleic acid molecule encoding a first peptide or fusion protein, a second nucleic acid molecule encoding a second peptide or fusion protein, and a third nucleic acid molecule encoding a third peptide or fusion protein.

[0100] One or more nucleic acids may be isolated and may be recombinant or synthetic. In some instances, one or more nucleic acids are chemically modified, for example, via the inclusion of modified nucleotides. One or more nucleic acids may contain at least one modified sugar moiety, at least one modified internucleotide bond, or at least one modified nucleobase. One or more nucleic acids may be nucleic acid analogs, which may be compounds having a nucleobase arrangement that mimics the nucleobase arrangement in DNA or RNA. Compared to naturally occurring nucleic acids, nucleic acid analogs may have a modified backbone.

[0101] In some instances, one or more nucleic acids are operatively linked to a heterologous promoter. In some instances, one or more nucleic acids are bound to a substrate or label or similar substance. Such modifications are common in the art and are known to those skilled in the art.

[0102] One or more nucleic acids may encode one or more genetic constructs. The genetic construct may be in the form of an expression cassette adapted for the expression of the encoded peptide in cells. The genetic construct may be introduced into cells without being incorporated into a vector. For example, a genetic construct, which may be a nucleic acid molecule, may be incorporated into a liposome or viral vector. Alternatively, purified nucleic acid molecules (e.g., histone-free DNA or naked DNA) may be directly inserted into cells by suitable means (e.g., direct endocytosis). The genetic construct may be introduced directly into the cells of a host subject (e.g., bacterial cells) via transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, or gene gun bombardment. Alternatively, the genetic construct may be contained within a recombinant vector for expression in suitable host cells.

[0103] Therefore, in the sixth aspect, one or more vectors are provided that contain one or more nucleic acids according to the fifth aspect.

[0104] Suitable vectors include lipid nanoparticles, micelles, exosomes, viral vectors, non-viral vectors, plasmids, granules, and bacteriophages. Those skilled in the art will understand that the nucleic acids of this disclosure can be combined with a variety of main-chain vectors for expression purposes. Vectors may include various other functional elements, including suitable promoters to initiate gene expression. For example, a vector may be designed to replicate autonomously in the cytosol of a host cell. In this case, elements that induce or regulate DNA replication may be required in the vector. Alternatively, a vector may be designed to integrate into the host cell's genome or not.

[0105] Therefore, in the seventh aspect, a host cell is provided, which contains one or more nucleic acids as described in the fifth aspect, or one or more vectors as described in the sixth aspect.

[0106] The host cell can be a bacterial cell, such as *Escherichia coli*. Alternatively, the host cell can be an animal cell, such as a mouse or rat cell. In some instances, the host cell is not a human cell. Using known techniques, the host cell can be transformed with the nucleic acids or vectors disclosed herein. The appropriate means for introducing genetic constructs into the host cell will depend on the cell type.

[0107] In the eighth aspect, one or more pharmaceutical compositions are provided, comprising a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third or fourth aspect, a peptide according to a kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, or one or more carriers of the sixth aspect.

[0108] The third or fourth aspect kit may contain separate pharmaceutical compositions, each containing a peptide within the third or fourth aspect kit.

[0109] A pharmaceutical composition may comprise one or more of the following: a pharmaceutically acceptable medium, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable stabilizer, or a pharmaceutically acceptable preservative, or any combination thereof. For it to be pharmaceutically acceptable, the substance or combination of substances must be suitable for the preparation of the pharmaceutical composition or dosage form. A pharmaceutical composition may comprise one or more of the following: a medium, excipient, flavoring agent, lubricant, solubilizer, suspending agent, dye, filler, flow aid, pressure aid, inert binder, sweetener, preservative, coating, or disintegrant.

[0110] The peptide of the first aspect, the fusion protein of the second aspect, the composition of the third or fourth aspect, the peptide in the kit of the third or fourth aspect, the nucleic acid of the fifth aspect, the carrier of the sixth aspect, or the pharmaceutical composition of the eighth aspect can be used to generate a protective immune response against one or more bacterial species that induce sepsis.

[0111] Therefore, in the ninth aspect, one or more immunogenic compositions are provided, comprising a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third or fourth aspect, a peptide of a kit according to the third or fourth aspect, one or more nucleic acids of the fifth aspect, or one or more vectors of the sixth aspect.

[0112] The third or fourth aspect kit may contain separate immunogenic compositions, each containing a peptide within the third or fourth aspect kit.

[0113] The ninth aspect is that the immunogenic composition can be a vaccine.

[0114] The immunogenic composition of the ninth aspect may comprise any peptide of the first aspect, a fusion protein of the second aspect, a composition of the third aspect, or a composition of the fourth aspect. In a particular embodiment, the immunogenic composition of the ninth aspect comprises a first peptide comprising the sequence according to SEQ ID NO: 3; a second peptide comprising the sequence according to SEQ ID NO: 13; and a third peptide comprising the sequence according to SEQ ID NO: 14. As discussed herein, these peptides may comprise further portions, such as cysteine ​​residues for conjugation to a carrier protein or β-alanine residues for labeling. An exemplary carrier protein is KLH. Therefore, the immunogenic composition of the ninth aspect may comprise KLH.

[0115] In other embodiments, the immunogenic composition of the ninth aspect may comprise any nucleic acid of the fifth aspect or the carrier of the sixth aspect. In such embodiments, the nucleic acid and the carrier encode an antigenic peptide. Thus, such immunogenic compositions may be delivery vehicles for delivering nucleic acids such as mRNA, such as lipid nanoparticles, wherein the nucleic acids encode peptides, fusion proteins, or combinations of multiple peptides and / or fusion proteins as disclosed herein.

[0116] Immunogenic compositions may contain excipients that can act as adjuvants. Thus, in one embodiment, the immunogenic composition may contain particulate adjuvants, such as liposomes or immunostimulatory complexes (ISCOM). Other examples of adjuvants include cholera toxin or squalene-like molecules. Any adjuvant can be used, such as aluminum hydroxide (alum), tetanus toxoid, or diphtheria toxin. The mediator can be suitably used as an adjuvant, and may include, but is not limited to, water, phosphate-buffered saline (PBS), polyols, or dextran solutions.

[0117] The compositions of the eighth or ninth aspect can be formulated for specific routes of delivery. For example, they can be formulated for intramuscular, subcutaneous, intravenous, oral, intranasal, or intradermal delivery. The compositions can be formulated for delivery to specific subjects, such as newborns, infants, children, women of childbearing age, pregnant women, fetuses, elderly subjects, or people with diabetes.

[0118] In the tenth aspect, the peptide of the first aspect, the fusion protein of the second aspect, the composition of the third or fourth aspect, the kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, one or more pharmaceutical compositions of the eighth aspect, or one or more immunogenic compositions of the ninth aspect are provided for stimulating an immune response.

[0119] Preferably, the immune response includes the production of GAPDH-specific antibodies against one or more species of bacteria that induce sepsis. The bacteria that induce sepsis may be GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, and / or Klebsiella pneumoniae. In one embodiment, the Staphylococcus species is Staphylococcus aureus. Therefore, the GAPDH for which the antibody is specific may be one or more of those GAPDHs having the amino acid sequences provided as SEQ ID NO: 4 to 8. In a particular embodiment, the immune response may include the production of antibodies specific to the respective GAPDHs of GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, and Klebsiella pneumoniae.

[0120] Since GAPDH is a ubiquitous protein and is conserved in sepsis-inducing bacteria, it can stimulate an immune response that provides protection against all different serotypes of sepsis-inducing bacteria, which is advantageous.

[0121] The tenth application can be for in vitro, in vivo, or ex vivo use.

[0122] The tenth aspect of use may be for in vitro or ex vivo antibody production. Such use may involve the interaction of peptides, fusion proteins, or compositions as disclosed herein with in vitro or ex vivo antibody-producing cells, enabling the production of GAPDH-specific antibodies against one or more species of bacteria that induce sepsis. Suitable antibody-producing cells and techniques for producing antibodies are described in the art and will be known to those skilled in the art.

[0123] In another implementation, the tenth aspect is for in vivo use, namely for stimulating an immune response in a subject. The immune response may be a protective immune response against one or more bacterial species that induce sepsis.

[0124] In the eleventh aspect, a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third or fourth aspect, a peptide of a kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more carriers of the sixth aspect, a pharmaceutical composition of the eighth aspect, or an immunogenic composition of the ninth aspect are provided for use as pharmaceutical agents.

[0125] As discussed above, the peptides in the kits of the third or fourth aspect can be used as pharmaceutical agents. Therefore, in one embodiment, a peptide is provided for use in a treatment method, comprising the sequence according to SEQ ID NO: 3, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 3, wherein the method comprises administering said peptide and further administering: Contains a peptide according to the sequence of SEQ ID NO: 13, or has one or more additions, substitutions or deletions of the sequence according to SEQ ID NO: 13; and / or A peptide comprising the sequence according to SEQ ID NO: 14, or having one or more added, substituted or deleted sequences according to SEQ ID NO: 14.

[0126] In another embodiment, a peptide is provided for use in a therapeutic treatment, comprising the sequence according to SEQ ID NO: 13, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 13, wherein the method comprises administering the peptide and further administering: A peptide comprising the sequence according to SEQ ID NO: 3, or having one or more additions, substitutions, or deletions of the sequence according to SEQ ID NO: 3; and A peptide comprising the sequence according to SEQ ID NO: 14, or having one or more added, substituted or deleted sequences according to SEQ ID NO: 14.

[0127] In a further embodiment, a peptide is provided for use in a treatment method, comprising the sequence according to SEQ ID NO: 14, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 14, wherein the method comprises administering the peptide and further administering: A peptide comprising the sequence according to SEQ ID NO: 3, or having one or more additions, substitutions, or deletions of the sequence according to SEQ ID NO: 3; and A peptide comprising the sequence according to SEQ ID NO: 13, or having one or more added, substituted or deleted sequences according to SEQ ID NO: 13.

[0128] In one embodiment, the peptide of the first aspect, the fusion protein of the second aspect, the composition of the third or fourth aspect, the peptide of the kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more carriers of the sixth aspect, the pharmaceutical composition of the eighth aspect, or the immunogenic composition of the ninth aspect can be used to treat infection caused by one or more species of bacteria that induce sepsis, prevent said infection, reduce the risk of said infection, or reduce the severity of said infection.

[0129] In one embodiment, a method is provided for treating infection caused by one or more species of bacteria that induce sepsis, preventing said infection, reducing the risk of said infection, or reducing the severity of said infection, the method comprising administering to a subject a therapeutically effective amount of a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third or fourth aspect, a peptide of a reagent kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more carriers of the sixth aspect, a pharmaceutical composition of the eighth aspect, or an immunogenic composition of the ninth aspect.

[0130] The "therapeutic effective amount" of a reagent is any amount that, when administered to a subject, is the amount of reagent required to treat an infection or related pathological condition, prevent it, reduce its risk, or reduce its severity, or to produce the desired effect.

[0131] The peptides, fusion proteins, compositions, nucleic acids, carriers, or immunogenic compositions disclosed herein can prevent systemic infections caused by one or more of at least five different pathogens, which are the most common causes of sepsis, preferably: GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, and Klebsiella pneumoniae. In one embodiment, the Staphylococcus species is Staphylococcus aureus. The peptides, fusion proteins, compositions, kits, nucleic acids, carriers, and immunogenic compositions disclosed herein can be used to treat infections caused by any one or more of the said bacteria, prevent said infections, reduce the risk of said infections, or reduce the severity of said infections. The peptides, fusion proteins, compositions, kits, nucleic acids, carriers, and immunogenic compositions disclosed herein can be used to treat infections caused by GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, and Klebsiella pneumoniae, prevent said infections, reduce the risk of said infections, or reduce the severity of said infections.

[0132] Specifically, the peptide of the first aspect, the fusion protein of the second aspect, the composition of the third or fourth aspect, the peptide of the kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more carriers of the sixth aspect, the pharmaceutical composition of the eighth aspect, or the immunogenic composition of the ninth aspect can be used to treat infectious diseases caused by bacterial species that induce sepsis, prevent said infectious diseases, reduce the risk of said infectious diseases, or reduce the severity of said infectious diseases.

[0133] The peptides, fusion proteins, compositions, kits, nucleic acids, carriers, pharmaceutical compositions, or immunogenic compositions disclosed herein may be used to treat, prevent, reduce the risk of, or reduce the severity of sepsis or any other disease, condition, or condition caused by or associated with an infection of a bacterial species that induces sepsis. These other diseases, conditions, or conditions include pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, and encephalitis.

[0134] Therefore, in one embodiment, a method is provided to treat, prevent, reduce the risk of, or reduce the severity of sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infection, soft tissue infection, gastrointestinal infection, bloodstream infection, or encephalitis, the method comprising administering to a subject a therapeutically effective amount of a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third or fourth aspect, a peptide of a reagent kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more carriers of the sixth aspect, a pharmaceutical composition of the eighth aspect, or an immunogenic composition of the ninth aspect.

[0135] Preterm birth and stillbirth can be caused by an exacerbated inflammatory response induced by bacterial infection. The most common factors in cases of bacterial-induced preterm birth and stillbirth are GBS, Escherichia coli, and Klebsiella pneumoniae, i.e., the sepsis-inducing bacteria described herein. Preterm birth and stillbirth can be caused by intrauterine infection due to bacteria (e.g., GBS, Escherichia coli, and Klebsiella species) ascending from the reproductive tract into the amniotic fluid. Immunization can be provided to unborn offspring through vaccination of the expectant mother. The fetus and newborn can therefore be protected from infection through maternal vaccination.

[0136] Therefore, in one embodiment, the peptides, fusion proteins, compositions, kits, nucleic acids, carriers, pharmaceutical compositions or immunogenic compositions disclosed herein are used to prevent or reduce the risk of preterm birth and / or stillbirth.

[0137] In one particular embodiment, the peptides, fusion proteins, compositions, kits, nucleic acids, vectors, pharmaceutical compositions, or immunogenic compositions of this disclosure are used to prevent or reduce the risk of infection-related preterm birth and / or stillbirth caused by GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, or Klebsiella pneumoniae. In one embodiment, the Staphylococcus species is Staphylococcus aureus. In another embodiment, the peptides, fusion proteins, compositions, kits, nucleic acids, vectors, pharmaceutical compositions, or immunogenic compositions of this disclosure are used to prevent or reduce the risk of infection-related preterm birth and / or stillbirth caused by GBS, Escherichia coli, or Klebsiella pneumoniae.

[0138] The administration of peptides, fusion proteins, compositions, peptides, nucleic acids, carriers, pharmaceutical compositions, or immunogenic compositions in kits can be directed at women of childbearing age. Boosters can be delivered in late pregnancy. Unborn infants (fetuses) benefit from passive immunization acquired when their mothers' antibodies cross the placenta to reach the developing child, especially in late pregnancy. As illustrated by examples, the vaccines of the present invention can also prevent or reduce the risk of preterm birth and stillbirth due to intrauterine infections caused by bacteria (e.g., GBS, Escherichia coli, and Klebsiella species) ascending from the reproductive tract into the amniotic fluid.

[0139] The subject to be treated can be any suitable subject, such as any subject susceptible to the diseases or conditions discussed herein. The subject can be a vertebrate, mammal, or livestock. Therefore, administration can be to any mammal, such as livestock (e.g., a horse), pet, or can be used in other veterinary applications. More preferably, the subject is a human.

[0140] Particularly relevant subject groups include those at greater risk of infection, such as immunocompromised individuals, newborns, infants, children, women of childbearing age, pregnant women, fetuses, elderly subjects, and individuals with diabetes.

[0141] Peptides, fusion proteins, compositions, peptides, nucleic acids, carriers, pharmaceutical compositions, or immunogenic compositions of kits can be administered before, during, or after a bacterial infection. The treatments disclosed herein can be prophylactic; that is, they can be used to prevent infection, reduce the risk of infection, or reduce the severity of infection, including preventing recurrence / recolonization of previous infections, reducing their risk, or reducing their severity. Peptides, fusion proteins, compositions, peptides, nucleic acids, carriers, or immunogenic compositions of kits disclosed herein can be administered to those who have already shown signs of infection, those considered immunocompromised, or those at greater risk of infection. Administration can be directed to seemingly healthy subjects as a purely preventative measure against the possibility of future such infections. For example, it can be administered as part of a general vaccination program or in programs targeting immunocompromised individuals, newborns, infants, children, women of childbearing age, pregnant women, fetuses, the elderly, and people with diabetes.

[0142] As used in this article, "immunocompromised" encompasses individuals with a genetic or acquired deficiency in the immune system, as well as those with reduced immune function due to their stage of life. For example, newborns, toddlers, children, women of childbearing age, pregnant women, fetuses, the elderly, and people with type 1 diabetes can all be considered immunocompromised.

[0143] As used herein, the terms “newborn” and “newborn” can refer to human children from birth to about one month of age. The terms apply to premature, post-term, and full-term infants. Before birth, the term “fetus” is used. As used herein, the terms “toddler” and “infant” can refer to young children from about one month to about one or two years of age (i.e., the age when a child is learning to walk and talk, at which point the term “walking child” may be used instead). As used herein, the term “child” refers to young children, covering those from walking children to about 12 years of age (i.e., pre-puberty). As used herein, the term “elderly” refers to subjects of advanced age. For example, it can refer to men and women aged 60 or older, 65 or older, 70 or older, 75 or older, or 80 or older. Non-human subjects in the corresponding later stages of life are also covered by this term. In one instance, as used herein, the term “diabetic patient” refers to an individual with type 1 diabetes (also known as juvenile diabetes or insulin-dependent diabetes) or type 2 diabetes. People with type 1 diabetes can be considered immunocompromised, and this invention is also relevant to people with type 2 diabetes because they may be at greater risk of infection.

[0144] Peptides, fusion proteins, compositions, peptides in kits, nucleic acids, carriers, pharmaceutical compositions, or immunogenic compositions may be administered by any suitable means, including injection or transmucosal administration. Administration may be intramuscular, subcutaneous, intravenous, oral, intranasal, or intradermal.

[0145] The route of administration can vary depending on the desired protective mechanism. For example, if the reagents disclosed herein are administered transmucosally, they can induce an IgA response at the mucosal surface. IgA can block the binding of bacterial GAPDH to the host cell epithelium. If the reagents are administered systemically, they can induce an IgG response that blocks the binding of bacterial GAPDH to TLR2 on the surface of B1 cells and prevents early IL-10 production through these cells.

[0146] When administering peptides from a third or fourth aspect kit, the peptides can be administered simultaneously or sequentially. Peptides can be delivered on the same day or on different days. In a particular embodiment, the peptides are administered as a mixture. In a more specific embodiment, the peptides are administered as a single composition.

[0147] It should be understood that the reagents disclosed herein can be used as monotherapy to treat, improve, or prevent infections or related pathological conditions caused by bacteria that induce sepsis. Alternatively, the reagents disclosed herein can be used as adjuncts to or in combination with known therapies. For example, known reagents for treating bacterial infections that induce sepsis. For example, peptides, fusion proteins, compositions, nucleic acids, carriers, pharmaceutical compositions, and immunogenic compositions can be used in combination with known reagents for treating neonatal sepsis caused by fungi or viruses. They can be used in combination with known antiretroviral reagents.

[0148] The reagents disclosed herein can be administered alone or concurrently with other existing vaccines. For example, administration can be concurrently with vaccines recommended for immunocompromised individuals, infants, children, women of childbearing age, pregnant women, the elderly, and people with diabetes (e.g., tetanus and diphtheria vaccines).

[0149] There are no limitations on which peptide, fusion protein, composition, kit peptide, nucleic acid, carrier, pharmaceutical composition, or immunogenic composition described herein should be administered to which patient. Rather, it is contemplated that any product described herein can be administered to any patient as described herein. In fact, the inventors expressly intend that this disclosure covers every and every combination of peptide, fusion protein, composition, kit peptide, nucleic acid, carrier, pharmaceutical composition, or immunogenic composition with the indicated patient population. This disclosure therefore includes every and every possible combination of therapeutic agents with the indicated patient population.

[0150] It should be understood that the required amount of peptides, fusion proteins, compositions, nucleic acids, carriers, pharmaceutical compositions, or immunogenic compositions is determined by their bioactivity and bioavailability, which in turn depend on the administration modality, physicochemical properties, and whether it is used as a monotherapy or in combination therapy. Administration frequency is also affected by the half-life of the agent in the subject to be treated. The optimal dose to be administered can be determined by those skilled in the art and will vary depending on the specific agent used, the strength of the pharmaceutical composition, the administration modality, and the progression of the bacterial infection. Additional factors depending on the specific subject to be treated may necessitate dose adjustments, including the subject's age, weight, sex, diet, and timing of administration.

[0151] Sequence comparisons can be performed using readily available sequence alignment programs. These publicly available and commercially available computer programs can calculate sequence identity between two or more sequences.

[0152] Technicians should understand how to calculate the percentage identity between two nucleic acid sequences or two amino acid sequences. To calculate the percentage identity, the two sequences must first be aligned, followed by the calculation of the sequence identity value. The percentage identity of the two sequences depends on the following and can take different values: (i) the method used to align the sequences, such as the Needleman-Wunsch algorithm (e.g., applied via Needle (EMBOSS) or Stretcher (EMBOSS), the Smith-Waterman algorithm (e.g., applied via Water (EMBOSS), or the LALIGN application (e.g., applied via Matcher (EMBOSS)); and (ii) the parameters used by the alignment method, such as local alignment versus global alignment, the matrix used, and the parameters applied to gaps.

[0153] Once alignment has been performed, there are different ways to calculate the percentage identity between two sequences. For example, the number of identity sequences can be divided by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-empty positions; or (iv) the number of equivalent positions excluding protruding ends. Furthermore, it should be understood that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences, the higher the expected sequence identity.

[0154] The percentage identity between two nucleic acid sequences can then be calculated by such alignment as (N / T)*100, where N is the number of positions where the sequences share the same residues, and T is the total number of positions compared, including vacancy but excluding overhangs.

[0155] Sequence alignment can be paired sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In one instance, the identity between two amino acid sequences can be calculated using the Needle (EMBOSS) service with default parameters such as matrix (BLOSUM62), vacancy open (10), vacancy extension (0.5), terminal vacancy penalty (false), terminal vacancy open (10), and terminal vacancy extension (0.5). In another instance, the identity between two amino acid sequences can be calculated using the Matcher (EMBOSS) service with default parameters such as matrix (BLOSUM62), vacancy open (14), vacancy extension (4), and substitution match (1). In one instance, the identity between two nucleic acid sequences can be calculated using the service Needle (EMBOSS), which is set with default parameters such as matrix (DNAfull), vacancy open (10), vacancy extension (0.5), end vacancy penalty (false), end vacancy open (10), and end vacancy extension (0.5). In another instance, the identity between two nucleic acid sequences can be calculated using the service Matcher (EMBOSS), which is set with default parameters such as matrix (DNAfull), vacancy open (16), vacancy extension (4), and substitution matching (1).

[0156] In one particular embodiment, the sequence identity disclosed herein can be calculated based on a global alignment of relevant features, such as a comparison of the full length of the antigenic portion of a peptide with the full reference sequence enumerated herein. As an example, sequence identity can be calculated between the full length of a portion of a peptide or fusion protein (which corresponds to a portion of bacterial GAPDH) and any sequence within any of SEQ ID NO: 4 to 8.

[0157] All features described herein (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined with any of the foregoing aspects in any combination, except for at least some mutually exclusive combinations of such features and / or steps.

[0158] To provide a better understanding of the invention and to show how its embodiments can be implemented, reference will now be made to embodiments that are not intended to limit the invention in any way. Example

[0159] Overview Peptides designed to induce an immune response against bacterial GAPDH were obtained. Cysteine ​​was added to all peptides to promote KLH anchoring. Additionally, β-alanine was incorporated into the structure of all peptides in the final formulation to facilitate quality control analysis of peptide production and conjugation.

[0160] Surprisingly, the inventors observed that the specific formulation designated herein as PNV resulted in a synergistic immunogenic effect. The immunogenicity of the formulation against the bacterium GAPDH was higher than that observed with various peptides. This synergistic effect did not occur with the other formulations tested.

[0161] In summary, the experimental data in this paper confirm that: a) Synergistic effects induced by specific combinations of the three peptides; b) Random substitutions and deletions of amino acids within peptides, resulting in stable and immunogenic peptides; c) This particular formulation – consisting of three peptides conjugated individually with KLH in a 1:1: (w / w) ratio – as a prophylactic vaccine has shown superior immunogenicity and protective effect compared to any other peptide combination tested (with or without a different carrier protein).

[0162] Figure 1 Immunogenicity of different conjugated peptide formulations against different bacterial GAPDHs is shown. Serum anti-GAPDH IgG titers obtained from mice by ELISA, the mice being immunized with different formulations of GAPDH peptides conjugated to KLH or CRM, are shown. Adult BALB / c mice (6–8 weeks old) received three sc immunizations with the indicated formulations, with administrations 3 weeks apart. Each formulation contained 100 µg of each conjugated peptide suspended in physiological saline (NaCl 0.9%) and 125 µg of aluminum hydroxide (Alhydrogel 2%) as adjuvant. One week after the last administration, blood was collected and serum IgG titers, i.e., serum dilution values, were determined by ELISA. 450nm ≤ 0.1. The plate was coated with GAPDH of the bacteria indicated on the x-axis.

[0163] Figure 2The immunogenicity of PNV was shown to be higher than the sum of the immunogenicities of the individual peptides. Serum anti-GAPDH IgG titers obtained from mice immunized with PNV or various peptides conjugated to KLH are shown by ELISA. Adult BALB / c mice (6–8 weeks old) received three sc immunizations with PNV or 100 µg of the indicated conjugated peptide [with 125 µg of aluminum hydroxide (Alhydrogel 2%) suspended in physiological saline (NaCl 0.9%) as adjuvant], with 3-week intervals between administrations. One week after the last administration, blood was collected, and serum IgG titers, i.e., serum dilution values, were determined by ELISA. 450nm ≤ 0.1. The plate was coated with GAPDH of the bacteria indicated in the figure. P-values ​​are indicated. ***P ≤ 0.0001; **P ≤ 0.001.

[0164] Figure 3 PNV protects adult mice from bacterial infection. Survival curves are shown after adult mice were infected with the indicated bacteria, either immunized with PNV (green line) or sham-immunized with adjuvant alone (red line). Adult BALB / c mice (6–8 weeks old) received three sc immunizations with PNV or adjuvant alone, with a 3-week interval between administrations. One week after the last immunization, the mice were immunized with 10 7 CFU of Staphylococcus aureus, 10 7 CFU Klebsiella pneumoniae, 10 8 CFU (Colombia pneumoniae), or 10 7 CFU of E. coli was challenged intraperitoneally (ip). At least three independent experiments were performed. P-values ​​are indicated in the graph. The number of surviving animals relative to the total number of infected animals is shown in parentheses. Differences between treatments were evaluated using a log-rank test.

[0165] Figure 4 The maternal vaccination with PNV protects offspring from bacterial infection. Survival curves after infection are shown for pups born to mothers immunized with PNV (green line) or sham-immunized (red line). Adult C57BL / 6 females (6–8 weeks) were sc immunized with PNV or adjuvant alone (sham immunization). Animals received two administrations prior to mating, with a 3-week interval between administrations. A booster immunization was administered on day 14 of gestation. Neonates were given 10 mg of PNV 48 hours after birth. 5 CFU's GBS, 10 6 CFU of Staphylococcus aureus, or 5 x 10 4CFU of *E. coli* was used for sc infection. At least three independent experiments were performed. P-values ​​are indicated in the graph. The number of surviving animals relative to the total number of infected animals is shown in parentheses. Differences between treatments were evaluated using a log-rank test.

[0166] Example 1 - Development of Immunogenic Agents Neutralization of bacterial GAPDH has proven to be a good strategy for preventing infections in mice caused by GBS, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus pneumoniae. A major obstacle to targeting bacterial GAPDH is its relatively high homology with human GAPDH. To overcome this obstacle, the inventors have selected peptides that are exposed on the surface of bacterial GAPDH but are not present in human GAPDH at all. A list of peptides that can target different bacterial GAPDHs without cross-reactivity with human proteins is provided in WO2015 / 189422 A1.

[0167] Six different formulations were generated. Formulation #1 consists of three different peptides representing bacterial GAPDH antigens; formulation #2 consists of two different peptides representing bacterial GAPDH antigens; formulation #3 consists of two different peptides representing bacterial GAPDH antigens; formulation #4 consists of two fusion proteins, each containing two peptides representing bacterial GAPDH antigens; formulation #5 consists of a first peptide according to SEQ ID NO: 3, a second peptide according to SEQ ID NO: 13, and a third peptide according to SEQ ID NO: 14; and formulation #6 consists of four different peptides representing bacterial GAPDH antigens.

[0168] The peptide is chemically synthesized and conjugated to a carrier protein. Keyhole hemocyanin (KLH) or cross-reactive material 197 (CRM) is used as the carrier protein at a 1:1 (w / w) ratio. For conjugation, an additional cysteine ​​residue is added to the C-terminus of the peptide's amino acid sequence to allow chemical conjugation to the carrier protein. Formulations #1 to #5 include KLH as the carrier protein. Formulation #6 includes CRM as the carrier protein.

[0169] Formulations were prepared containing a previously determined amount (100 µg / peptide) of each conjugated peptide in physiological saline (NaCl 0.9%) with 0.125 mg aluminum hydroxide (Alhydrogel 2%, Croda) as an adjuvant. Mice were then immunized subcutaneously (sc) three times with three weeks between administrations of the formulations. Serum anti-GAPDH IgG titers observed after immunization with different formulations are plotted on... Figure 1 middle.

[0170] Interest was found in a particular peptide because it contains a conserved domain in different bacterial GAPDHs. The chemical synthesis of this peptide (TQTEITAVGDLQLVKTVA – SEQ ID NO: 1) yielded an unstable product unsuitable for mouse immunization. Considering that this peptide might constitute an important target for neutralizing common domains in different bacterial GAPDHs, the inventors attempted to synthesize different variants containing random deletions or inclusions of amino acids outside the conserved domain. One of these variants yielded a soluble and stable product (SEQ ID NO: 3), which can be conjugated to KLH and used for mouse immunization. Additionally, the inventors considered chimeras of different GAPDH peptides obtained by splicing two peptides from different bacterial GAPDHs into the same sequence. Formulation #4 consists of two chimeras: the first chimera is formed by fusing a peptide exposed on Staphylococcus aureus GAPDH with a peptide exposed on GBS GAPDH; the second chimera has peptides corresponding to Escherichia coli and Klebsiella pneumoniae GAPDHs.

[0171] like Figure 1 Different formulations induced varying immunogenicity in mice, as observed in the study. Notably, formulation #5 induced higher immunogenicity in mice, as observed through higher anti-GAPDH IgG titers. Introducing new peptides or changing the carrier protein did not improve the immunogenicity of formulation #5. The use of KLH and the specific peptide combination contained in formulation #5 yielded optimal results.

[0172] Given that formulation #5 demonstrated the best immunogenicity in mice, the inventors introduced an additional amino acid, β-alanine (βA), immediately following the terminal cysteine ​​residue in each peptide. The addition of βA improved the analytical procedure, allowing for better quality control measurements. This final formulation was designated as the Paragon Novel Vaccine (PNV). The final amino acid sequences of the peptides are listed in Table 1. The addition of the additional amino acid did not alter the immunogenicity of this formulation, as PNV immunization resulted in the same anti-GAPDH IgG titer as formulation #5. Figure 2 ).

[0173] Example 2 – Confirmation of Synergistic Effect The inventors tested the immunogenicity of each conjugated peptide in PNV individually (Table 1). For this purpose, as mentioned above, different groups of adult mice received three sc administrations of each peptide in physiological saline with 2% Alhydrogel as an adjuvant. Interestingly, the immunogenicity of each peptide against different bacteria GAPDH was significantly lower than that against PNV. In fact, the peptides in the PNV formulation acted synergistically to induce a higher anti-GAPDH IgG titer than the sum of the IgG titers observed for each peptide individually. Figure 2). Figure 2 Serum anti-GAPDH IgG titers are expressed as total counts rather than logarithmic counts. 10 Scaled presentation to allow for the observation of synergistic effects.

[0174] Example 3 - Prevention of Infection Finally, the inventors tested the efficacy of PNV in preventing infections caused by different bacteria that secrete GAPDH. Figure 3 As described in the study, vaccination of adult mice with PNV significantly increased survival after infection with Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, and Klebsiella pneumoniae, compared to control animals receiving adjuvants alone. GBS is almost exclusively associated with neonatal infections. GBS GAPDH has also been described as an immunosuppressive protein strongly associated with neonatal susceptibility to these infections. Because PNV also targets GBS GAPDH, the inventors tested the efficacy of PNV as a maternal vaccine to prevent neonatal bacterial infections caused by GBS. Figure 4 As observed, PNV successfully protected newborns from lethal GBS infection. Furthermore, because Staphylococcus aureus and Escherichia coli are important neonatal pathogens, the inventors demonstrated that maternal vaccination with PNV also protected mouse pups from lethal infections caused by these two bacterial pathogens (Figure 5).

[0175] In summary, these results demonstrate that PNV contains specific formulations of peptides exposed to bacterial GAPDH, which possess synergistic immunogenic capabilities and can effectively protect adult mice from lethal infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae, as well as newborn mice from infections caused by GBS, Staphylococcus aureus, and Escherichia coli.

Claims

1. A peptide comprising the amino acid sequence according to SEQ ID NO: 3 (TQTTEITAVGDQLVKTVA).

2. The peptide of claim 1, wherein the length of the peptide is equal to or less than 150 residues, 100 residues, 50 residues or 30 residues.

3. The peptide according to claim 1 or claim 2, wherein the sequence flanked by SEQ ID NO: 3 is derived from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

4. The peptide according to any of the preceding claims, wherein the sequence side-joined with SEQ ID NO: 3 is derived from SEQ ID NO:

5.

5. The peptide according to any of the preceding claims, wherein the peptide comprises fewer than 8, 7, 6, 5, 4 or 3 adjacent residues from SEQ ID NO:

9.

6. The peptide according to any of the preceding claims, wherein the peptide comprises an N-terminal or C-terminal cysteine.

7. The peptide according to any of the preceding claims, wherein the peptide comprises β-alanine.

8. The peptide according to any of the preceding claims, wherein the peptide comprises SEQ ID NO: 12(TQTTEITAVGDQLVKTVA(bALA)C).

9. The peptide according to any of the preceding claims, wherein the peptide has 0-12 additional amino acids at the N-terminus and / or C-terminus side of SEQ ID NO: 3 or SEQ ID NO:

12.

10. The peptide according to any of the preceding claims, wherein the peptide is according to SEQ ID NO:

12.

11. The peptide according to any of the preceding claims, wherein the peptide is linked to a carrier protein.

12. The peptide according to any of the preceding claims, wherein the peptide comprises a cysteine ​​residue, and the carrier protein is linked to the peptide via the cysteine ​​residue.

13. A fusion protein comprising the peptide according to any one of claims 1 to 12.

14. The fusion protein of claim 13, wherein the fusion protein comprises more than one peptide according to any one of claims 1 to 12.

15. The fusion protein according to claim 13 or claim 14, wherein the fusion protein comprises the amino acid sequence according to SEQ ID NO: 13 (EVKEGGFEVNGKFIKVSA) and / or the amino acid sequence according to SEQ ID NO: 14 (DVTVEQVNEAMKNASNESF).

16. A composition or kit comprising: The peptide according to any one of claims 1 to 12, or the fusion protein according to any one of claims 13 to 15, and A peptide comprising the amino acid sequence according to SEQ ID NO: 13, or having one or more added, substituted or deleted sequences according to SEQ ID NO: 13; and / or comprising the amino acid sequence according to SEQ ID NO: 14, or having one or more added, substituted or deleted sequences according to SEQ ID NO:

14.

17. A composition or kit comprising a first peptide according to any one of claims 1 to 12, a second peptide comprising the amino acid sequence according to SEQ ID NO: 13, and a third peptide comprising the amino acid sequence according to SEQ ID NO:

14.

18. The composition or kit according to claim 16 or claim 17, wherein: The peptide containing SEQ ID NO: 13 contains SEQ ID NO: 15 (EVKEGGFEVNGKFIKVSA(bALA)C); and / or The peptide containing SEQ ID NO: 14 contains SEQ ID NO: 16 (DVTVEQVNEAMKNASNESF(bALA)C).

19. The composition or kit according to any one of claims 16 to 18, comprising the peptide of SEQ ID NO: 13: (i) A length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues; and / or (ii) A sequence comprising a side sequence of SEQ ID NO: 13, which is derived from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and / or (iii) Contains a sequence side-connected to SEQ ID NO: 13, which is derived from SEQ ID NO: 7; and / or (iv) Contains fewer than 8, 7, 6, 5, 4, or 3 adjacent residues from SEQ ID NO: 9; and / or (v) having 0-12 additional amino acids at the N-terminus and / or C-terminus side of SEQ ID NO: 13 or SEQ ID NO:

15.

20. The composition or kit according to any one of claims 16 to 19, comprising the peptide of SEQ ID NO: 14: (i) A length equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues; and / or (ii) A sequence comprising a side sequence of SEQ ID NO: 14, which is derived from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and / or (iii) Contains a sequence side-connected to SEQ ID NO: 14, which is derived from SEQ ID NO: 6; and / or (iv) Contains fewer than 8, 7, 6, 5, 4, or 3 adjacent residues from SEQ ID NO: 9; and / or (v) having 0-12 additional amino acids at the N-terminus and / or C-terminus side of SEQ ID NO: 14 or SEQ ID NO:

16.

21. The composition or kit according to any one of claims 16 to 20, wherein: The peptide containing SEQ ID NO: 13 is according to SEQ ID NO: 15; and / or The peptide containing SEQ ID NO: 14 is according to SEQ ID NO:

16.

22. A composition or kit comprising: The first peptide comprises the sequence according to SEQ ID NO: 3, or has one or more added, substituted or deleted sequences according to SEQ ID NO: 3; A second peptide comprising, or having one or more added, substituted, or deleted sequences according to SEQ ID NO: 13; and The third peptide comprises the sequence according to SEQ ID NO: 14, or has one or more added, substituted or deleted sequences according to SEQ ID NO:

14.

23. The composition or kit according to any one of claims 16 to 22, wherein: The peptide containing SEQ ID NO: 3 contains β-alanine; The peptide containing SEQ ID NO: 13 contains β-alanine; and / or The peptide containing SEQ ID NO: 14 contains β-alanine.

24. The composition or kit according to any one of claims 16 to 23, wherein: The peptide containing SEQ ID NO: 3 contains an N-terminal or C-terminal cysteine ​​residue; The peptide containing SEQ ID NO: 13 contains an N-terminal or C-terminal cysteine ​​residue; and / or The peptide containing SEQ ID NO: 14 contains an N-terminal or C-terminal cysteine ​​residue.

25. The composition or kit according to any one of claims 16 to 24, wherein: The peptide containing SEQ ID NO: 3 is according to SEQ ID NO: 12; The peptide containing SEQ ID NO: 13 is based on SEQ ID NO: 15; and The peptide containing SEQ ID NO: 14 is according to SEQ ID NO:

16.

26. The composition or kit according to any one of claims 16 to 25, wherein... A peptide containing SEQ ID NO: 3 is linked to a carrier protein; and / or The peptide containing SEQ ID NO: 13 is linked to a carrier protein; and / or The peptide containing SEQ ID NO: 14 is linked to a carrier protein.

27. The composition or kit according to any one of claims 16 to 26, wherein... The peptide containing SEQ ID NO: 3 contains a cysteine ​​residue, and a carrier protein is linked to the peptide via the cysteine ​​residue; and / or The peptide containing SEQ ID NO: 13 contains a cysteine ​​residue, and a carrier protein is linked to the peptide via the cysteine ​​residue; and / or The peptide containing SEQ ID NO: 14 contains a cysteine ​​residue, and the carrier protein is linked to the peptide via the cysteine ​​residue.

28. The composition or kit according to any one of claims 16 to 27, wherein the peptide comprising SEQ ID NO: 3 is a portion of a fusion protein, the peptide comprising SEQ ID NO: 13 is a portion of a fusion protein, and / or the peptide comprising SEQ ID NO: 14 is a portion of a fusion protein.

29. The composition or kit according to any one of claims 16 to 28, wherein: The peptide containing SEQ ID NO: 3 is based on SEQ ID NO: 12 and is linked to a carrier protein via a C-terminal cysteine ​​residue; The peptide comprising SEQ ID NO: 13 is according to SEQ ID NO: 15 and is linked to a carrier protein via a C-terminal cysteine ​​residue; and The peptide containing SEQ ID NO: 14 is according to SEQ ID NO: 16 and is linked to the carrier protein via a C-terminal cysteine ​​residue.

30. The peptide according to claim 11 or claim 12, or the composition or kit according to any one of claims 26, 27 and 29, wherein the carrier protein is KLH.

31. The peptide, composition, or kit according to claim 30, wherein the KLH is one or more subunits of KLH.

32. One or more nucleic acids encoding a peptide according to any one of claims 1 to 12, a fusion protein according to any one of claims 13 to 15, or a peptide of a composition or kit according to any one of claims 16 to 31.

33. One or more vectors comprising one or more nucleic acids according to claim 32.

34. A cell comprising one or more nucleic acids according to claim 32 or one or more vectors according to claim 33.

35. One or more pharmaceutical compositions comprising a peptide according to any one of claims 1 to 12, 30 or 31, a fusion protein according to any one of claims 13 to 15, a composition according to any one of claims 16 to 31, a peptide of a kit according to any one of claims 16 to 31, one or more nucleic acids according to claim 32, or one or more carriers according to claim 33.

36. One or more immunogenic compositions comprising a peptide according to any one of claims 1 to 12, 30 or 31, a fusion protein according to any one of claims 13 to 15, a composition according to any one of claims 16 to 31, a peptide of a kit according to any one of claims 16 to 31, one or more nucleic acids according to claim 32, or one or more vectors according to claim 33.

37. The peptide according to any one of claims 1 to 12, 30 or 31, the fusion protein according to any one of claims 13 to 15, the composition according to any one of claims 16 to 31, the peptide of the kit according to any one of claims 16 to 31, one or more nucleic acids according to claim 32, one or more carriers according to claim 33, one or more pharmaceutical compositions according to claim 35, or one or more immunogenic compositions according to claim 36 for use in stimulating an immune response.

38. The use according to claim 37, wherein the immune response is directed against one or more species of bacteria that induce sepsis.

39. A peptide according to any one of claims 1 to 12, 30 or 31, a fusion protein according to any one of claims 13 to 15, a composition according to any one of claims 16 to 31, a peptide of a kit according to any one of claims 16 to 31, one or more nucleic acids according to claim 32, one or more carriers according to claim 33, one or more pharmaceutical compositions according to claim 35, or one or more immunogenic compositions according to claim 36, used as a pharmaceutical agent.

40. A peptide according to any one of claims 1 to 12, 30 or 31, a fusion protein according to any one of claims 13 to 15, a composition according to any one of claims 16 to 31, a peptide of a kit according to any one of claims 16 to 31, one or more nucleic acids according to claim 32, one or more carriers according to claim 33, one or more pharmaceutical compositions according to claim 35, or one or more immunogenic compositions according to claim 36, for preventing infection by one or more species of bacteria that induce sepsis, reducing the risk of said infection, or reducing the severity of said infection.

41. The use according to claim 38, or the use of a peptide, fusion protein, composition, kit peptide, one or more nucleic acids, one or more carriers, one or more pharmaceutical compositions, or one or more immunogenic compositions according to claim 40, wherein one or more species of the bacteria inducing sepsis are GBS, Escherichia coli, Staphylococcus species, Streptococcus pneumoniae, and / or Klebsiella pneumoniae.

42. A peptide according to any one of claims 1 to 12, 30, or 31; a fusion protein according to any one of claims 13 to 15; a composition according to any one of claims 16 to 31; a peptide of a kit according to any one of claims 16 to 31; one or more nucleic acids according to claim 32; one or more carriers according to claim 33; one or more pharmaceutical compositions according to claim 35; or one or more immunogenic compositions according to claim 36, wherein the peptide is used for: Prevent, reduce the risk of, or reduce the severity of any one or more of the following: sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infection, soft tissue infection, gastrointestinal infection, bloodstream infection, and encephalitis, or To prevent or reduce the risk of premature birth or stillbirth.

43. A peptide, fusion protein, composition, kit peptide, one or more nucleic acids, one or more carriers, one or more pharmaceutical compositions, or one or more immunogenic compositions for use according to any one of claims 39 to 42, wherein the subject is an immunocompromised person, a newborn, an infant, a child, a woman of childbearing age, a pregnant woman, a fetus, a diabetic patient, and / or an elderly person.

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