WO2025051845A1

WO2025051845A1 - Products and methods for inducing immune responses to bacteria

Info

Publication number: WO2025051845A1
Application number: PCT/EP2024/074811
Authority: WO
Inventors: Pedro Jorge Fonseca Madureira; Marta Virgínia Mota VIEIRA; Liliana Almeida CURADO; Carla Andreia Fidalgo TEIXEIRA; Ana Patrícia Figueiredo FIDALGO; Filipa Gláucia Rodrigues LEMOS; Cristiana Andreia Pereira NOGUEIRA; Pedro Miguel Dias CASTANHEIRA
Original assignee: Immunethep Sa
Current assignee: Immunethep Sa
Priority date: 2023-09-06
Filing date: 2024-09-05
Publication date: 2025-03-13
Anticipated expiration: 2026-03-06

Abstract

The invention relates to peptides, fusion proteins, and compositions suitable for inducing immune responses to bacteria. The invention also relates to the treatment or prevention of infection by sepsis-inducing bacteria, and also to the treatment and prevention of pathologies caused by such bacteria. Such pathologies include sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, encephalitis, premature birth, and stillbirth.

Description

PRODUCTS AND METHODS FOR INDUCING IMMUNE RESPONSES TO BACTERIA

FIELD OF THE INVENTION

The invention relates to peptides, fusion proteins, and compositions suitable for inducing immune responses to bacteria. The invention also relates to the treatment or prevention of infection by sepsisinducing bacteria, and also to the treatment and prevention of pathologies caused by such bacteria. Such pathologies 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 major cause of death worldwide. A recent study estimated that in 2019, 7.7 million people died from complications associated with bacterial infections. Among these, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus agalactiae (commonly referred as Group B Streptococcus, GBS) were responsible for 53.6% of these deaths.

The present inventors have previously described that neutralization of extracellular bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a good strategy to prevent or treat infections caused by the above-mentioned bacteria (e.p. WO2O15/189422 At).

Extracellular bacterial GAPDH is an IL-10-inducing protein that causes generalized immunosuppression in the host at veiy early time points after infection (Madureira, P., et al., PLoS Pathog, 2011. 7(11): p. 01002363). Extracellular bacterial GAPDH has a high degree of identity among the aforementioned bacterial species. Nevertheless, humans also possess GAPDH. Bacterial and human GAPDH can share 20-40 % identity. Thus, the inventors disclosed a list of peptides that can be used as targets for immunotherapies aiming at neutralizing bacterial GAPDHs, without any cross-reactivity to the human homolog (WO2O15/ 189422 Al). These peptides are exposed on bacterial GAPDH but completely absent from human GAPDH. From each bacterial GAPDH, the inventors selected a list of 7 to 12 peptides, ranging from 9 to 22 amino acids in length that could match these criteria.

The inventors also previously demonstrated that IgG-dependent antibody neutralization of bacterial GAPDH can provide protection against infection (Madureira, P., et al., PLoS Pathog, 2011. 7(11): p. 01002363).

There remains a need for vaccine compositions that are efficacious for a broad range of bacteria.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a peptide that comprises amino acid sequence according to SEQ ID NO: 3 (TQTTEITAVGDQLVKTVA).

In a second aspect, there is provided a fusion protein comprising a peptide according to the first aspect.

In a third aspect, there is provided a composition or kit that comprises a peptide of the first aspect or a fusion protein of the second aspect. In an embodiment, the composition or kit comprises a peptide of the first aspect or a fusion protein of the second aspect, and a peptide that comprises an amino acid sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and/or a peptide that comprises an amino acid sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions. In an embodiment, there is provided a composition or kit that comprises a first peptide which is a peptide of the first aspect, a second peptide that comprises an amino acid sequence according to SEQ ID NO: 13, and a third peptide that comprises an amino acid sequence according to SEQ ID NO: 14.

In a fourth aspect, there is provided a first peptide comprising a sequence according to SEQ ID NO: 3 or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions; a second peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and a third peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions.

In an 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.

In a fifth aspect, there is provided one or more nucleic acids encoding a peptide according to the first aspect, a fusion protein according to the second aspect, or peptides of a composition or kit according to the fourth aspect or fifth aspect.

In a sixth aspect, there is provided one or more vectors comprising one or more nucleic acids of the fifth aspect.

In a seventh aspect, there is provided a cell comprising one or more nucleic acids of the fifth aspect or one or more vectors of the sixth aspect.

In an eighth aspect, there is provided one or more pharmaceutical compositions comprising a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third aspect or fourth aspect, the peptides of a kit according to the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, or one or more vectors of the sixth aspect.

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

In a tenth aspect, there is provided use of a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third aspect or fourth aspect, the peptides of a kit according to the third aspect 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 the eighth aspect, or one or more immunogenic compositions of the ninth aspect for stimulating an immune response.

In a eleventh aspect, there is provided a peptide according to the first aspect, a fusion protein according to the second aspect, a composition according to the third aspect or fourth aspect, the peptides of a kit according to the third aspect 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 the eighth aspect, or one or more immunogenic compositions of the ninth aspect for use as a medicament.

In an embodiment, the peptide according to the first aspect, fusion protein according to the second aspect, composition according to the third aspect or fourth aspect, peptides of a kit according to the third aspect 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 the eighth aspect, or one or more immunogenic compositions of the ninth aspect is for use in preventing, reducing the risk of, or reducing the severity of an infection by one or more species of sepsis-inducing bacteria.

In some embodiments, the one or more species of sepsis-inducing bacteria is GBS, E. coli, Staphylococcus spp., S. pneumoniae, and/or K pneumoniae.

In an embodiment, the peptide according to the first aspect, fusion protein according to the second aspect, composition according to the third aspect or fourth aspect, peptides of a kit according to the third aspect 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 the eighth aspect, or one or more immunogenic compositions of the ninth aspect is for use in: preventing, reducing the risk of, or reducing the severity of any one or more of sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinaiy tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, and encephalitis, or preventing or reducing the risk of premature birth or stillbirth.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Immunogenicity of different conjugated peptide formulations towards different bacterial GAPDH.

Figure 2: Paragon novel vaccine (PNV) immunogenicity is higher than the sum of the immunogenicity for the individual peptides.

Figure 3: PNV protects adult mice from bacterial infections.

Figure 4: Maternal vaccination with PNV protects offspring from bacterial infections.

DETAILED DESCRIPTION

The inventors’ previous work provides 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 herein). The inventors have now expanded upon this work and have generated an improved peptide that is suitable for use in such compositions. Furthermore, the inventors provide herein synergistic compositions of peptides that are suitable for the aforementioned purposes.

The inventors identified that a single peptide in their previous work, TQTEITAVGDLQLVKTVA (SEQ ID NO: 1), contains a domain that is common to Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus agalactiae. This domain is TQT- XXXXXXXX-QLVK (X representing any amino acid - SEQ ID NO: 2). The chemical synthesis of this peptide did not yield a stable product. Interestingly, the stochastic introduction, substitution, and/or deletion of specific amino acids outside the conserved domain allows the synthesis and purification of a highly stable, soluble peptide. A final peptide, TQTTEITAVGDQLVKTVA (SEQ ID NO: 3), was obtained.

A peptide comprising 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 to E. coli GAPDH and K pneumoniae GAPDH (Figure 2). Furthermore, the TQT peptide was found to be synergistic in combination with other peptides, and hence suitable for use in compositions for inducing a broad-spectrum immune response to sepsisinducing bacteria. As a part of a synergistic formulation, IgG responses were induced to all sepsisinducing bacteria that were tested, including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Streptococcus agalactiae (Figure 1 - Formulation #5 contains TQT).

Thus, in a first aspect, there is provided a peptide that comprises amino acid sequence according to SEQ ID NO: 3. The region of the peptide that is SEQ ID NO: 3 should be capable of acting as an antigen when administered to a subject. As such, in some embodiments the peptide of the first aspect is equal to or less than 150 residues in length. This increases the chance that the peptide will not adopt a structure that obscures the antigen. In other embodiments, the peptide of the first aspect is equal to or less than too residues, 50 residues, or 30 residues in length. The peptide of the first aspect may, in some examples, comprise no more than 12, 10, 5, 4, 3, 2, or 1 additional residues at the N-terminal and/or C- terminal end of SEQ ID NO: 3. In some embodiments, the peptide comprises only SEQ ID NO: 3 and also residues or moieties that are required for non- antigenic aspects of the composition, for instance labelling and/or conjugation. In an example, the peptide may comprise only SEQ ID NO: 3, a betaalanine or other label, and a cysteine for conjugation to a carrier protein. The peptide may comprise a beta-alanine and a cysteine at the N-terminal or C-terminal end of the peptide, wherein the cysteine is the terminus of the peptide.

The peptide of the first aspect may comprise sequences that flank SEQ ID NO: 3 on either or both sides. In some examples, these sequences may be derived from bacterial GAPDH, such as a sepsis-inducing bacterial species. For instance, the sequences may be derived from any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 3 may be at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 3 may match a sequence within any one of SEQ ID NOs: 4 to 8 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions.

In a particular example, 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:

... IIGSHFGSVFDATOTTEITAVGDOLVKTVA f SEO ID NO: to)

In another example, which may be an alternative or in addition to the above, the C-terminal end of SEQ ID NO: 3 may be attached to a 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:

TQTTEITAVGDOLVKTVAWYDNEYGFVTOL... (SEQ ID NO: 11)

The sequence or sequences flanking SEQ ID NO: 3 that are derived from SEQ ID NO: 5 maybe at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. The sequences flanking SEQ ID NO: 3 may match a sequence within SEQ ID NO: 5 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions.

The sequence or sequences of SEQ ID NO: 5 that flank SEQ ID NO: 3 may lead to a peptide that is equal to or less than 150 residues, 100 residues, 50 residues, or 30 residues in length. The peptide may also include residues or moieties that are required for non-antigenic aspects of the composition, for instance labelling and/or conjugation.

It is desirable for the peptide to not include self- antigens associated with the subject to be treated. Thus, in some embodiments, the peptide of the first aspect does not comprise human self-antigens. This is achieved, in some examples, by avoiding the inclusion of any contiguous sequence of 8 amino acids from within SEQ ID NO: 9. The peptide of the first aspect may comprise less than 7, 6, 5, 4, or 3 residues of contiguous sequence from within SEQ ID NO: 9.

The peptide of the first aspect may be an isolated peptide. The isolated peptide may be isolated from any additional sequences and/or substances with which it is associated in nature. The isolated peptide may be a part of a purified composition, wherein the other substantial components are characterised. The isolated peptide may be linked or fused to other residues or other polypeptide sequences.

The isolated peptide may optionally be linked to moieties or residues that assist with the labelling, identification, or quantification of the peptide. For instance, a beta- alanine residue may be included in the isolated peptide. In some examples, a beta- alanine residue is attached directly to either the first or last residue of SEQ ID NO: 3.

The isolated peptide may optionally be linked to moieties or residues that assist with the conjugation of the peptide to other moieties, residues, or polypeptides. For instance, a cysteine residue may be included in the isolated peptide. In particular examples, a cysteine residue may be the N-terminal or C- terminal residue of the peptide of the first aspect. The cysteine may be suitable for the conjugation of a carrier protein to the peptide of the first aspect.

In some embodiments, the peptide of the first aspect is linked to a carrier protein. Examples of carrier proteins include keyhole limpet haemocyanin (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 natural sources, and purified KLH may comprise one or more KLH subunits. Thus, when acting as a carrier protein, the KLH may be or may comprise one or more subunits of KLH. The carrier protein may be or may comprise a plurality of subunits of KLH.

The peptide of the first aspect may 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.

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 from 1:2 to 1:1. In a particular embodiment, the ratio is 1:1. In an example, the peptide is conjugated to KLH via a terminal cysteine residue at a ratio of 1:1.

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

The isolated peptide of the first aspect may be part of a fusion protein and may be linked to other antigenic sequences. Thus, in a second aspect, there is provided a fusion protein that comprises amino acid sequence according to SEQ ID NO: 3.

The fusion protein may comprise any peptide as disclosed in relation to the first aspect.

In some examples, the fusion protein may comprise more than one copy of SEQ ID NO: 3. Alternatively, or in addition, the fusion protein may comprise another peptide or peptides designed to induce an immune response to sepsis-inducing bacteria. The other peptide or peptides may be one or more peptides as defined or described in WO2O15/ 189422 Al (herein incorporated by reference). In some examples, the fusion peptide may comprise amino acid sequence according to EVKEGGFEVNGKFIKVSA (SEQ ID NO: 13) and/or DVTVEQVNEAMKNASNESF (SEQ ID NO: 14). In a fusion protein comprising two or more peptides the linkage may be direct and so have no amino acids in-between the peptides being linked. Alternatively, the linkage may be indirect and so may have one or more amino acid residues, or other moieties, between the peptides being linked. A pattern of one or more of the described peptides may be repeated to form the larger peptide/small protein. The repetitions may be directly adjacent to each other, in a so-called tandem repeat, or they may be spaced apart in each case. Alternatively, the linked peptides may appear in a random order.

The peptide of the first aspect or fusion protein of the second aspect may be present in a composition comprising other peptides or in a kit comprising other peptides. Thus, in a third aspect of the invention, there is provided a composition or kit comprising a peptide of the first aspect or a fusion protein of the second aspect.

The composition or kit may comprise other peptides, for instance other peptides comprising antigens derived from bacterial GAPDH proteins. The other peptide or peptides may be one or more peptides as defined or described in WO2O15/189422 Al. In some examples, the composition or kit may comprise peptides comprising amino acid sequence according to SEQ ID NO: 13 and/or SEQ ID NO: 14.

The peptides within the composition or kit may be conjugated to a carrier protein. Examples of carrier proteins include KLH, CRM, and TT. In a particular embodiment, the carrier protein is KLH. The carrier proteins may be conjugated to the peptides 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. The carrier proteins maybe conjugated to the peptides at a ratio of 1:1 (w/w). In a particular embodiment, the peptides are conjugated to KLH at a ratio of 1:1.

Any details or features disclosed herein in relation to compositions or kits of the fourth aspect may also be applied to compositions or kits of the third aspect.

The present inventors have identified that certain combinations of antigens have synergistic effects. Illustrative data are presented in Figure 2, which demonstrate that an exemplary formulation (referred to as “PNV”) leads to a greater response when compared to the response individually induced by any of the three antigens within PNV. For instance, each of the three antigens induces only a low IgG titre to S. aureus GAPDH but, in combination, the IgG title is high (Figure 2).

Table 1 discloses exemplary peptides comprising the relevant antigens. The antigens are SEQ ID NOs: 3, 13, and 14 (respectively). The peptides are SEQ ID NOs: 12, 15, and 16 (respectively).

Table 1: Paragon Novel Vaccine (PNV) peptide sequence

Amino Acid Sequence Peptide Designation Carrier Protein

TQTTEITAVGDQLVKTVA(pA)C TQT

EVKEGGFEVNGKFIKVSA(PA)C STREP KLH

DVTVEQVNEAMKNASNESF(PA)C GRAMP

When compared to other formulations of bacteria GAPDH antigens, PNV was found to induce high titres across a range of bacterial species (Figure 1 - see Formulation #5). Hence, this formulation is surprisingly effective compared to formulations that comprise other combinations of bacteria GAPDH antigens. The inventors further demonstrated that an exemplary composition comprising the aforementioned antigens can be used to protect adult mice from bacterial infections (Figure 3) and can also be used for maternal vaccination to protect offspring from bacterial infections (Figure 4).

Thus, in a fourth aspect of the invention, there is provided a composition or kit comprising: a first peptide comprising a sequence according to SEQ ID NO: 3 or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions; a second peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and a third peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions.

Each of the first, second, and third peptides may comprise one or more additions, substitutions, or deletions compared to the recited sequence. Such modifications may be tolerated if they do not alter the combined immunogenicity of the antigens, for instance when tested according to the experiments illustrated in Figure 2. In some examples, the peptides comprise no more than five, four, three, two, or one additions, substitutions, or deletions. In some examples, the deletions are truncations and hence reduce the size of the peptides. In examples, the peptides comprise five, four, three, two, or one deletions at the N-terminal end or C-terminal end of the recited sequences to result in truncated peptides. In some examples, the first, second, and/or third peptides comprise the recited sequences with no additions, substitutions, or deletions. In some examples, the first peptide comprises the consensus sequence illustrated in SEQ ID NO: 2, and so retains this sequence even where one or more additions, substitutions, or deletions are included.

The first peptide may be any as disclosed in relation to the first aspect of the present disclosure. The first peptide may be comprised by any fusion protein as discussed in relation to the second aspect of the present disclosure.

In particular, the first peptide may be equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length. The first peptide may, in some examples, comprise no more than 12, 10, 5, 4, 3, 2, or 1 additional residues at the N-terminal and/or C-terminal end of SEQ ID NO: 3. In some embodiments, the peptide comprises only SEQ ID NO: 3 and also residues or moieties that are required for non- antigenic aspects, for instance labelling and/or conjugation. In an example, the peptide may comprise only SEQ ID NO: 3, a beta-alanine or other label, and a cysteine for conjugation to a carrier protein. The peptide may comprise a beta-alanine and a cysteine at the N-terminal or C-terminal end of the peptide, wherein the cysteine is the terminus of the peptide. The peptide of the first aspect may comprise sequences that flank SEQ ID NO: 3 on either or both sides. These sequences may be derived from bacterial GAPDH, such as a sepsis-inducing bacterial species. For instance, the sequences may be derived from any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 3 maybe at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 3 may match a sequence within any one of SEQ ID NOs: 4 to 8 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions. In a particular example, 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. In another example, which may be an alternative or in addition to the above, the C-terminal end of SEQ ID NO: 3 may be attached to a 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. The sequence or sequences flanking SEQ ID NO: 3 that are derived from SEQ ID NO: 5 may be at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. The sequences flanking SEQ ID NO: 3 may match a sequence within SEQ ID NO: 5 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions. The sequence or sequences of SEQ ID NO: 5 that flank SEQ ID NO: 3 may lead to a peptide that is equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length. The peptide may also include residues or moieties that are required for non- antigenic aspects, for instance labelling and/or conjugation.

The region of the second peptide that is SEQ ID NO: 13 should be capable of acting as an antigen when administered to a subject. To increase this possibility, in some embodiments the second peptide is equal to or less than 150 residues in length. This increases the chance that the peptide will not adopt a structure that obscures the antigen. In other embodiments, the second peptide is equal to or less than too residues, 50 residues, or 30 residues in length. The second peptide may, in some examples, comprise no more than 12, 10, 5, 4, 3, 2, or 1 additional residues at the N-terminal and/or C-terminal end of SEQ ID NO: 13. In some embodiments, the second peptide comprises only SEQ ID NO: 13 and also residues or moieties that are required for non- antigenic aspects, for instance labelling and/or conjugation. In an example, the second peptide may comprise only SEQ ID NO: 13, a beta-alanine or other label, and a cysteine for conjugation to a carrier protein. The second peptide may comprise a betaalanine and a cysteine at the N-terminal or C-terminal end of the peptide, wherein the cysteine is the terminus of the peptide.

The region of the third peptide that is SEQ ID NO: 14 should be capable of acting as an antigen when administered to a subject. To increase this possibility, in some embodiments the third peptide is equal to or less than 150 residues in length. This increases the chance that the peptide will not adopt a structure that obscures the antigen. In other embodiments, the third peptide is equal to or less than too residues, 50 residues, or 30 residues in length. The third peptide may, in some examples, comprise no more than 12, 10, 5, 4, 3, 2, or 1 additional residues at the N-terminal and/or C-terminal end of SEQ ID NO: 14. In some embodiments, the third peptide comprises only SEQ ID NO: 14 and also residues or moieties that are required for non-antigenic aspects, for instance labelling and/or conjugation. In an example, the third peptide may comprise only SEQ ID NO: 14, a beta-alanine or other label, and a cysteine for conjugation to a carrier protein. The third peptide may comprise a beta-alanine and a cysteine at the N-terminal or C-terminal end of the peptide, wherein the cysteine is the terminus of the peptide.

The second peptide may comprise sequences that flank SEQ ID NO: 13 on either or both sides. In some examples, these sequences are derived from bacterial GAPDH, such as a sepsis-inducing bacterial species. For instance, the sequences may be derived from any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 13 may be at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 13 may match a sequence within any one of SEQ ID NOs: 4 to 8 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions.

In a particular example, the N-terminal end of SEQ ID NO: 13 maybe 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:

... YDTTOGRFDGTVEVKEGGFEVNGKFIKVSA (SEQ ID NO: 17)

In another example, which may be in addition to the above, the C-terminal end of SEQ ID NO: 13 may be attached to a 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:

EVKEGGFEVNGKFIKVSAERDPEOIDWATD... (SEQ ID NO: 18)

The sequence or sequences flanking SEQ ID NO: 13 that are derived from SEQ ID NO: 7 maybe at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7. The sequences flanking SEQ ID NO: 13 may match a sequence within SEQ ID NO: 7 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions. The sequence or sequences of SEQ ID NO: 7 that flank SEQ ID NO: 13 may lead to a peptide that is equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length. The peptide may additionally include residues or moieties that are required for non- antigenic aspects, for instance labelling and/or conjugation.

The third peptide may comprise sequences that flank SEQ ID NO: 14 on either or both sides. In some examples, these sequences are derived from bacterial GAPDH, such as a sepsis-inducing bacterial species. For instance, the sequences may be derived from any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 14 may be at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 4 to 8. The sequences flanking SEQ ID NO: 14 may match a sequence within any one of SEQ ID NOs: 4 to 8 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions.

In a particular example, the N-terminal end of SEQ ID NO: 14 maybe 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:

... LTELTWLEKODVTVEQVNEAMKNASNESF (SEQ ID NO: 19)

In another example, which may be in addition to the above, the C-terminal end of SEQ ID NO: 14 may be attached to a 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:

DVTVEQVNEAMKNASNESFGYTEDEIVSS- (SEQ ID NO: 20)

The sequence or sequences flanking SEQ ID NO: 14 that are derived from SEQ ID NO: 6 maybe at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6. The sequences flanking SEQ ID NO: 14 may match a sequence within SEQ ID NO: 6 with 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1, or no substitutions, additions, or deletions. The sequence or sequences of SEQ ID NO: 6 that flank SEQ ID NO: 14 may lead to a peptide that is equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length. The peptide may additionally include residues or moieties that are required for non- antigenic aspects, for instance labelling and/or conjugation.

It is desirable for the peptides to not include self- antigens associated with the subject to be treated. Thus, in some embodiments, the first, second, and/or third peptides of the fourth aspect do not comprise human self-antigens. This is achieved, in some examples, by avoiding the inclusion of any contiguous sequence of 8 amino acids from within SEQ ID NO: 9. The peptides may comprise less than 7, 6, 5, 4, or 3 residues of contiguous sequence from within SEQ ID NO: 9.

The first, second, and/ or third peptides may be isolated peptides. The isolated peptides may be isolated from any sequences and/or substances with which they are associated in nature. The isolated peptides may be a part of a purified composition, wherein the other substantial components are characterised. The isolated peptides may be linked or fused to other residues or other polypeptide sequences.

The isolated first, second, and/ or third peptides may optionally be linked to moieties or residues that assist with the labelling, identification, or quantification of the peptides. For instance, a beta-alanine residue may be included in each of the isolated peptides. In some examples, a beta-alanine residue is attached directed to either the first or last residue of SEQ ID NO: 3, SEQ ID NO: 13, and/or SEQ ID NO: 14-

The isolated first, second, and/ or third peptides may optionally be linked to moieties or residues that assist with the conjugation of the peptides to other moieties, residues, or polypeptides. For instance, a cysteine residue may be included in the isolated peptides. In particular examples, a cysteine residue may be the N-terminal or C-terminal residue of the first, second, and/ or third peptides. The cysteine may be suitable for the conjugation of a carrier protein to the peptides of the fourth aspect.

In some embodiments, the peptides within the composition or kit of the fourth aspect are linked to carrier proteins. Examples of carrier proteins include KLH, CRM, and TT. In a particular embodiment, the carrier protein is KLH. As discussed, the KLH maybe or may comprise one or more subunits of KLH. The carrier protein may be or may comprise a plurality of subunits of KLH.

The first, second, and/or third peptides may be linked to the carrier protein via the aforementioned cysteine residue. In a particular embodiment, the first, second, and/or third peptides are linked to KLH via a C-terminal cysteine residue.

The carrier proteins may be conjugated to the peptides 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. The carrier proteins maybe conjugated to the peptides at a ratio of 1:1 (w/w). In a particular embodiment, the peptides are conjugated to KLH at a ratio of 1:1.

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

In an 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 the cysteine is the C- terminus. The second peptide may be of a sequence according to only SEQ ID NO: 15. The second peptide maybe of a sequence according to SEQ ID NO: 15, wherein the first residue of SEQ ID NO: 15 is the N-terminus and the last residue of SEQ ID NO: 15 is the C-terminus. The second peptide may be conjugated to a carrier protein, such as KLH, via the cysteine.

In an 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 the cysteine is the C- terminus. The third peptide maybe of a sequence according to only SEQ ID NO: 16. The third peptide may be of a sequence according to SEQ ID NO: 16, wherein the first residue of SEQ ID NO: 16 is the N- terminus and the last residue of SEQ ID NO: 16 is the C-terminus. The third peptide may be conjugated to a carrier protein, such as KLH, via the cysteine.

In some embodiments, the compositions and kits of the third and fourth aspects include only the recited peptides and do not include other peptides.

In a particular embodiment, there is provided a composition or kit that comprises three types of peptide and no other types of peptide, wherein the three types of peptide are: a first peptide that comprises SEQ ID NO: 3; a second peptide that comprises SEQ ID NO: 13; and a third peptide that comprises SEQ ID NO: 14.

In a particular embodiment, there is provided a composition that comprises: a first peptide that comprises SEQ ID NO: 3 and is conjugated to a carrier protein, such as KLH; a second peptide that comprises SEQ ID NO: 13 and is conjugated to a carrier protein, such as KLH; and a third peptide that comprises SEQ ID NO: 14 and is conjugated to a carrier protein, such as

KLH.

In a particular embodiment, there is provided a composition that comprises: a first peptide that comprises SEQ ID NO: 3 and a terminal cysteine, and is conjugated to a carrier protein, such as KLH; a second peptide that comprises SEQ ID NO: 13 and a terminal cysteine, and is conjugated to a carrier protein, such as KLH; and a third peptide that comprises SEQ ID NO: 14 and a terminal cysteine, and is conjugated to a carrier protein, such as KLH.

In a particular embodiment, there is provided a composition that comprises: a first peptide according to SEQ ID NO: 12, wherein the recited beta-alanine is optional; a second peptide according to SEQ ID NO: 15, wherein the recited beta-alanine is optional; and a third peptide according to SEQ ID NO: 16, wherein the recited beta-alanine is optional.

In a particular embodiment, there is provided a composition that comprises: a first peptide according to SEQ ID NO: 12, wherein the recited beta-alanine is optional, wherein the first peptide is conjugated to a carrier protein, such as KLH, via the recited cysteine; a second peptide according to SEQ ID NO: 15, wherein the recited beta-alanine is optional, wherein the second peptide is conjugated to a carrier protein, such as KLH, via the recited cysteine; and a third peptide according to SEQ ID NO: 16, wherein the recited beta-alanine is optional, wherein the third peptide is conjugated to a carrier protein, such as KLH, via the recited cysteine.

In a particular embodiment, there is provided a composition that comprises: a first peptide that consists of an amino acid sequence according to SEQ ID NO: 12, wherein the first peptide is conjugated to KLH via the recited cysteine; a second peptide that consists of an amino acid sequence according to SEQ ID NO: 15, wherein the second peptide is conjugated to KLH via the recited cysteine; and a third peptide that consists of an amino acid sequence according to SEQ ID NO: 16, wherein the third peptide is conjugated to KLH via the recited cysteine.

The amino acid sequence of GAPDH from GBS (UniProt Accession No. Q8E3E8) is as follows:-

MWKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPNMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGQFVKVSA EREPANIDWATDGVEIVLEATGFFASKEKAEQHIHENGAKKWITAPGGNDVKTWFNTNHDILDGTETVI SGASC TTNCLAPMAKALQDNFGVKQGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVI PELNGK LDGAAQRVPVPTGSVTELVATLEKDVTVEEVNAAMKAAANDSYGYTEDPIVSSDIVGI SYGSLFDATQTKVQTVDG NQLVKWSWYDNEMSYTSQLVRTLEYFAKIAK (SEQ ID NO: 4)

The amino acid sequence of GAPDH from E. coli (UniProt Accession No. D5D2F1) is as follows:-

MSKVGINGFGRIGRLVLRRLLEVKSNIDWAINDLTSPKILAYLLKHDSNYGPFPWSVDYTEDSLIVNGKSIAVYA EKEAKNI PWKAKGAEI IVECTGFYTSAEKSQAHLDAGAKKVLISAPAGEMKTIVYNVNDDTLDGNDTIVSVASCTT NCLAPMAKALHDSFGIEVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENI I PHTTGAAKAIGLVI PELSGKLKG HAQRVPVKTGSVTELVSILGKKVTAEEVNNALKKATNNNESFGYTDEEIVSSDI IGSHFGSVFDATQTEITAVGDL QLVKTVAWYDNEYGFVTQLIRTLEKFAKL (SEQ ID NO: 5)

The amino acid sequence of GAPDH from S. aureus (UniProt Accession No. A6QF81) is as follows:-

MAVKVAINGFGRIGRLAFRRIQEVEGLEWAVNDLTDDDMLAHLLKYDTMQGRFTGEVEWDGGFRVNGKEVKSFS EPDASKLPWKDLNIDWLECTGFYTDKDKAQAHIEAGAKKVLISAPATGDLKTIVFNTNHQELDGSETWSGASCT TNSLAPVAKVLNDDFGLVEGLMTTIHAYTGDQNTQDAPHRKGDKRRARAAAENI I PNSTGAAKAIGKVI PEIDGKL DGGAQRVPVATGSLTELTWLEKQDVTVEQVNEAMKNASNESFGYTEDEIVSSDWGMTYGSLFDATQTRVMSVGD RQLVKVAAWYDNEMSYTAQLVRTLAYLAELSK (SEQ ID NO: 6)

Although only the sequence of GAPDH from S. aureus is provided here, all of the available GAPDH sequences from Staphylococcus spp. possess over 98% sequence similarity.

The amino acid sequence of GAPDH from S. pneumoniae (UniProt Accession No. Q97NL1) is as follows

MWKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPVMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGKFIKVSA ERDPEQIDWATDGVEIVLEATGFFAKKEAAEKHLKGGAKKWITAPGGNDVKTWFNTNHDVLDGTETVI SGASCT TNCLAPMAKALQDNFGWEGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVI PELNGKL DGSAQRVPTPTGSVTELVAVLEKNVTVDEVNAAMKAASNESYGYTEDPIVSSDIVGMSYGSLFDATQTKVLDVDGK QLVKWSWYDNEMSYTAQLVRTLEYFAKIAK (SEQ ID NO: 7)

The amino acid sequence of GAPDH from K. pneumoniae (UniProt Accession No. B5XRG0) is as follows:-

MSKLGINGFGRIGRLVLRRLLEVDSSLEWAINDLTSPKVLAYLLKHDSNYGPFPWSVDFTEDALIVNGKTITVYA EKEAQHI PWQAAGAEVIVECTGFYTSAEKSQAHIQAGARKVLI SAPAGEMKTIVYNVNDDTLTPDDTI I SVASCTT NCLAPMAKVLQDAFGITVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENVI PHTTGAAKAIGLVI PALSGKLKG HAQRVPTKTGSVTELVSVLEKKVTADEVNQAMKQAAEGNESFGYTEEEIVSSDI IGSHFGSIYDATQLEIVEAGGV QLVKTVAWYDNEYGFVTQLIRVLEKFAR (SEQ ID NO: 8)

The amino acid sequence of human GAPDH (UniProt Accession No. P04406) is as follows:-

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINGNPITI FQERDPSKIKWGDAGAEYWESTGVFTTMEKAGAHLQGGAKRVI I SAPSADAPMFVMGVNHEKYDNSLKI I SNASC TTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNI I PASTGAAKAVGKVI PELNGKL TGMAFRVPTANVSWDLTCRLEKPAKYDDIKKWKQASEGPLKGILGYTEHQWSSDFNSDTHSSTFDAGAGIALN DHFVKLI SWYDNEFGYSNRWDLMAHMASKE (SEQ ID NO: 9)

The present disclosure extends to nucleic molecules that encode the peptides, fusion proteins, and peptides within compositions and kits of the invention.

Hence, according to a fifth aspect, there is provided one or more nucleic acids encoding a peptide according to the first aspect, a fusion protein according to the second aspect, the peptides and/or fusion proteins comprised by a composition or kit of the third aspect, or the peptides and/or fusion proteins comprised by a composition or kit of the fourth aspect.

A nucleic acid encoding a peptide according to the first aspect or a fusion protein according to the second aspect may be a single nucleic acid molecule.

The one or more nucleic acids of the fifth aspect may be one nucleic acid molecule that encodes all of the peptides or fusion proteins within compositions or kits of the third or fourth aspects. The peptides and/ or fusion proteins may be encoded in a manner such that they are translated as separate molecules. The peptides and/or fusion proteins may be separated by cleavable sequences. The single nucleic acid may be cleaved or a polypeptide that is a precursor to the peptides and/or fusion proteins may be cleaved.

The one or more nucleic acids of the fifth aspect maybe a plurality of nucleic acids wherein each individual nucleic acid molecule encodes one of the peptides or fusion proteins within the compositions or kits of the third or fourth aspects. Thus, the 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.

The one or more nucleic acids may be isolated and may be recombinant or synthetic. In some examples, the one or more nucleic acids are chemically modified, for example, via the inclusion of a modified nucleotide. The one or more nucleic acids may comprise at least one modified sugar moiety, at least one modified internucleoside linkage, or at least one modified nucleobase. The one or more nucleic acids may be nucleic acid analogues, which maybe compounds that have an arrangement of nucleobases that mimic the arrangement of nucleobases in DNA or RNA. The nucleic acid analogue may have a modified backbone compared to naturally occurring nucleic acids.

In some examples, the one or more nucleic acids are operably linked to a heterologous promoter. In some examples, the one or more nucleic acids are bound to a substrate or label or such like. Such modifications are usual in the art and will be known to the skilled person.

The one or more nucleic acids may encode one or more genetic constructs. A genetic construct may be in the form of an expression cassette, which may be suitable for expression of an encoded peptide in a cell. A genetic construct may be introduced into a cell without it being incorporated in a vector. For instance, a genetic construct, which may be a nucleic acid molecule, may be incorporated within a liposome or a viral vector. Alternatively, a purified nucleic acid molecule (for example, histone-free DNA or naked DNA) may be inserted directly into a cell by suitable means, for example, direct endocytotic uptake. A genetic construct may be introduced directly into cells of a host subject (for example, a bacterial cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, or ballistic bombardment. Alternatively, the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.

Hence, in a sixth aspect, there is provided one or more vectors comprising one or more nucleic acids according to the fifth aspect.

The suitable vectors include lipid nanoparticles, micelles, exosomes, viral vectors, non-viral vectors, plasmids, cosmids, and phage. The skilled person will appreciate that the nucleic acids of the present disclosure may be combined with many types of backbone vector for expression purposes. Vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression. For instance, the vector may be designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may be required in the vector. Alternatively, the vector may be designed such that it integrates into the genome of a host cell or such that it does not integrate into the genome of a host cell.

Accordingly, in a seventh aspect, there is provided a host cell comprising one or more nucleic acids of the fifth aspect or one or more vectors according to the sixth aspect.

The host cell maybe a bacterial cell, for example E. coli. Alternatively, the host cell maybe an animal cell, for example a mouse or rat cell. In some examples, the host cell is not a human cell. The host cell may be transformed with nucleic acids or vectors disclosed herein, using known techniques. Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.

In an eighth aspect, there is provided one or more pharmaceutical compositions comprising a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third aspect or fourth aspect, the peptides of a kit according to the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, or one or more vectors of the sixth aspect. A kit of the third or fourth aspect may comprise separate pharmaceutical compositions each comprising a peptide within the kits of the third or fourth aspect.

The pharmaceutical composition may comprise one or more of a pharmaceutically acceptable vehicle, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable stabilizer, or a pharmaceutically acceptable preservative, or any combination thereof. To be pharmaceutically acceptable, a substance or combination of substances must be suitable for the formulation of pharmaceutical compositions or a medicament. The pharmaceutical composition may comprise one or more of a vehicle, excipient, flavouring agent, lubricant, solubiliser, suspending agent, dye, filler, glidant, compression aid, inert binder, sweetener, preservative, coating, or disintegrating agent.

The peptides of the first aspect, fusion proteins of the second aspect, compositions of the third aspect or fourth aspect, peptides within kits of the third or fourth aspect, nucleic acids of the fifth aspect, vectors of the sixth aspect, or pharmaceutical compositions of the eighth aspect may be used to generate a protective immune response to one or more sepsis-inducing bacterial species.

Thus, in a ninth aspect, there is provided one or more immunogenic compositions comprising a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third aspect or fourth aspect, the peptides of a kit according to the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, or one or more vectors of the sixth aspect.

A kit of the third or fourth aspect may comprise separate immunogenic compositions each comprising a peptide within the kits of the third or fourth aspect.

The immunogenic composition of the ninth aspect may be a vaccine.

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

In other embodiments, the immunogenic composition of the ninth aspect may comprise any nucleic acid of the fifth aspect or vector of the sixth aspect. In such embodiments, the nucleic acids and vectors encode antigenic peptides. Thus, such immunogenic compositions may be delivery vehicles, such as lipid nanoparticles, that deliver nucleic acids, such as mRNA, that encode peptides, fusion proteins, or compositions of multiple peptides and/or fusion proteins as disclosed herein.

The immunogenic composition may comprise an excipient, which may act as an adjuvant. Thus, in an embodiment, immunogenic composition may comprise a microparticulate adjuvant, for example a liposome or an immune stimulating complex (ISCOM). Other examples of adjuvants include cholera toxin or a squalene-like molecule. Any adjuvant may be used, such as, for example, aluminium hydroxide (alum), tetanus toxoid, or diphtheria toxin. A vehicle may suitably be used for the adjuvant, which may include, but is not limited to, water, phosphate buffered saline (PBS), a polyol, or a dextrose solution.

The compositions of the eighth or ninth aspect may be formulated for a particular route of delivery. For instance, they may be formulated for intramuscular, subcutaneous, intravenous, oral, intranasal, or intradermal delivery. The compositions may be formulated for delivery to a particular subject, for instance formulated for delivery to neonates, babies, children, women of fertile age, pregnant women, foetuses, elderly subjects, or diabetic humans.

In a tenth aspect, there is provided a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third aspect or fourth aspect, a kit of the third aspect 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 for use in stimulating an immune response.

Preferably, the immune response includes the production of antibodies that are specific to GAPDH of one or more species of sepsis-inducing bacteria. The sepsis-inducing bacteria may be GBS, E. coli, Staphylococcus spp., S. pneumoniae, and/ or K. pneumoniae. In an embodiment, the Staphylococcus spp. is S. aureus. Thus, the GAPDHs for which the antibodies have specificity may be one or more of those having the amino acid sequences provided as SEQ ID NOs: 4 to 8. In a particular embodiment, the immune response may include the production of antibodies specific for the GAPDH of each of GBS, E. coli, Staphylococcus spp., S. pneumoniae, and K. pneumoniae.

Because GAPDH is a ubiquitous protein and conserved amongst the sepsis-inducing bacteria, an immune response may be stimulated that provides protection against all of the different serotypes of sepsisinducing bacteria, which is advantageous.

The use of the tenth aspect may be an in vitro, in vivo, or ex vivo use.

The use of the tenth aspect may be an in vitro or ex vivo use for the production of antibodies. Such uses may involve the interaction of a peptide, fusion protein, or composition as disclosed herein with antibodyproducing cells in vitro or ex vivo, such that antibodies that are specific for GAPDH of one or more species of sepsis-inducing bacteria may be produced. Suitable antibody-producing cells and techniques for producing antibodies are described in the art and will be known to the skilled person.

In another embodiment, the use of the tenth aspect is an in vivo use, i.e. for stimulating an immune response in a subject. The immune response maybe a protective immune response to one or more sepsisinducing bacterial species.

In an eleventh aspect, there is provided a peptide of the first aspect, a fusion protein of the second aspect, a composition of the third aspect or fourth aspect, the peptides of a kit of the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, a pharmaceutical composition of the eighth aspect, or an immunogenic composition of the ninth aspect for use as a medicament.

As discussed above, the peptides of a kit of the third or fourth aspect may be for use as a medicament. Thus, in an embodiment, there is provided a peptide comprising a sequence according to SEQ ID NO: 3, or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions, for use in a method of treatment, wherein the method comprises administering said peptide and also administering: a peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and/or a peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions. In another embodiment, there is provided a peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions, for use in a method of treatment, wherein the method comprises administering said peptide and also administering: a peptide comprising a sequence according to SEQ ID NO: 3 or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions; and a peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions.

In a further embodiment, there is provided a peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions, for use in a method of treatment, wherein the method comprises administering said peptide and also administering: a peptide comprising a sequence according to SEQ ID NO: 3 or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions; and a peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions.

In an embodiment, the peptide of the first aspect, fusion protein of the second aspect, composition of the third aspect or fourth aspect, peptides of a kit of the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, pharmaceutical composition of the eighth aspect, or immunogenic composition of the ninth aspect may be for use in treating, preventing, reducing the risk of, or reducing the severity of infection with one or more species of sepsis-inducing bacteria.

In an embodiment, there is provided a method of treating, preventing, reducing the risk of, or reducing the severity of infection with one or more species of sepsis-inducing bacteria, 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 aspect or fourth aspect, the peptides of a kit of the third aspect or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, a pharmaceutical composition of the eighth aspect, or an immunogenic composition of the ninth aspect.

A “therapeutically effective amount” of an agent is any amount which, when administered to a subject, is the amount of agent that is needed to treat, prevent, reduce the risk of, or reduce the severity of an infection or associated pathology, or produce the desired effect.

The peptides, fusion proteins, compositions, nucleic acids, vectors, or immunogenic compositions disclosed herein can prevent systemic infections caused by one or more of at least five different pathogens, being the most common causes of sepsis, preferably: GBS, E. coli, Staphylococcus spp., S. pneumoniae, and K. pneumoniae. In an embodiment, the Staphylococcus spp. is S. aureus. The peptides, fusion proteins, compositions, kits, nucleic acids, vectors, and immunogenic compositions disclosed herein may be for use in treating, preventing, reducing the risk of, or reducing the severity of infection caused by any one or more of said bacteria. The peptides, fusion proteins, compositions, kits, nucleic acids, vectors, and immunogenic compositions disclosed herein may be for use in treating, preventing, reducing the risk of, or reducing the severity of infection caused by GBS, E. coli, Staphylococcus spp., S. pneumoniae, and K. pneumoniae.

In particular, the peptide of the first aspect, fusion protein of the second aspect, composition of the third aspect or fourth aspect, peptides of a kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, pharmaceutical composition of the eighth aspect, or immunogenic composition of the ninth aspect may be for use in the treating, preventing, reducing the risk of, or reducing the severity of infectious diseases caused by a species of sepsis-inducing bacteria.

A peptide, fusion protein, composition, kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition as disclosed herein can be for use in treating, preventing, reducing the risk of, or reducing the severity of sepsis or any other disease, disorder, or condition caused by or associated with an infection with a species of sepsis-inducing bacteria. These other diseases, disorders or conditions include pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, and encephalitis.

Thus, in an embodiment, there is provided a method of treating, preventing, reducing the risk of, or reducing the severity of sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, or encephalitis, the method comprising administering, to a subject, a therapeutically effective amount of a peptide of the first aspect, fusion protein of the second aspect, composition of the third aspect or fourth aspect, peptides of a kit of the third or fourth aspect, one or more nucleic acids of the fifth aspect, one or more vectors of the sixth aspect, pharmaceutical composition of the eighth aspect, or immunogenic composition of the ninth aspect.

Pre-term births and stillbirths can be caused by an exacerbated inflammatoiy response induced by bacterial infections. In the cases of bacterial-induced pre-term births and stillbirths, the most common agents are GBS, E. coli and K. pneumoniae, i.e., the sepsis-inducing bacteria described herein. Premature births and stillbirths can be caused by in utero infections due to the ascending of bacteria (such as GBS, E. coli and Klebsiella spp.) from the genital tract into the amniotic fluid. By vaccinating the expectant mother, immunity is provided in the unborn offspring. Foetuses and neonates can thus be protected against infection through a maternal vaccination.

Thus, in an embodiment, a peptide, fusion protein, composition, kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition of the present disclosure is used for preventing or reducing the risk of a premature birth and/or stillbirth.

In a particular embodiment, the peptide, fusion protein, composition, kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition of the present disclosure is used to prevent or reduce the risk of a premature birth and/or stillbirth associated with infections caused by GBS, E. coli, Staphylococcus spp., S. pneumoniae, or K. pneumoniae. In an embodiment, the Staphylococcus spp. is S. aureus. In another embodiment, the peptide, fusion protein, composition, kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition of the present disclosure is used to prevent or reduce the risk of a premature birth and/or stillbirth associated with infections caused by GBS, E. coli, or K. pneumoniae.

Administration of the peptide, fusion protein, composition, peptides of a kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition may be to women of fertile age. A boost may be delivered in the third trimester of gestation. Unborn infants (foetuses) benefit from passive immunity acquired when their mothers’ antibodies cross the placenta to reach the developing child, especially in the third trimester. As illustrated by the Examples, a vaccine of the invention can also prevent or the reduce the risk of premature births and stillbirths caused by in utero infections due to the ascending of bacteria (such as GBS, E. coli and Klebsiella spp.) from the genital tract into the amniotic fluid.

The subjects to be treated may be any suitable, for instance any susceptible to the diseases or conditions discussed herein. The subject may be a vertebrate, mammal, or domestic animal. Hence, the administration maybe to any mammal, for example livestock (for example, a horse), pets, or maybe used in other veterinary applications. Most preferably, the subject is a human being.

Particularly relevant groups of subjects include people that are at greater risk of infection. For instance, immunocompromised people, neonates, babies, children, women of fertile age, pregnant women, foetuses, elderly subjects, and diabetic humans.

The peptide, fusion protein, composition, peptides of a kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition may be administered before, during, or after onset of the bacterial infection. The presently disclosed treatments may be prophylactic; that is to say they may be used to prevent, reduce the risk of, or reduce the severity of an infection, including preventing, reducing the risk of, or reducing the severity of a relapse/recolonisation of a previous infection. The peptides, fusion proteins, compositions, peptides of a kit, nucleic acids, vectors, or immunogenic compositions as disclosed herein may be administered to those already showing signs of an infection, those considered to be immunocompromised, or those at greater risk of an infection. Administration can be to apparently healthy subjects as a purely preventative measure against the possibility of such an infection in future. For example, it can be administered as part of a general vaccination programme or a programme targeted to the immunocompromised, neonates, babies, children, women of fertile age, pregnant women, foetuses, the elderly, and diabetics.

As used herein, “immunocompromised” encompasses both those with genetic or acquired immune system defects, and also those who have less immune function due to their life stage. For instance, neonates, babies, children, women of fertile age, pregnant women, foetuses, the elderly, and type I diabetics may all be considered to be immunocompromised.

The terms “neonate” and “newborn”, as used herein, can refer to a human child from birth to around one month old. The terms apply to premature infants, postmature infants and full-term infants. Before birth, the term “foetus” is used. The terms “baby” and “infant”, as used herein, can refer to young children between the ages of around one month and around one or two years of age (i.e., the age when a child learns to walk and talk, when the term “toddler” may be used instead). The term “child”, as used herein, refers to young children, covering those from toddlers to around 12 years of age, i.e., the pre-teens. The term “elderly”, as used herein, refers to subjects of advanced age. For example, it can refer to men and women aged 60 or over, 65 or over, 70 or over, 75 or over, or 80 or over. Non-human subjects in the corresponding later years of life are also encompassed by this term. In an example, the term “diabetic”, as used herein, refers to a person suffering from diabetes mellitus type I (also known as juvenile or insulindependent diabetes) or diabetes mellitus type II. Type I diabetics may be considered to be immunocompromised and the invention is also relevant to type II diabetics because they may be at greater risk of infection.

The peptide, fusion protein, composition, peptides of a kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition may be administered by any suitable means, including by injection or mucosally. The administration may be intramuscularly, subcutaneously, intravenously, orally, intranasally, or intradermally.

The route of administration maybe varied depending on the mechanism of protection that is desired. For instance, if the presently disclosed agents are administered mucosally, they may induce an IgA response at the mucosal surface. IgA may block binding of the bacterial GAPDH to the host cell epithelium. If the agents are administered systemically, they may induce the production of an IgG response, which may block the binding of bacterial GAPDH to TLR2 on the surface of Bl cells and prevent early IL-10 production by these cells. When the administration is of the peptides of a kit of the third or fourth aspect, the peptides may be administered simultaneously or sequentially. The peptides may be delivered on the same day or on separate days. In particular embodiments, the peptides are administered as a mixture. In more particular embodiments, the peptides are administered as a single composition.

It will be appreciated that the agents disclosed herein may be used in a monotherapy, for treating, ameliorating, or preventing an infection with sepsis-inducing bacteria or an associated pathology. Alternatively, the agents disclosed herein may be used as an adjunct to, or in combination with, known therapies. For example, known agents for treating sepsis-inducing bacterial infections. For example, the peptides, fusion proteins, compositions, nucleic acids, vectors, pharmaceutical compositions, and immunogenic compositions may be used in combination with known agents for treating neonatal sepsis caused by fungi or viruses. They can be used in combination with known anti-retroviral agents.

The agents disclosed herein can be administered alone or simultaneously with other existing vaccines. For example, administration may be simultaneous with vaccines recommended for the immunocompromised, babies, children, women of fertile age, pregnant women, the elderly, and diabetics (such as, for example, tetanus and diphtheria vaccine).

There is no restriction on which peptide, fusion protein, composition, peptides of a kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition as described herein should be administered to which patient. Rather, it is intended that any of the products described herein can be administered to any patient as described herein. It is expressly intended by the inventors, in fact, that each and every combination of peptide, fusion protein, composition, peptides of a kit, nucleic acid, vector, pharmaceutical composition, or immunogenic composition, and indicated patient group, is encompassed by the present disclosure. The disclosure thus includes each and every possible combination of therapeutic agent and indicated patient group.

It will be appreciated that the amount of peptide, fusion protein, composition, nucleic acid, vector, pharmaceutical composition, or immunogenic composition that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the bacterial infection. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Sequence comparisons can be conducted with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

The skilled technician will appreciate how to calculate the percentage identity between two nucleic acid or two amino acid sequences. In order to calculate the percentage identity, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, the Needleman-Wunsch algorithm (e.g. as applied by Needle(EMBOSS) or Stretcher(EMBOSS), the Smith-Waterman algorithm (e.g. as applied by Water(EMBOSS)), or the LALIGN application e.g. as applied by Matcher(EMBOSS); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to gaps. Having made the alignment, there are different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

A calculation of percentage identities between two nucleic acid sequences may then be calculated from such an alignment as (N/T)*too, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.

The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In an example, the identity between two amino acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (14), gap extend (4), alternative matches (1). In an example, the identity between two nucleic acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).

In a particular embodiment, the sequence identities disclosed herein may be calculated based on a global alignment of the relevant feature, for instance the comparison of a complete length of the antigenic portion of a peptide to a complete reference sequence recited herein. As an example, the sequence identities may be calculated between the complete length of the portion of a peptide or fusion protein that corresponds to a portion of a bacterial GAPDH and any sequence within any one of SEQ ID NOs: 4 to 8.

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

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way.

EXAMPLES

Summary

Peptides designed to induce immune responses to bacterial GAPDH were obtained. A cysteine was added to all the peptides to facilitate anchoring to KLH. Also, a beta-alanine was introduced in the structure of all the peptides in the final formulation to facilitate quality control analysis of peptide production and conjugation.

Surprisingly, the inventors observed that a specific formulation, designated herein as PNV, led to a synergistic immunogenic effect. The immunogenicity of the formulation towards the bacterial GAPDHs was higher than that observed with the individual peptides. This synergistic effect did not happen with other formulations tested.

In summary, the experimental data herein demonstrate: a) A synergistic effect induced by a specific combination of three peptides; b) The aleatoiy substitution and deletion of amino acids within a peptide that leads to a stable and immunogenic peptide; c) That the use of this specific formulation - composed of three peptides individually conjugated with KLH at a ratio of 1:1: (w/w) - as a preventive vaccine, showed better immunogenicity and protective effect than any other tested combination of peptides with or without a different carrier protein.

Figure 1 shows the immunogenicity of different conjugated peptide formulations towards different bacterial GAPDH. Illustrated are serum anti-GAPDH IgG titers obtained by ELISA from mice immunized with the different formulations of GAPDH peptides conjugated with KLH or CRM. Adult BALB/c mice (6-8 weeks) received three sc immunizations, with a 3-week interval between administrations, with the indicated formulations. Each formulation contained 1OO pg of each conjugated peptide and 125 pg of aluminium hydroxide as adjuvant (Alhydrogel 2%) suspended in saline solution (NaCl 0.9%). One week after the last administration blood was collected and serum IgG titres were determined by ELISA as the value of serum dilution were Abs₄5onm was < 0.1. Plates were coated with the GAPDH of the bacteria indicated in the abscises.

Figure 2 shows that PNV immunogenicity is higher than the sum of the immunogenicity for the individual peptides. Illustrated are serum anti-GAPDH IgG titers obtained by ELISA from mice immunized with PNV or the individual peptides conjugated with KLH. Adult BALB/c mice (6-8 weeks) received three sc immunizations, with a 3-week interval between administrations, with PNV or with 100 pg of the indicated conjugated peptides [with 125 pg of aluminium hydroxide as adjuvant (Alhydrogel 2%) suspended in saline solution (NaCl 0.9%]. One week after the last administration blood was collected and serum IgG titres were determined by ELISA as the value of serum dilution were Abs₄5onm was < 0.1. Plates were coated with the GAPDH of the bacteria indicated in the graphs. P values are indicated. ***P< 0.0001; **P< 0.001.

Figure 3 shows that PNV protects adult mice from bacterial infections. Illustrated are survival curves after infection with the indicated bacteria of adult mice immunized with PNV (green line) or sham- immunized (red line) with the adjuvant alone. Adult BALB/c mice (6-8 weeks) received three sc immunizations, with a 3-week interval between administrations, with PNV or with the adjuvant alone. One week after the last immunization mice were challenged intra-peritoneally (ip) with 10“ CFU of S. aureus, with 10“ CFU of K. pneumoniae, with 10⁸ CFU of S. pneumoniae, or with 10“ CFU of E. coli. At least three independent experiments were performed. P values are indicated in the graphs. In parenthesis are shown the number of animals that survived vs the total number of animals infected. Differences between treatments were assessed using the log-rank test.

Figure 4 shows that maternal vaccination with PNV protects offspring from bacterial infections. Illustrated are survival curves after infection of pups born from PNV-immunized (green line) or sham- immunized (red line) dams. Adult C57BL/ 6 females (6-8 weeks) were sc immunized with PNV or with the adjuvant alone (sham-immunized). The animals received two administrations before mating, with a three-week interval between administrations. A boost immunization was administered at gestational day 14. Neonates were infected sc 48 hours after birth with 101 CFU of GBS, with to⁶ CFU of S. aureus, or with 5 x 104 CFU of E. coli. At least three independent experiments were performed. P values are indicated in the graphs. In parenthesis are shown the number of animals that survived vs the total number of animals infected. Differences between treatments were assessed using the log-rank test. Example 1 — Discovery of an immunogenic formulation

Neutralization of bacterial GAPDH proved to be a good strategy to prevent infections in mice caused by GBS, E. coli, S. aureus, K. pneumoniae, and S. pneumoniae. The major obstacle in 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 completely absent from human GAPDH. A list of peptides that can be targeted on the different bacterial GAPDH without cross-reactivity with the human protein is provided in WO2O15/189422 Al.

Six different formulations were generated. Formulation #1 is composed of three different peptides representing bacterial GAPDH antigens; formulation #2 is composed of two different peptides representing bacterial GAPDH antigens; formulation #3 is composed of two different peptides representing bacterial GAPDH antigens; formulation #4 is composed of two fusion proteins each comprising two peptides representing bacterial GAPDH antigens; formulation #5 is composed 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 is composed of four different peptides representing bacterial GAPDH antigens.

The peptides were chemically synthetized and conjugated to a carrier protein. Keyhole Limpet Haemocyanin (KLH) or Cross-Reactive Material 197 (CRM) were used as carrier proteins in a ratio of 1:1 (w/w). For conjugation, an additional cysteine was added to the C-terminus of the peptide amino acid sequences to allow chemical conjugation with the carrier proteins. Formulations #1 to #5 included KLH as a carrier protein. Formulation #6 included CRM as a carrier protein.

The formulations were generated to contain a previously determined amount of each conjugated peptide (lOOug/peptide) in saline solution (NaCl 0.9%) with 0.125 mg of aluminum hydroxide (Alhydrogel 2%, Croda) as an adjuvant. Mice were then immunized subcutaneously (sc) with the formulations three times, with a 3-week interval between administrations. The serum anti-GAPDH IgG titers observed after immunization with the different formulations are depicted in Figure 1.

A particular peptide was of interest because it contains a domain that is conserved in different bacterial GAPDHs. The chemical synthesis of this peptide (TQTEITAVGDLQLVKTVA - SEQ ID NO: 1) yielded a non-stable product that was not possible to use in mice immunizations. Considering that this peptide may constitute an important target for the neutralization of a common domain in GAPDH of different bacteria, the inventors tried to synthesize different variations of it, containing random deletions or inclusion of amino acids outside the conserved domain. One of these variations yielded a soluble and stable product (SEQ ID NO: 3) that could be conjugated with KLH and used for mice immunizations. In addition, the inventors also considered chimeras of different GAPDH peptides, obtained by putting together in the same sequence two peptides from different bacterial GAPDHs. Formulation #4 is constituted by two chimeras, the first one with a peptide exposed on GBS GAPDH fused to a peptide exposed on S. aureus GAPDH. The second chimera with peptides corresponding to E. coli and K. pneumoniae GAPDH.

As observed in Figure 1, different formulations resulted in different immunogenicity in mice. Interestingly, Formulation #5 was the one that induced higher immunogenicity in mice, as observed by the higher anti-GAPDH IgG titres. Introducing a new peptide 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 the best results.

Considering that Formulation #5 was the one that showed the best immunogenicity in mice, the inventors introduced an extra amino acid, p-alanine, (PA) next to the terminal cysteine in each peptide. The addition of pA improves analytic procedures, allowing better quality control measurements. This final formulation is designated as Paragon Novel Vaccine (PNV). The final amino acid sequences of the peptides are listed in Table i. The addition of an extra amino acid did not change the immunogenicity of this formulation, as PNV immunization resulted in the same anti-GAPDH IgG titres as Formulation #5 (Figure 2).

Example 2 — Demonstration of synergy

The inventors tested the immunogenicity of each of the individual conjugated peptides in PNV (Table 1). For that, different groups of adult mice received three sc administrations with each peptide in saline solution plus Alhydrogel 2% as adjuvant, as referred above. Interestingly, the immunogenicity obtained for each peptide towards the different bacterial GAPDH was significantly lower than the one obtained for PNV. In fact, peptides together in PNV formulation act synergistically to induce higher anti-GAPDH IgG titres than the sum of the IgG titres observed for each of the individual peptides (Figure 2). Serum anti-GAPDH IgG titres in Figure 2 are presented in total numbers, rather than in logio scale to enable the observation of the synergistic effect.

Example 3 — Prevention of infection

Finally, the inventors tested the efficacy of PNV to prevent infections caused by the different GAPDH- excreting bacteria. As depicted in Figure 3, vaccination of adult mice with PNV significantly increased survival of mice upon infections with S. aureus, E. coli, S. pneumoniae, and K. pneumoniae and when compared with control animals that received the adjuvant alone. GBS is almost exclusively associated with neonatal infections. GBS GAPDH was also described as an immunosuppressive protein strongly associated with neonatal susceptibility to these infections. As PNV also targets GBS GAPDH, the inventors tested the efficacy of PNV as a maternal vaccine to prevent neonatal bacterial infections caused by GBS. As observed in Figure 4, PNV successfully protected neonates from lethal GBS infection. Moreover, and as S. aureus and E. coli are important neonatal pathogens, the inventors showed that maternal vaccination with PNV also protected mice pups from lethal infections with these two bacterial pathogens (Figure 5).

Altogether, these results show that PNV contains a specific formulation of peptides exposed on bacterial GAPDH that possess synergistic immunogenic abilities and can effectively protect adult mice from lethal infections caused by E. coli, K. pneumoniae, S. aureus and S. pneumoniae, and neonatal mice from infections caused by GBS, S. aureus and E. coli.

Claims

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

2. A peptide according to claim t, wherein the peptide is equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length.

3. A peptide according to claim 1 or claim 2, wherein sequences flanking SEQ ID NO: 3 are derived from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

4. A peptide according to any preceding claim, wherein the sequences flanking SEQ ID NO: 3 are derived from SEQ ID NO: 5.

5. A peptide according to any preceding claim, wherein the peptide comprises less than 8, 7, 6, 5, 4, or 3 contiguous residues from within SEQ ID NO: 9.

6. A peptide according to any preceding claim, wherein the peptide comprises an N-terminal or C- terminal cysteine.

7. A peptide according to any preceding claim, wherein the peptide comprises a beta alanine.

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

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

10. A peptide according to any preceding claim, wherein the peptide is according to SEQ ID NO: 12.

11. A peptide according to any preceding claim, wherein the peptide is linked to a carrier protein.

12. A peptide according to any preceding claim, 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 a peptide according to any one of claims 1 to 12.

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

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

16. A composition or kit that comprises a peptide according to any one of claims 1 to 12 or a fusion protein according to any one of claims

13 to 15, and a peptide that comprises an amino acid sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and/or a peptide that comprises an amino acid sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO:

14 with one or more additions, substitutions, or deletions.

17- A composition or kit that comprises a first peptide according to any one of claims 1 to 12, a second peptide that comprises an amino acid sequence according to SEQ ID NO: 13, and a third peptide that comprises an amino acid sequence according to SEQ ID NO: 14.

18. A composition or kit according to claim 16 or claim 17, wherein: the peptide comprising SEQ ID NO: 13 comprises SEQ ID NO: 15

(EVKEGGFEVNGKFIKVSA(bALA)C); and/or the peptide comprising SEQ ID NO: 14 comprises SEQ ID NO: 16

(DVTVEQVNEAMKNASNESF(bALA)C).

19. A composition or kit according to any one of claims 16 to 18, wherein the peptide comprising SEQ ID NO: 13:

(i) is equal to or less than 150 residues, too residues, 50 residues, or 30 residues in length; and/ or

(ii) comprises sequences flanking SEQ ID NO: 13 that are 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) comprises sequences flanking SEQ ID NO: 13 that are derived from SEQ ID NO: 7; and/or

(iv) comprises less than 8, 7, 6, 5, 4, or 3 contiguous residues from within SEQ ID NO: 9; and/or

(v) has from 0-12 additional amino acids at the N-terminal and/or C-terminal side of SEQ ID: 13 or SEQ ID NO: 15.

20. A composition or kit according to any one of claims 16 to 19, wherein the peptide comprising SEQ ID NO: 14:

(ii) comprises sequences flanking SEQ ID NO: 14 that are 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) comprises sequences flanking SEQ ID NO: 14 that are derived from SEQ ID NO: 6; and/or

(v) has from 0-12 additional amino acids at the N-terminal and/or C-terminal side of SEQ ID: 14 or SEQ ID NO: 16.

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

22. A composition or kit comprising : a first peptide comprising a sequence according to SEQ ID NO: 3 or a sequence according to SEQ ID NO: 3 with one or more additions, substitutions, or deletions; a second peptide comprising a sequence according to SEQ ID NO: 13 or a sequence according to SEQ ID NO: 13 with one or more additions, substitutions, or deletions; and a third peptide comprising a sequence according to SEQ ID NO: 14 or a sequence according to SEQ ID NO: 14 with one or more additions, substitutions, or deletions.

23. A composition or kit according to any one of claims 16 to 22, wherein: the peptide comprising SEQ ID NO: 3 comprises a beta alanine; the peptide comprising SEQ ID NO: 13 comprises a beta alanine; and/or the peptide comprising SEQ ID NO: 14 comprises a beta alanine.

24. A composition or kit according to any one of claims 16 to 23, wherein: the peptide comprising SEQ ID NO: 3 comprises an N-terminal or C-terminal cysteine; the peptide comprising SEQ ID NO: 13 comprises an N-terminal or C-terminal cysteine; and/or the peptide comprising SEQ ID NO: 14 comprises an N-terminal or C-terminal cysteine.

25. A composition or kit according to any one of claims 16 to 24, wherein: 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.

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

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

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

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

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

31. A 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 the peptide according to any one of claims 1 to 12, the fusion protein according to any one of claims 13 to 15, or the peptides of the composition or kit according to any one of claims 16 to 31.

33. One or more vectors comprising the one or more nucleic acids of claim 32.

34. A cell comprising the one or more nucleic acids of claim 32 or the one or more vectors of claim 33-

35. One or more pharmaceutical compositions comprising 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, or the one or more vectors of claim 33.

36. One or more immunogenic compositions comprising 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, or the one or more vectors of claim 33.

37. Use of 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, the one or more vectors of claim 33, the one or more pharmaceutical compositions of claim 35, or the one or more immunogenic compositions of claim 36 for stimulating an immune response.

38. A use of claim 37, wherein the immune response is directed to one or more species of sepsisinducing bacteria.

39. 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, the one or more vectors of claim 33, the one or more pharmaceutical compositions of claim 35, or the one or more immunogenic compositions of claim 36 for use as a medicament.

40. 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, the one or more vectors of claim 33, the one or more pharmaceutical compositions of claim 35, or the one or more immunogenic compositions of claim 36 for use in preventing, reducing the risk of, or reducing the severity of an infection by one or more species of sepsis-inducing bacteria.

41. The use of claim 38 or the peptide, fusion protein, composition, peptides of a kit, one or more nucleic acids, one or more vectors, one or more pharmaceutical compositions, or one or more immunogenic compositions for use of claim 40, wherein the one or more species of sepsis-inducing bacteria is GBS, E. coli, Staphylococcus spp., S. pneumoniae, and/or K. pneumoniae.

42. 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 peptides of a kit according to any one of claims 16 to 31, the one or more nucleic acids of claim 32, the one or more vectors of claim 33, the one or more pharmaceutical compositions of claim 35, or the one or more immunogenic compositions of claim 36 for use in: preventing, reducing the risk of, or reducing the severity of any one or more of sepsis, pneumonia, meningitis, endocarditis, enterocolitis, urinary tract infections, soft tissue infections, gastrointestinal infections, bloodstream infections, and encephalitis, or preventing or reducing the risk of premature birth or stillbirth.

43. The peptide, fusion protein, composition, peptides of a kit, one or more nucleic acids, one or more vectors, one or more pharmaceutical compositions, or one or more immunogenic compositions for use of any one of claims 39 to 42, wherein the subject is immunocompromised, a neonate, a baby, a child, a woman of fertile age, a pregnant woman, a foetus, a diabetic, and/or an elderly person.